Denitrifying

Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 653 - 5
Crystallization and preliminary X-ray analysis of nitrous oxide reductase from Paracoccus pantotrophus; Jafferji A et al.; Nitrous oxide reductase is a periplasmic respiratory protein with a novel copper catalytic centre; it catalyses the terminal step, reduction of nitrous oxide to nitrogen, of the bacterial denitrification process . Nitrous oxide reductase from Paracoccus pantotrophus has been crystallized by the hanging-drop method . A prerequisite for crystallization was the oxidation of the enzyme with potassium ferricyanide in order to obtain homogenous oxidation states of the copper centres . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 116.4, b = 118.3, c = 267.0 A . Two homodimers, of approximate molecular weight 67 kDa per subunit, probably constitute the asymmetric unit and give a Matthews coefficient, V(m), of 3.4 A(3) Da(-1) and a solvent content of 59% by volume . The crystals diffract X-rays to 3.0 A resolution on an in-house source and are suitable for structure determination.

Biochemistry, 2000 Apr 25, 39(16), 4929 - 38
Unusual spectroscopic and electrochemical properties of the 2{4Fe-4S} ferredoxin of Thauera aromatica; Boll M et al.; A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica . It contains two {4Fe-4S} clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension . The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both {4Fe-4S} clusters from T . aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR . Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE . X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced {4Fe-4S} cluster . No typical S = (1)/(2) EPR signals were observed . At lower potentials (less than -500 mV), the more negative {4Fe-4S} cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin {4Fe-4S} cluster with a g(av) of 1.94 . However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals . At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2) . The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the {4Fe-4S} clusters . The observation of different spin states in T . aromatica ferredoxin is novel among CvFd-type ferredoxins.

J Biol Chem, 2000 Jul 21, 275(29), 21889 - 95
Nonaromatic products from anoxic conversion of benzoyl-CoA with benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase; Boll M et al.; The enzymes benzoyl-CoA reductase and cyclohex-1, 5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica . Reaction products obtained with {ring-(13)C(6)}benzoyl-CoA and {ring-(14)C}benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy . The main product obtained with titanium(III) citrate or with reduced {8Fe-8S}-ferredoxin from T . aromatica as electron donors was identified as cyclohexa-1, 5-diene-1-carbonyl-CoA . The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase . Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA . The potential required for the first electron transfer to the model compound S-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as -1.9 V versus a standard hydrogen electrode . The energetics of enzymatic ring reduction of benzoyl-CoA are discussed.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 909 - 15
Differentiation of newly described antarctic bacterial isolates related to Roseobacter species based on 16S-23S rDNA internal transcribed spacer sequences; Soller R et al.; The 16S-23S rDNA internal transcribed spacer (ITS) of Roseobacter denitrificans, Roseobacter litoralis, Ruegeria algicola and strains of the recently described species Antarctobacter heliothermus and Roseovarius tolerans were analysed in order to examine DNA sequence variations and to draw conclusions about inter- and intraspecific relationships . A . heliothermus included four strains with an ITS fragment length of 1092 bp . Roseovarius tolerans was described on the basis of eight strains . Five of these harboured two ITS fragments of different lengths (959 and about 1100 bp), while the others had one fragment of either 1083 bp (two strains) or 1165 bp (one strain) . ITS lengths of the related species Roseobacter denitrificans, Roseobacter litoralis and Ruegeria algicola were found to be 980, 984 and 1158 bp, respectively . Phylogenetic analyses of the DNA sequences allowed species affiliation of strains with sequence length differences of > 200 bp and recognition of relationships based on a well-supported ITS tree . The strains of A . heliothermus and Roseovarius tolerans each formed a monophyletic branch and they were separated from each other by Ruegeria algicola . This species was now clearly separated from Roseobacter denitrificans and Roseobacter litoralis, which corresponded to the new genus affiliation of Ruegeria algicola . These data were additionally supported by analyses of the structure, relative position and order of genes for tRNA(Ile) and tRNA(Ala) found within the ITS of each strain . Comparative DNA sequence analyses of ITS and 16S rDNA revealed limitations, on species and strain levels, with respect to the phylogenetic resolution of the 16S rDNA due to the limited number of informative (variable) sites, while ITS sequence analyses provided more variable and sufficiently conserved positions to discriminate between strains and to reconstruct their taxonomic relationships.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 547 - 50
Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain; Kelly DP et al.; Thiobacillus denitrificans is physiologically similar to the type species of the genus Thiobacillus, Thiobacillus Thioparus, and both are located in the beta-subclass of the Proteobacteria . T . denitrificans is distinguished from all other Thiobacillus species by its ability to grow as a facultatively anaerobic chemolithotroph, coupling the oxidation of inorganic sulfur compounds to the reduction of nitrate, nitrite and other oxidized nitrogen compounds to dinitrogen . A definitive description of this species is provided and strain NCIMB 9548T is designated as the type strain of the species, thereby correcting an earlier error in the literature.

Acta Microbiol Pol, 1999, 48(3), 297 - 306
Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures; Son TT et al.; Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa . In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions . As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively . Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively . Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L.

Appl Environ Microbiol, 2000 Apr, 66(4), 1595 - 601
Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions; Rockne KJ et al.; Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene . The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate . Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4 . Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay . Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite . Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent . No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls . Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added (14)C-labeled naphthalene . After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites . Nitrate consumption, along with the results from the (14)C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4 . Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius . This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.

Appl Environ Microbiol, 2000 Apr, 66(4), 1564 - 71
Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples; Michotey V et al.; Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples . The specificity and sensitivity of these primers were tested . Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques . Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method . Enumerations using both molecular techniques yielded similar results in seawater and sediment samples . However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.

Appl Environ Microbiol, 2000 Apr, 66(4), 1360 - 8
Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis; Fernandez L et al.; The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme . The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues . The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified . The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases . The protein was overproduced up to 170-fold in L . lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria . The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector . This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene . This confirms that EstA is the main enzyme responsible for esterase activity in L . lactis . Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids . Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently . Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes . We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.

Appl Environ Microbiol, 2000 Apr, 66(4), 1286 - 91
Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol; Shinoda Y et al.; Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp . strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively . Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process . Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions . Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group . Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis . It grew on phenol only under denitrifying conditions . Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.

J Bacteriol, 2000 Apr, 182(8), 2179 - 83
Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8; Ramirez S et al.; Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration . In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell . We describe the presence of two genes, narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters . The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters . To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes . Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability . Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type . These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters . The possible differential roles of these proteins in vivo are discussed.

FEBS Lett, 2000 Mar 24, 470(2), 216 - 20
Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui; Yoshimatsu K et al.; Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state . The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide . The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex . Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate . The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl.

Biochemistry, 2000 Mar 21, 39(11), 2989 - 96
Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a; Lee HM et al.; As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient . X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels . The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase {Yoshikawa et al . (1998) Science 280, 1723-1729} . In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis . Mutants were generated in eight different sites and the enzymes have been purified and characterized . The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans {Pfitzner et al . (1998) J . Biomembr . Bioenerg . 30, 89-93} . Each of the mutants in R . sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes . This includes H456A, where in the P . denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme . The only mutations that result in completely inactive enzyme in the set examined in the R . sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures . To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained . The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes . Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data . In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity . The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group . Unlike R52, however, Q471 does not appear to be critical for the enzyme function.

Microbiology, 2000 Feb, 146 ( Pt 2), 509 - 16
Transcriptional analysis of the nirS gene, encoding cytochrome cd1 nitrite reductase, of Paracoccus pantotrophus LMD 92.63; Saunders NF et al.; The gene for cytochrome cd1 nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS . This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d1 cofactor (nirE) . Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic . The lack of a transcript under aerobic conditions argues against a role for cytochrome cd1 in the previously proposed aerobic denitrification pathway in Pa . pantotrophus . A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified . The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant . The transcript start point for nirS has been determined by two complementary techniques, 5'-RACE (Rapid amplification of cDNA 5' ends) and primer extension . It is 29 bp upstream of the AUG of nirS . An anaerobox, which presumably binds Nnr, is centred a further 41.5 bp upstream of the transcript start . No standard sigma70 DNA sequence motifs can be identified, but a conserved sequence (T-T-GIC-C-G/C-G/C) can be found in approximately the same position (-16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide.

Anal Biochem, 2000 Mar 15, 279(2), 202 - 8
Analysis of nitrite/nitrate in biological fluids: denitrification of 2-nitropropane in F344 rats; Sohn OS et al.; 2-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats . To obtain evidence that denitrification of 2-NP also occurs in vivo, we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction . However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na(2)CO(3), and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H(3)PO(4) . With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment . Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 microM (PB-pretreated) and 940 microM (vehicle-pretreated controls) . The PB-pretreated and control rats, respectively, excreted 250 and 120 micromol nitrate/nitrite in the 24-h urine post 2-NP treatment . The modifications described make the method more specific, reproducible, and more widely applicable .

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 61 - 71
Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster; Hayashi NR et al.; Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila) . cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria . cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins . When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE . However, the cell extract had slightly higher activity than a cell extract of E . coli without cbbA . Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H . thermoluteolus was in class IIA . Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions . The activities of the two enzymes were regulated independently.

Appl Environ Microbiol, 2000 Mar, 66(3), 1209 - 12
Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans; Barak Y et al.; Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions . Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source . Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source . By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P . denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches . Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.

Appl Environ Microbiol, 2000 Mar, 66(3), 1167 - 74
Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis; Layton AC et al.; The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis . 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates . Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones . Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources . Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species . This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp . strain M3 isolated from this treatment plant . A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp . strain M3 . Phylogenetic analysis revealed that Hyphomicrobium sp . strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II . Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp . strain M3 . The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity) . Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure . Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold . Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

Appl Environ Microbiol, 2000 Mar, 66(3), 1147 - 51
Transcriptional analysis of the tutE tutFDGH gene cluster from Thauera aromatica strain T1; Coschigano PW; The denitrifying strain T1, identified as Thauera aromatica, is able to grow with toluene serving as its sole carbon source . Previous work identified two genes, tutD and tutE, that are involved in toluene metabolism . Two small open reading frames, tutF and tutG, which may also play a role in toluene metabolism, were also identified . The present work examines the transcriptional organization and regulation of these toluene utilization genes . Northern analysis indicates that the four genes are organized into two operons, tutE and tutFDG, and that both operons are regulated in response to toluene . Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the tutE translational start and 76 bp upstream of the tutF translational start . Furthermore, a fifth gene, tutH, has been identified immediately downstream of tutG . It is transcribed from the same start site as tutFDG and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da . The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins.

Biochemistry, 2000 Feb 15, 39(6), 1356 - 63
Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans . Evidence from FTIR difference spectroscopy and site-directed mutagenesis; Behr J et al.; By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates {Behr, J., Hellwig, P., Mantele, W., and Michel, H . (1998) Biochemistry 37, 7400-7406} . To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I . The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy . Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared . Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase . The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.

Eur J Biochem, 2000 Feb, 267(4), 993 - 1000
Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase; Pacheco-Moises F et al.; Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied . Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin . Among the oxyanions, sulfite induced the higher increase in ATPase activity . Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax . There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme . Dithiothreitol did not affect the ATPase activity . ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin . In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin . It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.

Chemosphere, 2000 Mar, 40(5), 549 - 55
Biological denitrification in a closed seawater system; Grguric G et al.; Build-up of high nitrate concentrations in closed seawater systems where primary productivity is undesirable and water changes are impractical presents unique problems . Nitrate concentration in Ocean Tank at the New Jersey State Aquarium reached 9500 microM after 6 years of operation . A biological denitrification system was installed in 1998 and nitrate concentration in the aquarium decreased to 7000 microM within the first 100 days of operation . The system offers additional benefits by increasing the pH and alkalinity of seawater and providing a reducing environment to balance the oxidizing disinfection environment in the aquarium . The initial performance of the denitrification system was monitored and two semi-empirical models were developed: one based on the actual methanol additions, and another based on the daily amounts of nitrogen gas removed . The first model predicts a net nitrate decrease of 39 microM/day in the aquarium . The second model predicts a net decrease of 25 microM/day, in good agreement with the empirical value of 23 microM/day . This indicates that nitrogen gas removal is the controlling factor during denitrification in this facility, and the second model can be used to predict and optimize the operation of the system.

Appl Environ Microbiol, 2000 Feb, 66(2), 493 - 8
Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions; Chayabutra C et al.; A strategy for sequential hydrocarbon bioremediation is proposed . The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand . The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate . To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation . The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first . Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting . The effects of different nitrate and nitrite concentrations were examined next . When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations . However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth . Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter) . Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions . Sequential hexadecane biodegradation by P . aeruginosa was then investigated . The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations . The culture was then supplemented with nitrate and purged with nitrogen (N(2)) . Nitrate was consumed rapidly initially . The live cell concentration, however, also decreased . The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period . Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 23 - 9
Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression; Revers LF et al.; Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype . Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected . ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.

Arch Microbiol, 2000 Jan, 173(1), 58 - 64
Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria; Ehrenreich P et al.; The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor . Three distinct types of denitrifying bacteria were isolated in pure culture . A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C6H14) . Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C8H18) . A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C16H34) . Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains . Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen.

J Biol Chem, 2000 Jan 21, 275(3), 1691 - 8
"ADP sulfurylase" from Thiobacillus denitrificans is an adenylylsulfate:phosphate adenylyltransferase and belongs to a new family of nucleotidyltransferases; Bruser T et al.; During AMP-dependent sulfite oxidation by some sulfur bacteria, the liberation of sulfate from adenosine-5'-phosphosulfate (APS) is catalyzed by APS:phosphate adenylyltransferase (APAT) . Here we report the first biochemical and genetic characterization of APAT . We isolated this enzyme from the chemolithoautotroph Thiobacillus denitrificans and cloned the corresponding gene . The enzyme is homodimeric with 41,387-Da subunits and exhibits a specific activity of 2100 micromol min(-1) mg(-1) . The K(m) values are K(m(APS)) = 300 microM and K(m(P(i))) = 12 mM . Catalysis occurs by a ping-pong mechanism with a covalently bound AMP as reaction intermediate . The arsenolysis of APS, but not of ADP, CDP, GDP, UDP, or IDP, is also catalyzed, indicating a specific and unidirectional function . The former enzyme name ADP-sulfurylase implies that the reverse reaction is catalyzed; therefore, this name should not be used any longer . Histidine modification of APAT results in complete inactivation that can be suppressed by substrate addition . APAT is highly similar to galactose-1-phosphate uridylyltransferase and also related to Ap(4)A phosphorylase . Active site residues of galactose-1-phosphate uridylyltransferase are conserved in APAT and Ap(4)A phosphorylase, suggesting a histidine as the nucleotide-binding residue in all three enzymes, which together form a new family of nucleotidyltransferases.

Biosci Biotechnol Biochem, 1999 Nov, 63(11), 2020 - 2
The subunit structure of nitrite reductase purified from the denitrifier Achromobacter cycloclastes; Inatomi K; The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution . In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit . Signal-peptide cleavage sites in its N-terminal region are discussed.

Eur J Biochem, 2000 Jan, 267(2), 422 - 33
Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans; Schwarze C et al.; Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions . The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation . By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA . Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes . This reaction involves cytochrome c551 in a diffusional process . Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol . Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin . The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool . Chromatophores contain approximately 20 ubiquinone-10 molecules per RC . At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation . Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer . Results can be rationalized in the framework of a Q-cycle-type model.

J Biol Inorg Chem, 1999 Dec, 4(6), 749 - 58
Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp . strain J and the structure of novel Az-iso2; Suzuki S et al.; Two azurin-type blue copper proteins, which are related to the electron-transfer processes involving methylamine/methanol oxidation, have been spectroscopically and electrochemically characterized . The obligate methylotroph Methylomonas sp . strain J gives rise to two azurins (Az-isol and Az-iso2) with methylamine dehydrogenase (MADH-Mj) . The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620-630 nm in those of usual azurins . The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character . The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry . The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively . The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5 . However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1 . The structure of Az-iso2 has been determined and refined against 1.6 A X-ray diffraction data . The whole structure of Az-iso2 is quite similar to those of azurins reported already . The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different . These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.

Arch Microbiol, 2000 Nov, 174(5), 307 - 13
Evidence for a functional similarity between the two-component regulatory systems RegSR, ActSR, and RegBA (PrrBA) in alpha-Proteobacteria; Emmerich R et al.; The symbiotic bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti, and the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodovulum sulfidophilum, Roseobacter denitrificans and Rhodobacter sphaeroides possess homologous two-component regulatory systems, namely RegSR, ActSR, RegBA and PrrBA . The respective response regulators of these bacteria control expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance . We therefore asked whether the regulators are functionally exchangeable or whether they have disparate functions in the different species, despite the amino acid sequence similarity . In this study, we showed that purified B . japonicum RegR bound in vitro to genuine DNA targets for Rba . capsulatus RegA, and that RegA was phosphorylated in vitro when RegSc (a soluble variant of the sensor kinase RegS) was added to an Escherichia coli extract containing overexpressed RegA . In vivo, RegA and S . meliloti ActR activated transcription of the B . japonicum fixR-nifA operon, normally a target for RegR . The genes for both regulators, regA and actR, were able to complement a B . japonicum regR mutant with respect to the formation of a nitrogen-fixing symbiosis with soybean . Vice versa, RegR activated in Rba . capsulatus the expression of the photosynthesis operon puc, normally a target for RegA . In conclusion, the results show that B . japonicum RegR, Rba . capsulatus RegA, and S . meliloti ActR are functionally similar.

J Mol Biol, 2000 Jan 21, 295(3), 667 - 78
Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states; Harrenga A et al.; The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1 . 4 A for the reduced state . This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states . The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes . In general, the structure has the same features as that of known eukaryotic cytochromes c . However, the surface properties are very different . Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms . One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c . It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A . Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c . The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one .

FEBS Lett, 1999 Dec 3, 462(3), 416 - 20
Sequence conservation from human to prokaryotes of Surf1, a protein involved in cytochrome c oxidase assembly, deficient in Leigh syndrome; Poyau A et al.; The human SURF1 gene encoding a protein involved in cytochrome c oxidase (COX) assembly, is mutated in most patients presenting Leigh syndrome associated with COX deficiency . Proteins homologous to the human Surf1 have been identified in nine eukaryotes and six prokaryotes using database alignment tools, structure prediction and/or cDNA sequencing . Their sequence comparison revealed a remarkable Surf1 conservation during evolution and put forward at least four highly conserved domains that should be essential for Surf1 function . In Paracoccus denitrificans, the Surf1 homologue is found in the quinol oxidase operon, suggesting that Surf1 is associated with a primitive quinol oxidase which belongs to the same superfamily as cytochrome oxidase.

FEMS Microbiol Ecol, 2000 Jan 1, 31(1), 73 - 86
Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil; Chidthaisong A et al.; Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using {U-(14)C}glucose and {2-(14)C}acetate . The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate . Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not . These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations . Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O) . Addition of nitrate resulted in complete inhibition of CH(4) production from {U-(14)C}glucose and {2-(14)C}acetate . However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from {U-(14)C}glucose by only 70 and 65%, respectively . None of the electron acceptors significantly increased the production of (14)CO(2) from {U-(14)C}glucose, but all increased the production of (14)CO(2) from {2-(14)C}acetate . Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control . Addition of ferrihydrite and sulfate reduced (14)CH(4) production from {2-(14)C}acetate by 83 and 92%, respectively . Chloroform completely inhibited the methanogenic consumption of acetate . It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite . Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.

Biochim Biophys Acta, 2000 Jan 3, 1456(1), 1 - 4
Interaction between the formyl group of heme a and arginine 54 in cytochrome aa(3) from Paracoccus denitrificans; Riistama S et al.; The optical spectrum of heme a is red-shifted in aa(3)-type cytochrome c oxidases compared to isolated low-spin heme A model compounds . Early spectroscopic studies indicated that this may be due to hydrogen-bonding of the formyl group of heme a to an amino acid in the close vicinity . Here we show that most of the optical spectral shift of native heme a is due to a hydrogen-bonding interaction between the formyl group and arginine-54 in subunit I of cytochrome aa(3) from Paracoccus denitrificans, and that a smaller part is due to an electrostatic interaction between the D ring propionate of heme a and arginine-474.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14718 - 23
The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications; Giuffre A et al.; We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions . The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation . Thus, contrary to the eukaryotic enzyme, both T . thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at {NO} approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant . We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction . We propose that the NO reductase activity of T . thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure . These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases . Our findings represent functional evidence in support of this hypothesis.

J Biol Chem, 1999 Dec 31, 274(53), 37974 - 81
Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase; Kannt A et al.; The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential . The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O . Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species . A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3) . This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process . This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN . Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Mol Microbiol, 1999 Oct, 34(1), 24 - 36
Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein; Saunders NF et al.; The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively . The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of {4Fe-4S} clusters in many ferredoxin-like proteins . NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence . NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti . Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides . The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type . Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators . An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes . Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.

J Appl Microbiol, 1999 Sep, 87(3), 353 - 8
Effects of cultural conditions on denitrification by pseudomonas stutzeri measured by membrane inlet mass spectrometry
Firth JR, Edwards C.
Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter . Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium . MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen . A nitrate concentration of 15 mmol l-1 and a nitrite concentration of 5 mmol l-1 were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released . Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products . Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps . stutzeri is the amount of nitrite generated at the first enzymatic stage of the process . This appears to cause inhibition of the denitrification pathway above 5 mmol l-1 and at high enough concentrations (15 mmol l-1) restricts growth.

Microbiology, 1999 Nov, 145 ( Pt 11), 3265 - 71
Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate; Leutwein C et al.; Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate . This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica . Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds . The authors show here by experiments with cell extracts of toluene-grown T . aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible succinyl-CoA-dependent CoA-transferase . The product of the CoA-transferase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway . The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed . Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates . In accordance, the reaction of succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(-)-enantiomer.

Microbiology, 1999 Nov, 145 ( Pt 11), 3047 - 57
Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB; Page MD et al.; Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by omega insertion . Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids . All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa3 . Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not . The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant . In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT . Siderophore biosynthesis and/or secretion by P . denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD . These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG.

Appl Environ Microbiol, 1999 Dec, 65(12), 5493 - 9
Partitioning effects during terminal carbon and electron flow in sediments of a low-salinity meltwater pond near Bratina Island, McMurdo Ice Shelf, Antarctica; Mountfort DO et al.; A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5 degrees C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes . Addition of {2-(14)C}acetate to sediment samples resulted in the passage of label mainly to CO(2) . Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm(-3) h(-1) compared to 2.5 to 6 nmol cm(-3) h(-1)), the portion of methane production attributed to acetate cleavage was <2% . Substantial increases in the methane production rate were observed with H(2) (2.4-fold), and H(2) uptake was totally accounted for by methane production under physiological conditions . Formate also stimulated methane production (twofold), presumably through H(2) release mediated through hydrogen lyase . Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, approximately 0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM) . No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis . The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H(2) and CO(2) where chloride, but not sulfate, has a modulating role . Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20 degrees C.

Appl Environ Microbiol, 1999 Dec, 65(12), 5484 - 92
Biodegradation of free phytol by bacterial communities isolated from marine sediments under aerobic and denitrifying conditions; Rontani JF et al.; Biodegradation of (E)-phytol {3,7,11, 15-tetramethylhexadec-2(E)-en-1-ol} by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20 degrees C . This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10, 14-trimethylpentadecan-2-one and (E)-phytenic acid . The first step in both aerobic and anaerobic bacterial degradation of (E)-phytol involves the transient production of (E)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one . Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations . Since (E)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15 degrees C), the major part of (E)-phytol appeared to be biodegraded in situ via (E)-phytenic acid . (Z)- and (E)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core . This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating beta-decarboxymethylation and beta-oxidation reaction sequences induced by denitrifiers . Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (E)-phytol.

Biotechnol Bioeng, 2000 Jan 5, 67(1), 19 - 24
Modeling of biological processes using self-cycling fermentation and genetic algorithms; Pinchuk RJ et al.; Self-cycling fermentation (SCF) was coupled with a genetic algorithm (GA) to provide a simple system for evaluating biological models . The SCF provided the necessary system excitation and data "richness" required to completely define the fitted biological models . The solution scheme based on the GA avoided the computational difficulties often associated with calculus-based nonlinear regression techniques, resulting in rapid and accurate convergence . After validating the mathematical approach, data from the SCF obtained under denitrifying conditions were fitted successfully to an established model using the GA . Finally, data obtained in the SCF for the removal of phenol were used to compare multiple models . This work suggests that the SCF, in conjunction with the GA, provides a coherent system that can facilitate the characterization of biological systems .

Bioorg Med Chem, 1999 Oct, 7(10), 2215 - 9
Genetic engineering of Escherichia coli for the production of precorrin-3 in vivo and in vitro; Roessner CA et al.; The construction of a new recombinant strain of Escherichia coli in which two vitamin B12 biosynthetic genes, cobA and cobI, from Pseudomonas denitrificans are simultaneously overexpressed has resulted in the in vivo synthesis and accumulation of Factor III, an isobacteriochlorin not normally synthesized in E . coli . A lysate of the new strain can take the place of two lysates normally required to provide uroporphyrinogen III methyltransferase (cobA) and precorrin-2 methyltransferase (cobI) in an anaerobic five-enzyme synthesis of the early B12 intermediate, precorrin-3 (the reduced form of Factor III) from delta-aminolevulinic acid.

Biochemistry, 1999 Nov 16, 38(46), 15150 - 6
Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase; Ma J et al.; Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase . Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix . The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P . denitrificans oxidase) . Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase . The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase . Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center . The data suggest that E89II (E . coli numbering) is critical for the function of the heme copper oxidases . The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel . This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant . Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.

Analyst, 1999 Feb, 124(2), 129 - 34
Optical biosensing of nitric oxide using the metalloprotein cytochrome c'; Blyth DJ et al.; The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO) . The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements . Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre . Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period . The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO . However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%) . An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1.

J Biol Chem, 1999 Nov 19, 274(47), 33296 - 9
The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction; Harrenga A et al.; Cytochrome c oxidase catalyzes the reduction of oxygen to water . This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping") . The mechanism of proton pumping is still a matter of debate . Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase . Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K . No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed . The three histidine Cu(B) ligands are well defined in the oxidized and in the reduced states . These results are hardly compatible with the "histidine cycle" mechanisms formulated previously.

J Bacteriol, 1999 Nov, 181(22), 6907 - 13
Anaerobic growth of Paracoccus denitrificans requires cobalamin: characterization of cobK and cobJ genes; Shearer N et al.; A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated . This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media . Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway . Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase . The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P . denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions . Comparison of the cobK and cobJ genes with their orthologues suggests that P . denitrificans uses the aerobic pathway for cobalamin synthesis . It is paradoxical that under anaerobic growth conditions, P . denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis . Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media . These observations provide evidence that P . denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.

Arch Microbiol, 1999 Nov, 172(5), 303 - 12
Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria; Harms G et al.; The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud . Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks) . Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months . Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail . Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively . Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene . Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., alpha-terpinene . In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids . Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions . Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene . Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack . Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1 . Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the beta-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis . This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes.

Biochem Biophys Res Commun, 1999 Nov, 265(1), 177 - 83
The cbbQ genes, located downstream of the form I and form II RubisCO genes, affect the activity of both RubisCOs; Hayashi NR et al.; Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1 and cbbLS-2) and one form II type (cbbM) enzymes . The cbbQ-m gene was located downstream of cbbM, and showed high similarity to other cbbQ genes and nirQ/norQ genes in denitrifying bacteria . Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv . marinus closely resembled CbbQ from Thiobacillus intermedius and Thiobacillus neapolitannus and less closely resembled NirQ and NorQ . The cbbQ-m gene has been shown to activate the form II RubisCO in E . coli cells, and the cbbQ-t from Hydrogenophilus thermoluteolus could also activate the form II RubisCO . Both cbbQ genes have also been shown to activate the form I RubisCO from Hp . thermoluteolus in E . coli cells . However, the activation levels of two form I RubisCOs from Hv . marinus were smaller than that of form I RubisCOs from Hp . thermoluteolus . Form II RubisCO activated by CbbQ-m (QM) was purified from E . coli cells . The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ .

Arch Microbiol, 1999 Oct, 172(4), 204 - 12
Phototrophic utilization of toluene under anoxic conditions by a new strain of blastochloris sulfoviridis
Zengler K, Heider J, Rossello-Mora R, Widdel F.
The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene . When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed . Agar dilution series indicated the dominance of two types of phototrophic bacteria . One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene . The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate . One strain of the latter type, ToP1, was studied in detail . Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the alpha-subclass of Proteobacteria . However, the type strain (DSM 729) of Blc . sulfoviridis grew neither on toluene nor on benzoate . Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments . Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon . In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected . These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria . The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats . Counting series revealed that up to around 1% (1.8 x 10(5) cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene.

Biochim Biophys Acta, 1999 Oct 12, 1434(2), 248 - 59
A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization; Alves T et al.; Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions . The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps . N-terminal sequence comparison showed that the Ps . nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa . UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem . Monohaemic cytochrome c(552) from Ps . nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116 inverted question mark omitted inverted question mark000 min(-1) . Using this cytochrome the enzyme retained the same activity even at high ionic strength . There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Biochim Biophys Acta, 1999 Sep 1, 1413(1), 1 - 13
The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel; Pereira MM et al.; The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283) . In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised . It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type . This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase . The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3) . Subunit I of R . marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels) . However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R . marinus oxidase . Homology modelling of the R . marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre . Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV- . These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

J Microbiol Methods, 1999 Oct, 38(1-2), 91 - 9
Effects of storage on measurements of potential microbial activities in stream fine benthic organic matter; Bonin HL et al.; Sample storage can significantly influence measured microbial activities in stream fine benthic organic matter (FBOM), possibly confounding effects of sample variability and short-term changes in activity . Denitrification potential, acetylene reduction and respiration rates, mineralizable N and extractable ammonium concentrations, and beta-glucosidase and phosphatase enzyme activities of FBOM from first-order mountain streams in the western Oregon Cascade Mountains were assayed at various times after collection to determine potential storage effects . Denitrification potential, phosphatase activity, and extractable ammonium remained stable over a minimum of 11 h of storage at 5 degrees C . Mineralizable N concentrations, respiration rates, and beta-glucosidase activity all decreased within 12 h of collection . Results varied for acetylene reduction . Once assay conditions were established, denitrification potential and respiration rates were linear with incubation time . Based on paired t-tests, measures of acetylene reduction, denitrification potential, respiration rate, beta-glucosidase activity, and phosphatase activity were generally similar at a 1-wk interval within the same stream reaches.

Carbohydr Res, 1999 Jun 30, 319(1-4), 133 - 40
Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960; Parolis LA et al.; The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy . The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments . The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot . The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds . Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid.

J Bacteriol, 1999 Oct, 181(20), 6403 - 10
Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp . strain T; Krieger CJ et al.; The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp . strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate . Permeabilized cells of m-xylene-grown Azoarcus sp . strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate . In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate . Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp . strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption . Based on these findings, we propose that Azoarcus sp . strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA . A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.

Biospectroscopy, 1999, 5(5 Suppl), S19 - 32
Paramagnetic NMR studies of blue and purple copper proteins; Kolczak U et al.; 1H- and 13C-NMR spectroscopy is applied to investigate the CU(A) and type 1 active sites of copper proteins in solution . The analysis of hyperfine shifted 1H resonances allows the comparison of the electron spin density delocalization in the CU(A) site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin) . Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues . A reevaluation of the Cys H(beta) chemical shifts that is in agreement with the data published for both the P . denitrificans and the P . versutus Cu(A) soluble domains confirms the thermal accessibility of the 2B(3u) electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands . The 13C-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments . The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron.

FEMS Microbiol Ecol, 1999 Oct 1, 30(2), 113 - 123
Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group; Sorokin DY et al.; A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate . The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions . Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions . In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N(2)O as electron acceptor . The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions . Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N(2)O as electron acceptors . Others functioned best with N(2)O during anaerobic thiosulfate oxidation . The latter strains appeared to have a lower level of nitrite reductase activity . Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor . The strains had DNA-DNA similarity levels higher than 30% . Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica . As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed 'Flavobacterium lutescens' (ATCC 27951) is also a P . stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N(2)O . The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P . stutzeri 'superspecies', can play a much more significant role in the biogeochemical cycles than was previously recognized.

Biochim Biophys Acta, 1999 Oct 6, 1447(1), 57 - 63
Site-directed mutagenesis of the response regulator DmsR for the dmsCBA operon expression in Rhodobacter sphaeroides f . sp . Denitrificans: An essential residue of proline-130 in the linker; Yamamoto I et al.; DmsR protein is a member of the OmpR response regulator subfamily that activates the transcription of the dmsCBA operon in Rhodobacter sphaeroides f . sp . denitrificans . By site-directed mutagenesis some functional amino acid residues were investigated in DmsR, which consists of the N-terminal regulatory and the C-terminal DNA-binding domains and the linker connecting the two domains . The substitution of P130S in the linker caused decreases of both DNA-binding and transcriptional activator activities . Introducing additional substitutions of R129P or D131P to the DmsR-P130S derivative recovered both activities, demonstrating necessity of proline residue at one of the positions 129-131 in the linker . Substitutions of D12A, D55A, and K104M, at residues conserved in the phosphorylation region, caused no production of DMSO reductase, but retained DNA-binding ability, suggesting that unphosphorylated DmsR also has high affinity to its target nucleotide sequence of DNA . Substitutions in the C-terminal domain suggested the presence of a winged helix-turn-helix structure observed in the DNA-binding domain of the Escherichia coli OmpR.

J Bacteriol, 1999 Oct, 181(19), 6028 - 32
Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f . sp . denitrificans IL106; Sabaty M et al.; We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f . sp . denitrificans IL106 . A mutant with this enzyme deleted is unable to grow under denitrifying conditions . Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate . Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase . This demonstrates that R . sphaeroides f . sp . denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species . In contrast, we show that nitrite induces the synthesis of the nitrate reductase.

J Biol Chem, 1999 Oct 1, 274(40), 28606 - 11
H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans . Studies on topology and stoichiometry of the peripheral subunits; Yano T et al.; The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane . In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P . denitrificans NDH-1 in the membranes were investigated using immunological techniques . Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes . Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex . Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association . In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed . In addition, determination of stoichiometry of the peripheral subunits of the P . denitrificans NDH-1 was completed by radioimmunological methods . All the peripheral subunits are present as one molecule each in the enzyme complex . These results estimated the total number of cofactors in the P . denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x{2Fe-2S} and 6x{4Fe-4S}, provided that the NQO6 subunit bears one {4Fe-4S} cluster.

J Biol Chem, 1999 Oct 1, 274(40), 28598 - 605
Characterization of the putative 2x{4Fe-4S}-binding NQO9 subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans . Expression, reconstitution, and EPR characterization; Yano T et al.; Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2x{4Fe-4S} clusters, were investigated using recombinant expression techniques and EPR spectroscopy . The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification . Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm . The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S(2-) (2.0-2.2 mol each per mol of subunit) . However, the iron-sulfur content was considerably increased by in vitro reconstitution . The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S(2-) per molecule and exhibited optical absorption spectra similar to those of 2x{4Fe-4S} ferredoxins . Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K) . Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g(, perpendicular) = 2.05, approximately 1.93, and g(parallel, perpendicular) = 2.08, approximately 1.90, and respective slow (P((1)/(2)) = 8 milliwatts) and fast (P((1)/(2)) = 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g(parallel, perpendicular) = 2.05, approximately 1.93) with slow spin relaxation . When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g(parallel, perpendicular) = 2.08, approximately 1.90, with enhanced spin relaxation . In either case, our EPR data provide the first experimental evidence for the presence of two {4Fe-4S} iron-sulfur clusters in the NQO9 subunit.

Nature, 1999 Sep 9, 401(6749), 181 - 4
Coherent reaction dynamics in a bacterial cytochrome c oxidase; Liebl U et al.; Biological reactions in protein complexes involve structural dynamics spanning many orders of magnitude in time . In standard descriptions of catalysis by enzymes, the transition state between reactant and product is reached by thermal, stochastic motion . In the ultrashort time domain, however, the protein moiety and cofactor motions leading to altered conformations can be coherent rather than stochastic in nature . Such coherent motions may play a key role in controlling the accessibility of the transition state and explain the high efficiency of the reaction . Here we present evidence for coherent population transfer to the product state during an ultrafast reaction catalysed by a key enzyme in aerobic organisms . Using the enzyme cytochrome c oxidase aa3 from the bacterium Paracoccus denitrificans, we have studied haem dynamics during the photo-initiated ultrafast transfer of carbon monoxide from haem a3 to CuB by femtosecond spectroscopy . The ground state of the unliganded a3 species is populated in a stepwise manner in time, indicating that the reaction is mainly governed by coherent vibrations (47cm(-1)) . The reaction coordinate involves conformational relaxation of the haem group and we suggest that ligand transfer also contributes.

J Clin Microbiol, 1999 Oct, 37(10), 3374 - 9
Capillary electrophoresis-single-strand conformation polymorphism analysis for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis; Ghozzi R et al.; We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF) . Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes . The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer . Data were analyzed with GeneScan 672 software . This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers . Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism . The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 {M . N . Widjojoatmodjo, A . C . Fluit, and J . Verhoef, J . Clin . Microbiol . 32:3002-3007, 1994}) . This primer set differentiated the main CF pathogens from closely related species but did not distinguish P . aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans . Two hundred seven CF clinical isolates (153 of P . aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A . xylosoxidans) were tested with P11P-P13P . The CE-SSCP patterns obtained were identical to those for the corresponding reference strains . Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains . This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.

FEMS Microbiol Lett, 1999 Sep 1, 178(1), 147 - 53
Substrate range of benzylsuccinate synthase from Azoarcus sp . strain T; Beller HR et al.; Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria . In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp . strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane) . Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate . Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer {(R)-2-benzyl-3-carboxypropionic acid} . The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates . The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

J Bacteriol, 1999 Sep, 181(18), 5662 - 8
In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization; Johnson HA et al.; Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp . strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol . 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate . We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain . With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp . strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) {mg of protein}(-1) and an apparent K(m) for ethylbenzene of approximately 60 microM . The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene . Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis . Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone . When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase . Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp . strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.

FEBS Lett, 1999 Sep 17, 458(2), 83 - 6
Similarities and dissimilarities in the structure-function relation between the cytochrome c oxidase from bovine heart and from Paracoccus denitrificans as revealed by FT-IR difference spectroscopy; Hellwig P et al.; The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT-IR spectroscopic approach . A direct comparison to the electrochemically induced FT-IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes . These differences are partially attributed to interactions of subunits influencing the heme and protein modes . In the spectral regions characteristic for v(C=O) and v(COO-)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm-1 are observed . On this basis, the possible involvement of Asp-51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X-ray structures, is critically discussed.

Appl Environ Microbiol, 1999 Sep, 65(9), 4189 - 96
On the occurrence of anoxic microniches, denitrification, and sulfate reduction in aerated activated sludge; Schramm A et al.; A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge . Microsensor measurements (O(2), NO(2)(-), NO(3)(-), and H(2)S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale . Incubations of activated sludge with (15)NO(3)(-) and (35)SO(4)(2-) were used to determine denitrification and sulfate reduction rates on a batch scale . In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low . However, in two sludges anoxia in flocs coincided with significant denitrification rates . Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions . In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase . A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels . This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges.

Biophys J, 1999 Sep, 77(3), 1694 - 711
The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region; Horvath MP et al.; The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a . This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures . Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data . Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3) . Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore . The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 562 - 4
Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans; Koutny M et al.; The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S . (1997) Biochim . Biophys . Acta 1318, 202-216) does not involve the concentration of an electron donor . Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide . NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme . The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex .

FEBS Lett, 1999 Aug 13, 456(3), 365 - 9
Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase; Pfitzner U et al.; Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin . We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion . Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer . Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions . This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase . Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Biochemistry, 1999 Aug 17, 38(33), 10670 - 7
The calcium binding site in cytochrome aa3 from Paracoccus denitrificans; Riistama S et al.; A shift in the spectrum of heme a induced by calcium or proton binding, or by the proton electrochemical gradient, has been attributed to interaction of Ca2+ or H+ with the vicinity of the heme propionates in mitochondrial cytochrome c oxidase, and proposed to be associated with the exit path of proton translocation . However, this shift is absent in cytochrome c oxidases from yeast and bacteria {Kirichenko et al . (1998) FEBS Lett . 423, 329-333} . Here we report that mutations of Glu56 or Gln63 in a newly described Ca2+/Na+ binding site in subunit I of cytochrome c oxidase from Paracoccus denitrificans {Ostermeier et al . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 10547-10553} establish the Ca2+-dependent spectral shift in heme a . This shift is counteracted by low pH and by sodium ions, as was described for mammalian cytochrome c oxidase, but in the mutant Paracoccus enzymes Na+ is also able to shift the heme a spectrum, albeit to a smaller extent . We conclude that the Ca2+-induced shift in both Paracoccus and mitochondrial cytochrome aa3 is due to binding of the cation to the new metal binding site . Comparison of the structures of this site in the two types of enzyme allows rationalization of their different reactivity with cations . Structural analysis and data from site-directed mutagenesis experiments suggest mechanisms by which the cation binding may influence the heme spectrum.

Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 139 - 52
The functions of the flavin contact residues, alphaArg249 and betaTyr16, in human electron transfer flavoprotein; Dwyer TM et al.; Arg249 in the large (alpha) subunit of human electron transfer flavoprotein (ETF) heterodimer is absolutely conserved throughout the ETF superfamily . The guanidinium group of alphaArg249 is within van der Waals contact distance and lies perpendicular to the xylene subnucleus of the flavin ring, near the region proposed to be involved in electron transfer with medium chain acyl-CoA dehydrogenase . The backbone amide hydrogen of alphaArg249 is within hydrogen bonding distance of the carbonyl oxygen at the flavin C(2) . alphaArg249 may modulate the potentials of the two flavin redox couples by hydrogen bonding the carbonyl oxygen at C(2) and by providing delocalized positive charge to neutralize the anionic semiquinone and anionic hydroquinone of the flavin . The potentials of the oxidized/semiquinone and semiquinone/hydroquinone couples decrease in an alphaR249K mutant ETF generated by site directed mutagenesis and expression in Escherichia coli, without major alterations of the flavin environment as judged by spectral criteria . The steady state turnover of medium chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase decrease greater than 90% as a result of the alphaR249Ks mutation . In contrast, the steady state turnover of short chain acyl-CoA dehydrogenase was decreased about 38% when alphaR249K ETF was the electron acceptor . Stopped flow absorbance measurements of the oxidation of reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA product complex by wild type human ETF at 3 degrees C are biphasic (t(1/2)=12 ms and 122 ms) . The rate of oxidation of this reduced binary complex of the dehydrogenase by the alphaR249K mutant ETF is extremely slow and could not be reasonably estimated . alphaAsp253 is proposed to function with alphaArg249 in the electron transfer pathway from medium chain acyl-CoA dehydrogenase to ETF . The steady state kinetic constants of the dehydrogenase were not altered when ETF containing an alphaD253A mutant was the substrate . However, t(1/2) of the rapid phase of oxidation of the reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA charge transfer complex almost doubled . betaTyr16 lies on a loop near the C(8) methyl group, and is also near the proposed site for interflavin electron transfer with medium chain acyl-CoA dehydrogenase . The tyrosine residue makes van der Waals contact with the C(8) methyl group of the flavin in human ETF and Paracoccus denitrificans ETF (as betaTyr13) and lies at a 30 degrees C angle with the plane of the flavin . Human betaTyr16 was substituted with leucine and alanine residues to investigate the role of this residue in the modulation of the flavin redox potentials and in electron transfer to ETF . In betaY16L ETF, the potentials of the flavin were slightly reduced, and steady state kinetic constants were modestly altered . Substitution of an alanine residue for betaTyr16 yields an ETF with potentials very similar to the wild type but with steady state kinetic properties similar to betaY16L ETF . It is unlikely that the beta methyl group of the alanine residue interacts with the flavin C(8) methyl . Neither substitution of betaTyr16 had a large effect on the fast phase of ETF reduction by medium chain acyl-CoA dehydrogenase.

J Bacteriol, 1999 Aug, 181(16), 5099 - 102
Disruption of narG, the gene encoding the catalytic subunit of respiratory nitrate reductase, also affects nitrite respiration in Pseudomonas fluorescens YT101; Ghiglione JF et al.; The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette . In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P . fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase . Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions . These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

Appl Environ Microbiol, 1999 Aug, 65(8), 3599 - 604
Evidence for involvement of gut-associated denitrifying bacteria in emission of nitrous oxide (N(2)O) by earthworms obtained from garden and forest soils; Matthies C et al.; Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N(2)O)-emitting garden soils emitted 0.14 to 0.87 nmol of N(2)O h(-1) g (fresh weight)(-1) under in vivo conditions . L . rubellus obtained from N(2)O-emitting forest soil also emitted N(2)O, which confirmed previous observations (G . R . Karsten and H . L . Drake, Appl . Environ . Microbiol . 63:1878-1882, 1997) . In contrast, commercially obtained Lumbricus terrestris did not emit N(2)O; however, such worms emitted N(2)O when they were fed (i.e., preincubated in) garden soils . A . caliginosa, L . rubellus, and O . lacteum substantially increased the rates of N(2)O emission of garden soil columns and microcosms . Extrapolation of the data to in situ conditions indicated that N(2)O emission by earthworms accounted for approximately 33% of the N(2)O emitted by garden soils . In vivo emission of N(2)O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N(2)O . In the presence of nitrate, acetylene increased the N(2)O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N(2)O . In vivo emission of N(2)O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline . On a fresh weight basis, the rates of N(2)O emission of dissected earthworm gut sections were substantially higher than the rates of N(2)O emission of dissected worms lacking gut sections, indicating that N(2)O production occurred in the gut rather than on the worm surface . In contrast to living earthworms and gut sections that produced N(2)O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N(2)O-emitting earthworms produced N(2)O only under anoxic conditions . Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N(2)O by earthworms and contribute to the N(2)O that is emitted from certain terrestrial ecosystems.

Appl Environ Microbiol, 1999 Aug, 65(8), 3487 - 92
Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils; Murray RE et al.; Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes . When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46% . The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N(2)O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61% . The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter . We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration . Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.

Appl Environ Microbiol, 1999 Aug, 65(8), 3319 - 24
Anaerobic mineralization of quaternary carbon atoms: isolation of denitrifying bacteria on dimethylmalonate; Kniemeyer O et al.; The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source . Quantitative growth experiments showed a complete mineralization of dimethylmalonate . According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the alpha-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans(T)), and three strains isolated from freshwater ditches were affiliated with the beta-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae(T) and Acidovorax facilis(T), respectively) . Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 x 10(4) dimethylmalonate-utilizing cells ml(-1), representing up to 0.4% of the total culturable nitrate-reducing population.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1129 - 40
Taxonomic characterization of denitrifying bacteria that degrade aromatic compounds and description of Azoarcus toluvorans sp . nov . and Azoarcus toluclasticus sp . nov; Song B et al.; A taxonomic characterization of twenty-one strains capable of degrading aromatic compounds under denitrifying conditions, isolated from ten different geographical locations, was performed on the basis of general morphological and physiological characteristics, cellular fatty acids, DNA base composition, small ribosomal (16S) subunit DNA sequences, whole-cell protein patterns and genomic DNA fragmentation analysis, in addition to DNA similarity estimations using hybridization methods . The collection of strains was subdivided into a number of different groups . A first group, consisting of four strains, could be assigned to the previously described species Azoarcus tolulyticus . A second group (five strains) had DNA which reannealed highly to that of strains of the first group, and it is considered to represent a genomovar of A . tolulyticus . The third and fourth groups, composed of a total of five strains, represent a new species of Azoarcus, Azoarcus toluclasticus (group 3) and a genomovar of this species (group 4), respectively . Finally, the fifth group, with two strains, corresponds to another new species of the genus Azoarcus, Azoarcus toluvorans . In addition to these five groups, the collection includes five individual strains perhaps representing as many different new species . The above classification is partially consistent with the results of approaches other than DNA-DNA hybridization (electrophoretic patterns of whole-cell proteins and of the fragments obtained after digestion of total DNA with infrequently cutting restriction enzymes) . On the other hand, no correlation of these groupings was found in terms of the cellular fatty acid composition . It is also unfortunate that no simple sets of easily determinable phenotypic properties could be defined as being characteristic of each of the groups.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1045 - 51
Thauera mechernichensis sp . nov., an aerobic denitrifier from a leachate treatment plant; Scholten E et al.; A heterotrophic bacterial strain TL1T capable of aerobic denitrification was previously enriched in continuous culture from a landfill leachate treatment plant and isolated as a pure culture . The taxonomic position of this isolate within the beta-subclass of the Proteobacteria was determined by 16S rDNA sequence analysis and by conventional taxonomy including substrate spectrum, quinone type (ubiquinone Q-8) and cellular fatty acid composition . Detection of the specific polyamine 2-hydroxyputrescine supports the membership of strain TL1T in the beta-subclass of the Proteobacteria . The results of 16S rDNA sequencing showed that the strain clustered with, but was separate from, Thauera aromatica and Thauera selenatis . DNA-DNA hybridization experiments indicated that the new isolate represents a new species of the genus, for which the name Thauera mechernichensis is proposed; the type strain is DSM 12266T.

Biodegradation, 1999 Feb, 10(1), 75 - 82
Fate and behavior of organic compounds in an artificial saturated subsoil under controlled redox conditions: the sequential soil column system; Nay M et al.; A system was developed to investigate the fate and behavior of anthropogenic organic contaminants at concentrations present in polluted subsoils and aquifers . A sequential soil column system was constructed to simulate redox conditions from methanogenic, sulfate-reducing, denitrifying, to aerobic conditions which normally occur in a leachate pollution plume . This system allowed the simulation of subsurface pollution with a range of xenobiotics and the observation of the microbial response to this contamination . After an adaptation period of up to about 7 months, 2,4-dichlorophenol and 2-nitrophenol were eliminated and perchloroethene disappeared almost completely in the methanogenic column . Toluene was partially transformed under sulfate-reducing conditions, and nearly completely in the nitrate-reducing column . The same applied to naphthalene under denitrifying and aerobic conditions . Aerobically, a fraction of benzene was transformed, and 1,4-dichlorobenzene decreased to very low residual concentrations in one system . No significant transformation of 1,1-dichloroethene could be seen.

Biodegradation, 1999 Feb, 10(1), 15 - 25
Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments; Phelps CD et al.; We examined the extent of biodegradation of benzene, toluene, ethylbenzene and the three isomers of xylene (BTEX) as a mixture and from gasoline in four different sediments: the New York/New Jersey Harbor estuary (polluted); Tuckerton, N.J . (pristine); Onondaga Lake, N.Y . (polluted) and Blue Mtn . Lake, N.Y . (pristine) . Enrichment cultures were established with each sediment using denitrifying, sulfidogenic, methanogenic and iron reducing media, as well as site water . BTEX loss, as measured by GC-FID, was extensive in the sediments which had a long history of pollution, with all compounds being utilized within 21-91 days in the most active cultures, and was very slight or non-existent in the pristine sediments . Also, the pattern of loss was different under the various reducing conditions within each sediment and between sediments . For example benzene loss was only observed in sulfidogenic cultures from the NY/NJ Harbor sediments while toluene was degraded under all redox conditions . The loss of BTEX was correlated to the reduction of the various electron acceptors . In cultures amended with gasoline the degradation was much slower and incomplete . These results show that the fate of the different BTEX components in anoxic sediments is dependent on the prevailing redox conditions as well as on the characteristics and pollution history of the sediment.

J Biochem (Tokyo), 1999 Aug, 126(2), 408 - 12
The pH-dependent changes of intramolecular electron transfer on copper-containing nitrite reductase; Kobayashi K et al.; Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu . In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A . xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite . However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0 . A similar result was obtained for the enzyme from A . cycloclastes . The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction . This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite . The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer . The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr{H+}+{H+}2)/(K(0)+{H+}), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively . These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.

Biochemistry, 1999 Jul 27, 38(30), 9735 - 45
The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer; Dwyer TM et al.; Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells . The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring . We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond . The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol . The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD . However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein . These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region . Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone . The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol . However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions . This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF . ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (</=0.5% the wild-type rates) . These results indicate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stabilization of the anionic semiquinone and anionic hydroquinone oxidation states of ETF and that this hydrogen bond may provide a pathway for electron transfer between the ETF flavin and the flavin of ETF-QO.

Biochemistry, 1999 Jul 13, 38(28), 9000 - 12
Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy; Butler CS et al.; The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a {4Fe-4S} center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD) . A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies . The Mo(V) EPR signal of resting NAP (High g {resting}) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable . Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal . A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g {nitrate}) . Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme . The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2 . 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A . The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme . Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g {unsplit}) . This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD . In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals . Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively . EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV) . Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

Microbiology, 1999 Jun, 145 ( Pt 6), 1409 - 20
Mutational analysis of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter capsulatus; Shaw AL et al.; Four genes, dorC, dorD, dorB and dorR of the DMSO respiratory gene cluster of Rhodobacter capsulatus have been identified and sequenced . dorC encodes a pentahaem c-type cytochrome of the NirT class and the derived DorC protein sequence shows highest similarity to TorC from the Escherichia coli trimethylamine-N-oxide (TMAO) respiratory system . Mutagenesis of dorC resulted in the loss of a 46 kDa haem-staining polypeptide from membranes of R . capsulatus . dorD encodes a protein with highest sequence similarity to TorD from the E . coli TMAO respiratory system . DMSO reductase polypeptide (DorA) could not be detected in cell-free extracts of a dorD mutant and it is suggested that DorD has a role in stabilizing the DorA apo-protein prior to insertion of the pterin molybdenum cofactor . dorB encodes a protein with highest sequence similarity to NapD of Paracoccus denitrificans . Mutagenesis of dorB reduced the activity of DMSO reductase and led to the accumulation of a larger form of the enzyme that is presumed to represent a cytoplasmic precursor polypeptide . It is suggested that DorB has a role in the biogenesis of DMSO reductase prior to its secretion into the periplasm . dorR is transcribed in the opposite direction to dorC . The derived amino acid sequence of DorR indicates that it is a response regulator and mutation of dorR shows that it is essential for expression of the dorCDA operon . Expression of a chromosomal dorA::lacZ fusion was also dependent on the transcriptional regulator Fnr . The intergenic region between dorR and dorC contains four putative binding sites for DorR but no binding site for Fnr was identified.

Biochim Biophys Acta, 1999 Jul 13, 1432(2), 406 - 12
Molecular cloning, functional expression and purification of a glucan branching enzyme from Neisseria denitrificans(1); Buttcher V et al.; The nucleotide sequence containing the complete structural information for a glucan branching enzyme was isolated from a Neisseria denitrificans genomic library . The gene was expressed in Escherichia coli and the active recombinant protein was purified . The deduced protein of 762 amino acids with a calculated molecular weight of 86313 Da shows similarity to the primary protein sequences of other known glucan branching enzymes . Amino acid sequencing of the isolated protein by Edman degradation confirmed the deduced start codon of the structural gene of the glucan branching enzyme . The purified glucan branching enzyme has a stimulating effect on the Neisseria amylosucrase activity.

Eur J Biochem, 1999 Jul, 263(2), 420 - 9
6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase, enzymes of the benzoyl-CoA pathway of anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica; Laempe D et al.; Benzoyl-CoA is a common intermediate in the anaerobic bacterial metabolism of many aromatic substrates . Two enzymes and ferredoxin of the central benzoyl-CoA pathway in Thauera aromatica have been purified so far . Benzoyl-CoA reductase reduces the aromatic ring with reduced ferredoxin yielding cyclohexa-1,5-diene-1-carbonyl-CoA {Boll, M . & Fuchs, G . (1995) Eur . J . Biochem . 234, 921-933} . Dienoyl-CoA hydratase subsequently adds one molecule of water and thereby produces 6-hydroxycyclohex-1-ene-1-carbonyl-CoA {Laempe, D., Eisenreich, W., Bacher, A., & Fuchs, G . (1998) Eur . J . Biochem . 255, 618-627} . Here two new enzymes, which convert this intermediate to the noncyclic product 3-hydroxypimelyl-CoA, were purified from T . aromatica and studied . 6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase is an NAD(+)-specific beta-hydroxyacyl-CoA dehydrogenase that catalyzes 6-hydroxycyclohex-1-ene-1-carbonyl-CoA + NAD(+) --> 6-oxocyclohex-1-ene-1-carbonyl-CoA + NADH + H(+) . 6-Oxocyclohex-1-ene-1-carbonyl-CoA hydrolase acts on the beta-oxoacyl-CoA compound and catalyzes the addition of one molecule of water to the double bound and the hydrolytic C-C cleavage of the alicyclic ring, 6-oxocyclohex-1-ene-1-carbonyl-CoA + 2 H(2)O --> 3-hydroxypimelyl-CoA . The genes for both enzymes, had and oah, were cloned, had was overexpressed in Escherichia coli and the recombinant protein was purified . Hence, presumably all enzymes of the central benzoyl-CoA pathway of anaerobic aromatic metabolism from this organism have now been purified and studied and the corresponding genes have been cloned and sequenced.

J Bacteriol, 1999 Jul, 181(14), 4216 - 22
Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides; Graichen ME et al.; The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA . The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds . To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P . denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R . sphaeroides by using a broad-host-range vector . The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources . The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P . denitrificans . These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R . sphaeroides cannot synthesize TTQ . A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product . This finding suggests that active recombinant MADH is not expressed in E . coli because one of the accessory gene products is not functionally expressed . This study illustrates the potential utility of R . sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E . coli . These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

J Bacteriol, 1999 Jul, 181(14), 4205 - 15
Structural and functional analyses of photosynthetic regulatory genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus; Masuda S et al.; Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis . This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides . These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system . The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif . The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen . A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed . These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species . Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans . These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.

Biotechnol Bioeng, 1999 Aug 5, 64(3), 342 - 8
Biotransformation of carbon tetrachloride by various acetate- and nitrate-limited denitrifying consortia
Sherwood JL, Petersen JN, Skeen RS.
Fed-batch experiments were performed to determine the carbon tetrachloride (CT)-degrading ability of three denitrifying consortia cultured from sites not contaminated with CT . A mathematical model was used to quantify the rates of CT transformation by the consortia under both acetate-limiting and nitrate-limiting conditions . A rate constant for CT transformation on a cellular protein basis and the fraction of degraded CT transformed to chloroform (CF) were determined for each consortium by optimizing the model to fit the experimental data . The parameters for these experiments were statistically compared to those obtained for previous experiments with a denitrifying consortium cultured from an aquifer soil sample from the US Department of Energy Hanford site in southeastern Washington state . Results of F-test analysis indicated the rate of CT transformation and the production of CF both were functions of the limiting nutrient . Under nitrate-limited conditions, the rate constant for CT transformation for all four consortia was about 30 L/gmol/min and approximately 50% of the CT transformed was converted to CF . When acetate was the limiting nutrient, the rate constant for CT transformation was approximately 8 L/gmol/min and the CF yield decreased to about 25% . These results imply the ability to degrade CT may be inherent to some denitrifying organisms, regardless of previous exposure to CT .

Gene, 1999 Jul 8, 234(2), 275 - 83
Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides; Schlarb BG et al.; The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli . Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E . coli, but the yield was low . Expression in Paracoccus denitrificans yielded no holoprotein . When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P . laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species . Overexpressed proteins were compared to those isolated from P . laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.

Dig Dis Sci, 1999 Jun, 44(6), 1216 - 9
Vegetables, high nitrate foods, increased breath nitrous oxide; Mitsui T et al.; We have demonstrated nitrous oxide (N2O), a metabolite of the reduction of nitrate (NO(3)-) by microflora, in exhaled air . The purpose of this study is to examine the effect of ingesting vegetables, which contain high levels of NO(3)-, on breath N2O levels . We measured breath N2O in six healthy subjects aged 20-41 years at 15-min intervals after the following: (1) no ingestion; (2) ingestion of 180 g of vegetable juice; and (3) ingestion of 50 g of lettuce . N2O levels were measured with an infrared-photoacoustic (IR-PAS) analyzer . N2O was detectable in all subjects regardless of treatment protocol . Lettuce and vegetable juice significantly increased breath N2O levels . Total excretions in breath N2O were significantly higher in the lettuce group (P = 0.028) and the vegetable juice group (P = 0.046) than controls . Breath N2O increases after vegetable ingestion, probably due to denitrification of NO(3)- by normal microflora in the intestinal tract.

J Bacteriol, 1999 Jul, 181(13), 4129 - 32
Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans; Van Spanning RJ et al.; By using the 'lacZ gene, the activities of the nirI, nirS, and norC promoters were assayed in the wild type and in NNR-deficient mutants of Paracoccus denitrificans grown under various growth conditions . In addition, induction profiles of the three promoters in response to the presence of various nitrogenous oxides were determined . Transcription from the three promoters required the absence of oxygen and the presence both of the transcriptional activator NNR and of nitric oxide . The activity of the nnr promoter itself was halved after the cells had been switched from aerobic respiration to denitrification . This response was apparently not a result of autoregulation or of regulation by FnrP, since the nnr promoter was as active in the wild-type strain as it was in NNR- or FnrP-deficient mutants.

Chemosphere, 1999 Jul, 39(1), 45 - 54
Microbial dechlorination of polychlorinated biphenyls in anaerobic sewage sludge; Chang BV et al.; The potential of a chlorophenol (CP)-adapted consortium to dechlorinate polychlorinated biphenyls (PCBs) in sewage sludge was investigated . Results show that dechlorination rates differed significantly depending on sludge source and PCB congener . Higher total solid concentrations in sewage sludge and higher concentrations of chlorine in PCB resulted in slower dechlorination rates . No significant difference was found for 2,3,4,5-CB dechlorination from pH 6.0 to pH 8.0; however, dechlorination did not occur at pH 9.0 during a 41-day incubation period . Results show that at concentrations of 1 to 10 mg/L, the higher the PCB concentration, the faster the dechlorination rate . In addition, dechlorination rates were in the following order: methanogenic conditions > sulfate-reducing conditions > denitrifying conditions . The addition of acetate, lactate, pyruvate, and ferric chloride decreased lag times and enhanced dechlorination; however, the addition of manganese dioxide had an inhibitory effect . Dechlorination rates were also enhanced by the addition of PCB congeners, including 2,3,4-CB, 2,3,4,5-CB and 2,3,4,5,6-CB in mixture . Overall results show that the CP-adapted consortium has the potential to enhance PCB dechlorination . The optimal dechlorination conditions presented in this paper may be used as a reference for feasibility studies of PCB removal from sludge.

Biol Pharm Bull, 1999 May, 22(5), 532 - 4
Cycloprodigiosin hydrochloride obtained from Pseudoalteromonas denitrificans is a potent antimalarial agent; Kim HS et al.; Cycloprodigiosin hydrochloride (cPrG*HCl) is a stable fluorescent red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans . It was found that the compound was incorporated into Plasmodium falciparum cells upon incubation and exhibited a potent antimalarial activity with the concentration required for 50% of the activity being 11 nM, which is stronger than that of chloroquine, a well-known antimalarial agent . The compound did not affect growth rate of mammalian cells . Antimalarial activity of cPrG*HCl was also observed in vivo . These results indicate that cPrG*HCl is a potent antimalarial drug.

J Bacteriol, 1999 Jun, 181(12), 3658 - 65
Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli; Hartig E et al.; Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products . The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405 . We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase . The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast {Pichia} and plant {Nicotiana} nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family . The roles of NarX and NarL in nitrate respiration were studied with deletion mutants . NarL activated the transcription of narG, narK, and dnrE but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide . The promoters of narG, narK, and dnrE carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for Escherichia coli . The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions . Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of P . stutzeri for selective regulation of nitrate respiration.

J Biol Chem, 1999 Jun 18, 274(25), 17845 - 52
Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes . Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine; Inoue T et al.; The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively . The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins . However, not only a small change of the copper geometry, but concerted peptide bond flips are identified . The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein . When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state . A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A . A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.

Biochemistry, 1999 Jun 8, 38(23), 7565 - 71
Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form; Rost B et al.; The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1) . For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction . At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed . Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes . Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature . Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme . FT-IR difference spectra show band features similar to those for the fully reduced enzyme . Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer . These signals disappear when the temperature is lowered to 84 K . At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O . The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.

Curr Microbiol, 1999 May, 38(5), 300 - 8
Kinetic behavior of some polyphosphate-accumulating bacteria isolates in the presence of nitrate and oxygen; Merzouki M et al.; This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen . It is shown that species were able to respire both electron acceptors for phosphate accumulation . A . tumefaciens and A . dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A . hydrophila and K . oxytoca eliminated overall phosphate only in oxic conditions . A . dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas . The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A . dispar have a denitrification rate superior when the cells were adapted to nitrate.

Biochemistry, 1999 May 25, 38(21), 6935 - 42
Biochemical and electrochemical characterization of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans; Takagi K et al.; A new quinohemoprotein amine dehydrogenase from Paracoccus denitrificans IFO 12442 was isolated and characterized in views of biochemistry and electrochemistry . This enzyme exists in periplasm and catalyzes the oxidative deamination of primary aliphatic and aromatic amines . n-Butylamine or benzylamine as a carbon and energy source strongly induces the expression of the enzyme . Carbonyl reagents inhibit the enzyme activity irreversibly . This enzyme is a heterodimer constituted of alpha and beta subunits with the molecular mass of 59.5 and 36.5 kDa, respectively . UV-vis and EPR spectroscopy, and the quinone-dependent redox cycling and heme-dependent peroxidative stains of SDS-PAGE bands revealed that the alpha subunit contains one quinonoid cofactor and one heme c per molecule, while the beta subunit has no prosthetic group . The redox potential of the heme c moiety was determined to be 0.192 V vs NHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique . The analysis of the substrate titration curve allowed the evaluation of the redox potential of the quinone/semiquinone and semiquinone/quinol redox couples as 0.19 and 0.11 V, respectively.

Microb Ecol, 1999 May, 37(4), 263 - 275
Distribution of Microbial Physiologic Types in an Aquifer Contaminated by Crude Oil; Bekins BA et al.; > Abstract We conducted a plume-scale study of the microbial ecology in the anaerobic portion of an aquifer contaminated by crude-oil compounds . The data provide insight into the patterns of ecological succession, microbial nutrient demands, and the relative importance of free-living versus attached microbial populations . The most probable number (MPN) method was used to characterize the spatial distribution of six physiologic types: aerobes, denitrifiers, iron-reducers, heterotrophic fermenters, sulfate-reducers, and methanogens . Both free-living and attached numbers were determined over a broad cross-section of the aquifer extending horizontally from the source of the plume at a nonaqueous oil body to 66 m downgradient, and vertically from above the water table to the base of the plume below the water table . Point samples from widely spaced locations were combined with three closely spaced vertical profiles to create a map of physiologic zones for a cross-section of the plume . Although some estimates suggest that less than 1% of the subsurface microbial population can be grown in laboratory cultures, the MPN results presented here provide a comprehensive qualitative picture of the microbial ecology at the plume scale . Areas in the plume that are evolving from iron-reducing to methanogenic conditions are clearly delineated and generally occupy 25-50% of the plume thickness . Lower microbial numbers below the water table compared to the unsaturated zone suggest that nutrient limitations may be important in limiting growth in the saturated zone . Finally, the data indicate that an average of 15% of the total population is suspended.http://link.springer-ny.com/link/service/journals/00248/bibs/37n4p263.html

Arch Microbiol, 1999 Mar, 171(4), 230 - 6
Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium; Haggblom MM et al.; A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions . The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification . The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor . The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h . On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1-2 mm in diameter) in 2 to 3 weeks . Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO2 . 3-Chlorobenzoate was not degraded in the presence of oxygen . 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium . Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible . Degradation was specific to the positive of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate.

Eur J Biochem, 1999 Jun, 262(2), 507 - 15
Phenylacetyl-CoA:acceptor oxidoreductase, a membrane-bound molybdenum-iron-sulfur enzyme involved in anaerobic metabolism of phenylalanine in the denitrifying bacterium Thauera aromatica; Rhee SK et al.; Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine . In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (benzoylformate) . The enzyme that catalyzes the four-electron oxidation of phenylacetyl-CoA has been purified from this bacterium and studied . The enzyme preparation catalyzes the reaction phenylacetyl-CoA + 2 quinone + 2 H2O --> phenylglyoxylate + 2 quinone H2 + CoASH . Phenylacetyl-CoA:acceptor oxidoreductase is a membrane-bound molybdenum-iron-sulfur protein . The purest preparations contained three subunits of 93, 27, and 26 kDa . Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water . The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa . Phenylglyoxylyl-CoA, but not mandelyl-CoA, was observed as a free intermediate . All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl-CoA but not from phenylacetyl-CoA . The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen . This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha-oxidation of a CoA-activated carboxylic acid without utilizing molecular oxygen.

Mol Biol Evol, 1999 Apr, 16(4), 429 - 40
Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer; Figge RM et al.; Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3 . Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp . PCC6803 . In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined . Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3 . Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene . Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB . These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes . Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives . This specific relationship can therefore not reflect organismal evolution but must be the result of an interkingdom gene transfer, the direction of which cannot be determined with certainty at present . Contrary to this, the origin of the cytosolic Gap gene from trypanosomes can now be clearly defined as gamma-proteobacterial, since the newly established Ralstonia sequence (beta-proteobacteria) branches basally to the gamma-proteobacterial/trypanosomal assemblage.

J Biol Chem, 1999 May 21, 274(21), 14997 - 5004
Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase?
Nurizzo D, Cutruzzola F, Arese M, Bourgeois D, Brunori M, Cambillau C, Tegoni M.
The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fulop, V., Moir, J . W . B., Ferguson, S . J., and Hajdu, J . (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M . C., Mathieu, M., Cutruzzola, F., Bourgeois, D., Fulop, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C . (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzola, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C . , and Tegoni, M . (1998) Biochemistry 37, 13987-13996) . Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd . The four structures differ significantly in the c heme domains . Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd) . Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study . This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized . These results suggest that the d1 heme reduction is likely to be responsible of the movements . We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.

Protein Eng, 1999 Apr, 12(4), 305 - 11
Isolated transmembrane helices arranged across a membrane: computational studies; Tseitin VM et al.; A computational procedure for predicting the arrangement of an isolated helical fragment across a membrane was developed . The procedure places the transmembrane helical segment into a model triple-phase system 'water-octanol-water'; pulls the segment through the membrane, varying its 'global' position as a rigid body; optimizes the intrahelical and solvation energies in each global position by 'local' coordinates (dihedral angles of side chains); and selects the lowest energy global position for the segment . The procedure was applied to 45 transmembrane helices from the photosynthetic reaction center from Rhodopseudomonas viridis, cytochrome c oxidase from Paracoccus denitrificans and bacteriorhodopsin . In two thirds of the helical fragments considered, the procedure has predicted the vertical shifts of the fragments across the membrane with an accuracy of -0.15 +/- 3.12 residues compared with the experimental data . The accuracy for the remaining 15 fragments was 2.17 +/- 3.07 residues, which is about half of a helix turn . The procedure predicts the actual membrane boundaries of transmembrane helical fragments with greater accuracy than existing statistical methods . At the same time, the procedure overestimates the tilt values for the helical fragments.

Biochim Biophys Acta, 1999 May 5, 1411(2-3), 231 - 49
Bacterial nitric oxide synthesis; Cutruzzola F; The structure-function relationships in nitrite reductases, key enzymes in the dissimilatory denitrification pathway which reduce nitrite to nitric oxide (NO), are reviewed in this paper . The mechanisms of NO production are discussed in detail and special attention is paid to new structural information, such as the high resolution structure of the copper- and heme-containing enzymes from different sources . Finally, some implications relevant to regulation of the steady state levels of NO in denitrifiers are presented.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 645 - 51
A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb . nov; Rainey FA et al.; Comparison of both 16S rRNA coding sequences and DNA-DNA hybridization of ten strains of alpha-subclass of Proteobacteria currently classified as strains of Paracoccus denitrificans has shown that they fall into two groups which are distinct from each other at the species level . Comparison with published data on the cytochrome c profiles and other 16S rRNA coding sequences in the literature has confirmed these observations and enabled several other strains also to be assigned to these two groups . Group A comprises strains ATCC 17741T (the type strain of P . denitrificans), LMD 22.21T, DSM 413T, ATCC 19367, ATCC 13543, DSM 1404, DSM 1405, Pd 1222 (a genetic modification of DSM 413T) and NCIMB 8944 . Group B comprises ATCC 35512T (the original type strain of Thiosphaera pantotropha), LMD 82.5T, LMD 92.63, DSM 65, LMG 4218, IAM 12479, JCM 6892, DSM 11072, DSM 11073 and DSM 11104 . In light of these findings, it is proposed that: (1) strains of group A are retained as P . denitrificans, with ATCC 17741T as the type strain of the type species; and (2) all strains of group B are assigned to the new species combination Paracoccus pantotrophus comb . nov., with strain ATCC 35512T as the type strain . Comparative 16S rRNA sequence analysis and DNA-DNA hybridization of strains of Paracoccus versutus confirm that this species is distinct from both P . denitrificans and P . pantotrophus, but that its nearest phylogenetic neighbour is P . pantotrophus.

J Environ Sci Health B, 1999 May, 34(3), 491 - 507
Microbial dechlorination of 2,4,6-trichlorophenol in anaerobic sewage sludge; Chang BV et al.; The dechlorination of 2,4,6-trichlorophenol (TCP) in municipal sewage sludge with a chlorophenol (CP)-adapted consortium was investigated . Results show that dechlorination rates differed according to the source of the sludge samples used in the batch experiments . No significant differences in 2,4,6-TCP dechlorination were observed following treatment with inoculum at densities ranging from 10% to 50% (V/V), but a significant delay was noted at 5% (V/V) density . Overall, results show that the higher the 2,4,6-TCP concentration, the slower the dechlorination rate . The addition of acetate, lactate, pyruvate, vitamin B12 or manganese dioxide did not results in a significant change in 2,4,6-TCP dechlorination . Data collected from a bioreactor experiment revealed that pH 7.0 and a total solid concentration of 10 g/L were optimal for dechlorination . Dechlorination rates decreased significantly at higher agitation speeds . 2,4,6-TCP dechlorination was enhanced under methanogenic conditions, but it was inhibited under denitrifying and sulfate-reducing conditions.

Biosci Biotechnol Biochem, 1999 Mar, 63(3), 530 - 6
Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f . sp . denitrificans; Liu HP et al.; Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f . sp . denitrificans IL106 . Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products . Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions . A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells . Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation . These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.

J Biochem (Tokyo), 1999 May, 125(5), 864 - 8
Anaerobic purification and characterization of nitrous oxide reductase from Rhodobacter sphaeroides f . sp . denitrificans IL106; Sato K et al.; The nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f . sp . denitrificans IL106, has been purified under anaerobic conditions . The specific activity of the enzyme was 78 micromol nitrous oxide reduced per min per mg protein, which was approximately 80% higher than that of the aerobic form . The enzyme purified anaerobically retained most of its activity after aerobic storage at 4 degrees C for 2 months without any additives . Visible absorption spectra of the Rhodobacter nitrous oxide reductase resembled those of the enzymes from other origins . The enzyme retained its activity after reduction with sodium dithionite, and the enzyme activity could be determined using dithionite-reduced benzyl viologen . Turnover-dependent inactivation of the enzyme was suppressed by complete removal of oxygen from the reaction mixture, and promoted by zinc ions.

J Bacteriol, 1999 May, 181(9), 2914 - 21
Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans; Maehara A et al.; The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level . The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaCPd) cloned recently (S . Ueda, T . Yabutani, A . Maehara, and T . Yamane, J . Bacteriol . 178:774-779, 1996) . Gene walking around phaCPd revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaPPd gene, encoding GA16 protein, and the other was the phaRPd gene, encoding a protein that is putatively involved in the regulation of the expression of phaPPd . Overproduction of PhaPPd was observed in Escherichia coli carrying phaPPd, but the overproduction was not observed in the presence of phaRPd . Coexpression of phaPPd and PHA biosynthesis genes in E . coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB . GA16 protein was considered a phasin protein . The phaRPd gene had significant similarities to stdC, a possible transcriptional factor of Comamonas testosteroni, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.

J Bacteriol, 1999 May, 181(9), 2802 - 6
The periplasmic nitrate reductase in Pseudomonas sp . strain G-179 catalyzes the first step of denitrification; Bedzyk L et al.; Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria . Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined . To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp . strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis . Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor . No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region . Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide . Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate . These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.

Microbiology, 1999 Mar, 145 ( Pt 3), 633 - 41
Complex pattern formation by Pseudomonas strain KC in response to nitrate and nitrite; Emerson D; Pattern formation by enteric bacteria growing on semi-solid agar plates has recently been described, and in this paper a similar phenomenon is reported in an environmental isolate, Pseudomonas strain KC . This organism reproducibly formed complex patterns on 3-mm-thick, semi-solid agar (0.25%, w/v) mineral medium motility plates containing either 5 mM 2-oxoglutarate or 5 mM glycerol, and 2 mM NO2- or 3 mM NO3- . When the plates were inoculated at the centre and incubated in air at 30 degrees C, a growth zone formed that migrated slowly (<0.5 mm h(-1)) and uniformly outward . Within 24 h, a dense outer growth ring formed which then coalesced into discrete aggregates (approx . 0.5 mm in diameter) with uniform spacing; these aggregates moved radially at approximately 0.7 mm h(-1) and maintained their density and spacing, to form a spoke-like pattern . Microscopic observations revealed that cells within an intact aggregate were very motile, but did not appear to leave the aggregate . Pattern formation did not occur if the NO2- or NO3- concentration was altered, if the agar layer was thicker or thinner, if the plates were incubated under strictly denitrifying conditions, or if carbon sources that were chemoattractants, e.g . acetate, were used . Five other species of pseudomonads were tested under identical conditions, and none formed patterns . Oxygen microelectrode studies indicated that there was little or no oxygen within the aggregates . The growth of strain KC was progressively inhibited in imposed diffusion gradients of NO2- or NO3- of which NO2- was the more potent inhibitor . These results suggest that pattern formation by strain KC may reflect an adaptive response to adverse environmental conditions.

Microbiology, 1999 Mar, 145 ( Pt 3), 577 - 84
Determination of the Paracoccus denitrificans SOS box; del Rey A et al.; By gel retardation experiments with crude cell extracts of Paracoccus denitrificans it was demonstrated that a protein specifically binds to the promoter of the P . denitrificans recA gene . PCR mutagenesis of the recA promoter showed that the GAACN7GAAC motif is required for the formation of the DNA-protein complex . This protein also binds to the GTTCN7GTTC motif, which is present in the promoter of the P . denitrificans uvrA gene . Mutational analysis of the promoter regions of both P . denitrificans recA and uvrA genes indicated that the GAACN7GAAC and GTTCN7GTTC sequences are required for DNA-damage-mediated induction of these two genes in vivo . Furthermore, the P . denitrificans recA gene was DNA-damage-inducible when introduced into cells of the phylogenetically related phototrophic bacterium Rhodobacter sphaeroides, although this inducibility was lost in mutants in the GAACN7GAAC motif . These results indicate that P . denitrificans possesses the same SOS box as R . sphaeroides, which, in agreement with previous work, is proposed as being the GTTCN7GTTC motif.

FEBS Lett, 1999 Apr 1, 448(1), 157 - 9
Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c550; Koutny M et al.; A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1) has been purified from the anoxically grown cytochrome c550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N-terminal amino acid sequence) . Under our assay conditions, the half-saturation of electron transport occurred at about 10 microM pseudoazurin; the reaction was retarded by increasing ionic strength.

Biochim Biophys Acta, 1999 Apr 21, 1411(1), 114 - 20
Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase; Reincke B et al.; A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans . Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region . Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors . High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Eur J Biochem, 1999 May, 261(3), 767 - 74
The reduction state of the Q-pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans; Otten MF et al.; In this work we demonstrate how the reduction state of the Q-pool determines the distribution of electron flow over the two quinol-oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases . The dependence of the electron-flow rate to oxygen on the fraction of quinol in the Q-pool was determined in membrane fractions and in intact cells of the wild-type strain, a bc1-negative mutant and a quinol oxidase-negative mutant . Membrane fractions of the bc1-negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild-type strain or the quinol oxidase-negative mutant . In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher . In intact cells, the dependence was weaker . In uncoupled cells the dependence of the oxygen-consumption rates on the fractions of quinol in the Q-pool in the wild-type strain and in the two mutants came closer to that found for the membrane fractions . We also determined the dependence for membrane fractions of the wild-type in the absence and presence of antimycin A, an inhibitor of the bc1 complex . The dependence in the presence of antimycin A resembled that of the bc1-negative mutant . These results indicate that electron-flow distribution between the two quinol-oxidising branches in P . denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q-pool.

Mol Microbiol, 1999 Mar, 31(6), 1681 - 94
Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes; Vollack KU et al.; Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification . In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family . The fnrA gene encodes a genuine FNR-type regulator, which is expressed constitutively and controls the cytochrome cbb3-type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen-independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon) . The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family . Their main distinctive feature is the lack of cysteine residues for complexing the {4Fe-4S} centre of redox-active FNR-type regulators . However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature . We have studied the expression of the dnr genes under aerobic, oxygen-limited and denitrifying conditions . DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase . The dnrD gene is part of the 30 kb region carrying denitrification genes of P . stutzeri . Transcription of dnrD was activated in O2-limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA . In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD . dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase . The transcription of dnrS required FnrA in O2-limited cells . Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response . The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction . We found the highest amount of dnrE transcripts in aerobically nitrate-challenged cells . The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL . The multiplicity of FNR factors in P . stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate-activated metabolic processes.

Chemosphere, 1999 May, 38(11), 2561 - 8
Anaerobic formation and degradation of toxic aromatic compounds in agricultural and communal sewage deposits; Hofmann K et al.; Relatively high concentrations of phenol, p-cresol, phenylacetic acid and other aromatic compounds were found in agricultural and communal sewage deposits . These toxic aromatic compounds are products of the bacterial degradation of aromatic amino acids under anaerobic conditions . In laboratory experiments at 26 degrees C and under N2-atmosphere, the same aromatics were formed from the amino acid tyrosine and from gelatine in assays inoculated with sewage sludge . After exhaustion of tyrosine and gelatine, respectively, concentrations of the accumulated phenol and other aromatics remained stable for months, i.e., phenol, p-cresol, phenylacetic acid etc . are dead-end products of the bacterial metabolism under these conditions . After addition of sodium nitrate the aromatic compounds are metabolically decomposed by denitrification within weeks.

J Biol Chem, 1999 Apr 16, 274(16), 11383 - 9
The structure of an electron transfer complex containing a cytochrome c and a peroxidase; Pettigrew GW et al.; Efficient biological electron transfer may require a fluid association of redox partners . Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c . For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase . In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase . These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.

Syst Appl Microbiol, 1999 Feb, 22(1), 45 - 58
Taxonomic study of bacteria isolated from natural mineral waters: proposal of Pseudomonas jessenii sp . nov . and Pseudomonas mandelii sp . nov; Verhille S et al.; The taxonomic position of 23 strains isolated from mineral waters and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster IX, subclusters XIIIa and XIIIc of VERHILLE, S., ELOMARI, M., COROLER, L., IZARD, D., LECLERC, H . (Syst . Appl . Microbiol, 20, 137-149, 1997) has been genotypically further studied in the present work . On the basis of hybridization results, these strains were gathered into two new genomic groups for which we propose the names of Pseudomonas jessenii sp . nov . (Type strain CIP 105274) and Pseudomonas mandelii sp . nov . (Type strain CIP 105273) . Deoxyribonucleic acid relatedness levels showed homologies ranging from 78 to 100% for Pseudomonas jessenii and from 77 to 100% for Pseudomonas mandelii . Furthermore, hybrization rates with 66 representative well characterized species or only partially characterized species of the genus Pseudomonas were below 53%, with delta Tm values of 7 degrees C and more . The mol% G + C content ranged from 57 to 58 . The two new species presented basic morphological characteristics common to all pseudomonads . Various phenotypic features, such as denitrification, growth at 4 degrees C or 41 degrees C, trigonelline assimilation, alpha-L-glutamyl-L-histidine arylarmidase activity, growth on benzoate and meso-tartrate were found to differentiate Pseudomonas jessenii from Pseudomonas mandelii and from other Pseudomonas species . Pseudomonas jessenii encompassed a total of 9 strains from both phenotypic groups IX and XIIIa . Pseudomonas mandelii clustered a total of 13 strains from both phenotypic groups IX and XIIIc . Their clinical significance is unknown . The 16S rDNA of each type strain was sequenced and compared with the known sequences of the representative strains of the genus Pseudomonas . A phylogenetic tree was constructed to determine the intrageneric relationships within the genus Pseudomonas.

Appl Environ Microbiol, 1999 Apr, 65(4), 1681 - 7
Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments; Scala DJ et al.; Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves . Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers . Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold . The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% +/- 10% . Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs . A highly conserved region is proposed as the copper binding, catalytic site (CuZ) of the mature protein . Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups . Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.

Appl Environ Microbiol, 1999 Apr, 65(4), 1652 - 7
PCR detection of genes encoding nitrite reductase in denitrifying bacteria; Hallin S et al.; Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir . The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge . Sequence relationships of nir genes were also established . The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences . Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences . Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates . The primers did not amplify genes of nondenitrifying strains . The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples . PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case . The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene . The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find . We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

Biotechnol Bioeng, 1998 Dec 20, 60(6), 649 - 55
Experimental and modeling study of diauxic lag of Pseudomonas denitrificans switching from oxic to anoxic conditions; Liu PH et al.; We have shown that Pseudomonas denitrificans undergo a diauxie when switching from dissolved oxygen to nitrate as terminal electron acceptor . The length of time under aeration significantly affected the length of the diauxic lag, whereas the presence or absence of nitrate in the culture under aeration had a marginal effect . Nitrate consumption was very low during the lag period and then increased rapidly, coinciding with exponentially increasing biomass concentrations . Biochemical rate expressions that account for enzyme synthesis and activity in response to culture conditions and enzyme specific levels were developed . The new model successfully predicts the different lengths of diauxic lags observed in the experiments as well as the growth pattern and nitrate uptake .

Biotechnol Bioeng, 1998 Oct 5, 60(1), 61 - 9
Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans; Maehara A et al.; Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified . Gene dosage effects for PHA synthesis were investigated in recombinants of P . denitrificans with increased expression levels of each PHA synthetic enzyme . In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents {(g PHA) . (g total biomass)-1} and the highest rates of PHA accumulation {(g PHA) . (g residual biomass)-1 . h-1} among these recombinants . The PHA content and PHA accumulation rate (g PHA/g residual biomass . h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain . This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA . Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded . The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer .

Biotechnol Bioeng, 1998 Aug 20, 59(4), 393 - 9
Biodegradation of 1,1,1-trichloroethane by a carbon tetrachloride-degrading denitrifying consortium
Sherwood JL, Petersen JN, Skeen RS.
A denitrifying consortium capable of degrading carbon tetrachloride (CT) was shown to also degrade 1,1,1-trichloroethane (TCA) . Fed-batch experiments demonstrated that the specific rate of TCA degradation by the consortium was comparable to the specific rate of CT degradation (approximately 0.01 L/gmol/min) and was independent of the limiting nutrient . Although previous work demonstrated that 4-50% of CT transformed by the consortium was converted to chloroform (CF), no reductive dechlorination products were detected during TCA degradation, regardless of the limiting nutrient . The lack of chlorinated TCA degradation products implies that the denitrifying consortium possesses an alternate pathway for the degradation of chlorinated solvents which does not involve reductive dechlorination .

Biotechnol Bioeng, 1998 Jul 5, 59(1), 52 - 61
Kinetics of denitritification and denitratification in anoxic filters
Huang JS, Her JJ, Jih CG.
Denitritification and denitratification in anoxic filters were performed to generate experimental data . Also, a kinetic model of denitratification that accounts for intrinsic biokinetics and hydrodynamic behavior of the biofilter is proposed . In denitritification, the simulated results are in good agreement with the experimental data; and a higher nitrite influent concentration gives a higher nitrite reduction efficiency if the denitrifying loading is kept the same . In denitratification, the intermediate nitrite tends to accumulate, and a higher denitrifying loading results in a higher nitrite effluent concentration . By inserting biological and physical parameter values into the kinetic model, the variations in distributed fractions of nitrate-reductase (f) and nitrite-reductase (1-f) with different denitrifying loadings can be estimated by fitting in experimental data . The estimated f increased with an increase in denitrifying loading, implying that a higher denitrifying loading results in a higher nitrite effluent concentration . From parametric sensitivity analyses, the parameter f is more sensitive than other biological and physical parameters . Accordingly, the proposed kinetic model of denitratification can be used to predict the treatment performance of anoxic filters appropriately .

Biotechnol Bioeng, 1998 May 20, 58(4), 408 - 15
Denitrification and nitric oxide reduction in an aerobic toluene-treating biofilter; du Plessis CA et al.; The presence of significant denitrification activity in an aerobic toluene-treating biofilter was demonstrated under batch and flow-through conditions . N2O concentrations of 9.2 ppmv were produced by denitrifying bacteria in the presence of 15% acetylene, in a flow-through system with a bulk gas phase O2 concentration of >17% . The carbon source for denitrification was not toluene but a byproduct or metabolite of toluene catabolism . Denitrification conditions were successfully used for the reduction of 60 ppmv nitric oxide to 15 ppmv at a flow rate of 3 L min-1 (EBRT of 3 min) in a fully aerated, 17% v/v O2 (superficially aerobic) biofilter . Higher NO removal efficiency (97%) was obtained by increasing the toluene supply to the biofilter .

Biotechnol Bioeng, 1999 Feb, 62(4), 479 - 484
Selenium reduction by a denitrifying consortium; Rege MA et al.; A denitrifying bacterial consortium obtained from the Pullman, Washington wastewater treatment facility was enriched under denitrifying conditions and its ability to reduce selenite and selenate was studied . Replicate experiments at two different experimental conditions were performed . All experiments were performed under electron-acceptor limiting conditions, with acetate as the carbon source and nitrate the electron acceptor . In the first set of experiments, selenite was present, whereas, in the second set, selenate was added . A significant lag period of approximately 150 h was necessary before selenite or selenate reduction was observed . During this lag period, nitrate and nitrite use was observed . Once selenite or selenate reduction had started, nitrate and nitrite reduction was concomitant with selenium species reduction . Trace amounts of selenite were detected during the selenate reduction study . Analysis of the data indicates that, once selenium species reduction was induced, the rate of reduction was proportional to the selenium species concentration and to the biomass concentration . Furthermore, at similar biomass and contaminant concentrations, selenite reduction is approximately four times faster than selenate reduction .

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 4149 - 53
NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone; Schuler F et al.; The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain . The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits . A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone . Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases . Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben . We designed (trifluoromethyl)diazirinyl{3H}pyridaben ({3H}TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity . Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable . Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I . Immunoprecipitation of labeled membranes of P . denitrificans and T . thermophilus established photoaffinity labeling of the equivalent bacterial NQO6 . Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above . These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.

Eur J Biochem, 1999 Feb, 259(3), 651 - 9
Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015); Ferretti S et al.; Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes . No three-dimensional structural information has been published for this enzyme . We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer . Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes . We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule . The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1 . (mg of protein)-1; N2OR activity showed both base and temperature activation . The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm . On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared . Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm . As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated . Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.

Science, 1999 Mar 26, 283(5410), 2064 - 9
Arctic ozone loss due to denitrification
Waibel AE, Peter T, Carslaw KS, Oelhaf H, Wetzel G, Crutzen PJ, Poschl U, Tsias A, Reimer E, Fischer H.
Measurements from the winter of 1994-95 indicating removal of total reactive nitrogen from the Arctic stratosphere by particle sedimentation were used to constrain a microphysical model . The model suggests that denitrification is caused predominantly by nitric acid trihydrate particles in small number densities . The denitrification is shown to increase Arctic ozone loss substantially . Sensitivity studies indicate that the Arctic stratosphere is currently at a threshold of denitrification . This implies that future stratospheric cooling, induced by an increase in the anthropogenic carbon dioxide burden, is likely to enhance denitrification and to delay until late in the next century the return of Arctic stratospheric ozone to preindustrial values.

J Biol Chem, 1999 Mar 26, 274(13), 8428 - 36
Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase . Enhancement of adenosine N1-oxide reductase activity; Hilton JC et al.; The periplasmic DMSO reductase from Rhodobacter sphaeroides f . sp . denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R . sphaeroides or E . coli N-terminal signal sequence . Whereas the R . sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification . The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R . sphaeroides . The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO . Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum . The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states . This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.

Biochim Biophys Acta, 1999 Feb 10, 1430(1), 65 - 72
Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism; Moir JW; Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically . Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon {NO} . When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively . Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P . denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification . In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex . In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex . These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.

Biochim Biophys Acta, 1999 Jan 27, 1410(1), 71 - 6
Evaluation of relative contributions of two enzymes supposed to metabolise hydrogen peroxide in Paracoccus denitrificans; Koutny M et al.; A biosensor exploiting an electrochemically mediated enzyme-catalysed reaction was used to quantify relative contributions of cytoplasmic catalase and periplasmic cytochrome c peroxidase to the overall rate of hydrogen peroxide breakdown in cells of Paracoccus denitrificans . The effects of antimycin (an inhibitor of electron flow to cytochrome c peroxidase), the reaction rate versus substrate concentration profiles for the whole cells and subcellular fractions, and the time courses of oxygen concentration demonstrated a profound decrease in the capacity of cytochrome c peroxidase to reduce H2O2 under in vivo conditions . The reason is suggested to be a competition for available electrons between the enzyme and terminal oxidases metabolising oxygen produced by catalase.

J Bacteriol, 1999 Mar, 181(6), 1739 - 47
Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae; Heilbronn J et al.; An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized . The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors . Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent . The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products) . The structural gene for the tyrosine aminotransferase was cloned from K . pneumoniae and expressed in E . coli . The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E . coli, S . typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase . Structural motifs around key invariant residues placed the K . pneumoniae enzyme within the Ia subfamily of aminotransferases . Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases . The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

Chemosphere, 1999 Mar, 38(7), 1561 - 70
Photocatalytic degradation of 5-nitro-1,2,4-triazol-3-one NTO in aqueous suspension of TiO2 . Comparison with Fenton oxidation; Le Campion L et al.; 5-nitro-1,2,4-triazol-3-one (NTO) is a powerful insensitive explosive, present in industrial waste waters . A remediation method based on photochemical decomposition and Fenton oxidation of NTO has been evaluated by monitoring the mineralization of 14C-labelled NTO . The TiO2-catalyzed photodegradation (lambda > 290 nm, TiO2 0.4 g/l, NTO 150 mg/l)) leads to the complete mineralization of NTO in 3 hours . This degradation involves a simultaneous denitrification and ring scission of NTO leading to nitrites, nitrates and carbon dioxide . No significant photo-degradation of NTO was detected in the absence of the catalyst . Long term irradiation over one week, leads to a complete degradation of concentrated NTO (5 g/l), suggesting that this method could be useful to clean-up NTO wastes . Fenton oxidation offers an efficient cost-effective method for NTO remediation . This reaction is faster that the TiO2 catalyzed photolysis and find application on the mineralization of high concentrations of NTO (15 g/l) . Fenton oxidation provokes ring cleavage and subsequent elimination of the two carbon atoms of NTO as CO2 . During this reaction, the nitro group is completely transformed into nitrates.

Biochem J, 1999 Mar 15, 338 ( Pt 3), 701 - 8
The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis; Raux E et al.; MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem . In the present study, we have demonstrated complementation of defined cysG mutants of Sal . typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA . The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal . typhimurium and E . coli cysG) . MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag . After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+ . The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem . Moreover, despite the fact that S . cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal . typhimurium and Bacillus megaterium.

Appl Environ Microbiol, 1999 Mar, 65(3), 989 - 94
Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans; Barford CC et al.; Nitrogen stable-isotope compositions (delta15N) can help track denitrification and N2O production in the environment, as can knowledge of the isotopic discrimination, or isotope effect, inherent to denitrification . However, the isotope effects associated with denitrification as a function of dissolved-oxygen concentration and their influence on the isotopic composition of N2O are not known . We developed a simple steady-state reactor to allow the measurement of denitrification isotope effects in Paracoccus denitrificans . With {dO2} between 0 and 1.2 microM, the N stable-isotope effects of NO3- and N2O reduction were constant at 28.6 per thousand +/- 1.9 per thousand and 12.9 per thousand +/- 2.6 per thousand, respectively (mean +/- standard error, n = 5) . This estimate of the isotope effect of N2O reduction is the first in an axenic denitrifying culture and places the delta15N of denitrification-produced N2O midway between those of the nitrogenous oxide substrates and the product N2 in steady-state systems . Application of both isotope effects to N2O cycling studies is discussed.

DNA Res, 1998 Dec 31, 5(6), 365 - 71
Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes; Inatomi K; The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes . The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases . The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri . The gene product of A . cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites . The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site . The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.

Biochemistry, 1999 Feb 16, 38(7), 2206 - 12
Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin; Saxena K et al.; The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents . The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes . To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall . Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected . However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.

Biochemistry, 1999 Feb 16, 38(7), 1977 - 89
Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain; Roberts DL et al.; The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution . The overall fold is identical to that of the human enzyme, with the exception of a single loop region . Like the human structure, the structure of the P . denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit . Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein . Using the sequence and structures of the human and P . denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins . From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively . Mapping of ionic differences between the human and P . denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD) . Analysis of the ionic strength dependence of the electron transfer reaction between either human or P . denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P . denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength . This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins . Analysis of the electrostatic potentials of the human and P . denitrificans ETFs reveals that the P . denitrificans ETF is more negatively charged . This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs . Furthermore, by analysis of a model of the previously described human-P . denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.

Biochemistry, 1999 Feb 9, 38(6), 1685 - 94
Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes; Hellwig P et al.; Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach . Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively . Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm-1) yielded difference spectra for the number of redox potentials selected . With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained . The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors . The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock {(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York} . Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes such as proton transfer . A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.

Biodegradation, 1998, 9(3-4), 259 - 71
Technological aspects of the microbial treatment of sulfide-rich wastewaters: a case study; Sublette KL et al.; Thiobacillus denitrificans has been shown to be an effective biocatalyst for the treatment of a variety of sulfide-laden waste streams including sour water, sour gases, and refinery spent-sulfidic caustics . The term 'sour' originated in the petroleum industry to describe a waste contaminated with hydrogen sulfide or salts of sulfide and bisulfide . The microbial treatment of sour waste streams resulting from the production or refining of natural gas and crude oil have been investigated in this laboratory for many years . The application of this technology to the treatment of sour wastes on a commercially useful scale has presented several technical barriers including substrate inhibition (sulfide), product inhibition (sulfate), the need for septic operation, biomass recycle and recovery, mixed waste issues, and the need for large-scale cultivation of the organism for process startup . The removal of these barriers through process improvements are discussed in terms of a case study of the full-scale treatment of sulfide-rich wastewater . The ability of T . denitrificans to deodorize and detoxify an oil-field produced water containing sulfides was evaluated under full-scale field conditions at Amoco Production Co . Salt Creek Field in Midwest, WY . More than 800 m3/d of produced water containing 100 mg/L sulfide and total dissolved solids of 4800 mg/L were successfully biotreated in an earthen pit (3000 m3) over a six-month period . Complete removal of sulfides and elimination of associated odors were observed . The system could be upset by severe hydraulic disturbances; however, the system recovered rapidly when normal influent flow rates were restored.

Biodegradation, 1998, 9(3-4), 159 - 67
Microsensors as a tool to determine chemical microgradients and bacterial activity in wastewater biofilms and flocs; Santegoeds CM et al.; Microsensors used in microbial ecology are reviewed with emphasis on new sensor developments (NO3-, NO2-, NH4+, CO2, H2, H2S and CH4 microsensors as well as fiberoptical microsensors for O2, temperature and pH) . Examples of microsensor applications in biofilms and activated sludge flocs are presented, in which sulfate reduction and denitrification were studied.

FEMS Microbiol Rev, 1998 Dec, 22(5), 475 - 88
Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems; Hylemon PB et al.; Isoprenoic compounds play a major part in the global carbon cycle . Biosynthesis and mineralization by aerobic bacteria have been intensively studied . This review describes our knowledge on the anaerobic metabolism of isoprenoids, mainly by denitrifying and fermentative bacteria . Nitrate-reducing beta-Proteobacteria were isolated on monoterpenes as sole carbon source and electron donor . Thauera spp . were obtained on the oxygen-containing monoterpenes linalool, menthol, and eucalyptol . Several strains of Alcaligenes defragrans were isolated on unsaturated monoterpenes as growth substrates . A novel denitrifying beta-Proteobacterium, strain 72Chol, mineralizes cholesterol completely to carbon dioxide . Physiological studies showed the presence of several oxidative pathways in these microorganisms . Investigations by organic geochemists indicate possible contributions of anaerobes to early diagenetic processes . One example, the formation of p-cymene from monoterpenes, could indeed be detected in methanogenic enrichment cultures . In man, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol . During their enterohepatic circulation, bile acids are biotransformed by the intestinal microflora into a variety of metabolites . Known bacterial biotranformations of conjugated bile acids include: deconjugation, oxidation of hydroxy groups at C-3, C-7 and C-12 with formation of oxo bile acids and reduction of these oxo groups to either alpha- or beta-configuration . Quantitatively, the most important bacterial biotransformation is the 7 alpha-dehydroxylation of CA and CDCA yielding deoxycholic acid and lithocholic acid, respectively . The 7 alpha-dehydroxylation of CA occurs via a novel six-step biochemical pathway . The genes encoding several enzymes that either transport bile acids or catalyze various reactions in the 7 alpha-dehydroxylation pathway of Eubacterium sp . strain VPI 12708 have been cloned, expressed in Escherichia coli, purified, and characterized.

Acta Microbiol Pol, 1998, 47(3), 297 - 304
Adaptation of a phenol-degrading denitrifying bacteria to high concentration of phenol in the medium; Son TT et al.; The growth and uptake of phenol by 8 strains isolated from wastewater sediments in stationary cultures in medium with increasing concentrations of phenol (from 100 to 600 mg/L) under denitrifying conditions were studied . All the strains grew in media containing 250 mg phenol/L and only strains 101/1, 83/2 and 21/1/ in consecutive passages visibly increased both specific growth rate (mu day-1) as well as phenol-degrading activity (mg/L x day) . Consecutive passages of the culture in medium containing 400 mg phenol/L resulted in the elimination of 3 out of the 5 strains growing in the medium in the first passage . Only strain 101/1 demonstrated high specific growth rate and phenol-degrading activity in medium containing 600 mg phenol/L . In consecutive passages in medium containing 250, 400 and 600 mg phenol/L the specific growth (mu day-1) and phenol-degrading activity (mg/L x day) of P . aeruginosa 101/1 were 0.38 and 36; 0.12 and 19; 0.09 and 20, respectively.

Biochemistry, 1999 Jan 26, 38(4), 1176 - 84
The active site of Paracoccus denitrificans aromatic amino acid aminotransferase has contrary properties: flexibility and rigidity; Okamoto A et al.; Paracoccus denitrificans aromatic amino acid aminotransferase (EC 2 . 6.1.57; pdAroAT) binds with a series of aliphatic monocarboxylates attached to the bulky hydrophobic groups . To analyze the properties of the active site in this enzyme, we determined the tertiary structures of pdAroAT complexed with nine different inhibitors . Comparison of these active site structures showed that the active site of pdAroAT consists of two parts with contrary properties: rigidity and flexibility . The regions that interact with the carboxylates and methylene chains of the inhibitors gave essentially the same structures among these complexes, exhibiting the rigid property, which would involve fixing the substrate at the proper orientation for efficient catalysis . The region that interacts with the terminal hydrophobic groups of the inhibitors gave versatile structures according to the structures of the terminal groups, showing that this region is structurally flexible . This is mainly achieved by the conformational versatility of the side chains of Asp15, Lys16, Asn142, Arg292, and Ser296 . These residues formed in the active site hydrogen bond networks, which were adaptable for the structures of the terminal hydrophobic groups of the inhibitors, with a small deformation or partial destruction according to the shapes and sizes of the inhibitors . These observations illustrate how the flexibility and rigidity in the active site can be used for the substrate binding and recognition.

Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 899 - 902
Enhanced rate of intramolecular electron transfer in an engineered purple CuA azurin; Farver O et al.; The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple CuA site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear CuA center, because both type 1 and CuA centers are placed in the same location in the protein while all other structural elements remain the same . Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear CuA center in the purple azurin mutant . The rate constant of this intramolecular process, kET = 650 +/- 60 s-1 at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 +/- 20 s-1), in spite of a smaller driving force (0.69 eV for purple CuA azurin vs . 0.76 eV for blue copper azurin) . The reorganization energy of the CuA center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin . These results represent a direct comparison of electron transfer properties of the blue and purple CuA sites in the same protein framework and provide support for the notion that the binuclear purple CuA center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.

Sci Total Environ, 1998 Dec 11, 224(1-3), 177 - 80
Effects of mouth cleansing on the levels of exhaled nitrous oxide in young and older adults; Mitsui T et al.; Nitrous oxide (N2O) is produced by denitrification, i.e . by microbial reduction of nitrate (NO3-) . Our previous studies have established an analytical method for demonstrating the existence of N2O in exhaled air, and we showed that levels of N2O in exhaled air increase with age after puberty . However, the source of this change and its biological significance are still unclear . The purpose of this study was to examine whether the oral microorganisms are the main source of N2O . We measured exhaled N2O in 35 young adults (aged 19-29 years) and 34 older adults (aged 61-79 years) before and after mouth cleansing . N2O was measured using an infrared-photoacoustic analyzer equipped with an optical filter (UA0985, 2215 cm-1) . Participants were classified as producers and non-producers according to the levels of exhaled N2O relative to the level in the atmosphere . N2O production differed significantly between the young adult producers and the older adult producers . Mouth cleansing resulted in an immediate reduction in exhaled N2O in both groups . We only found seven (20.0%) producers in the young, and 32 (94.1%) producers in the older after mouth washing . The differences before and after mouth cleansing were significant in both groups (P < 0.01 in the young and P < 0.05 in the older) . The oral cavity is a major source of N2O . However, since approximately half-levels of N2O were still observed in exhaled air after mouth, cleansing, there may exist another N2O source in the human body.

Biochemistry, 1998 Dec 22, 37(51), 17726 - 34
Extended metal environments of cytochrome c oxidase structures; Karlin S et al.; The metals of the cytochrome c oxidase structures of the bovine heart mitochondrion (PDB code 1occ) and of the soil bacterium Paracoccus denitrificans (1arl) include a dicopper center (CuA), magnesium, two proximal hemes, a copper (CuB) atom, and a calcium . The mitochondrial structure also possesses a bound distant zinc ion . The extended environments of the metal sites are analyzed emphasizing residues of the second shell in terms of polarity, hydrophobicity, secondary structure, solvent accessibility, and H-bonding networks . A significant difference in the CuA metal environments concerns D-51 I in 1occ, absent from 1arl . The D-51 I appears to play an important role in the proton pumping pathway . Our analysis uncovers several statistically significant residue clusters, including a cysteine-histidine-tyrosine cluster overlapping the CuA-Mg complex; a histidine-acidic cluster enveloping the environment of Mg, the two hemes, and CuB; and on the protein surface a mixed charge cluster, which may help stabilize the quaternary structure and/or mediate docking to cytochrome c . These clusters may constitute possible pathways for electron transfer, for O2 diffusion, and for H2O movement . Many hydrogen bonding relations along the interface of subunits I and II demarcate this surface as a potential participant in proton pumping.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 19 - 24
Effect of nitrogen oxides on expression of the nir and nor genes for denitrification in Pseudomonas aeruginosa; Arai H et al.; The activity of the promoters involved in transcription of the genes (nirS, nirQ and norC) required for anaerobic reduction of nitrite and nitric oxide was investigated in NIR- and NOR-deficient mutants of Pseudomonas aeruginosa . The transcriptional activity of these three promoters was induced by nitrite in a wild-type strain and the activity was low in an nirS mutant . In norCBD and nirQOP mutants, which were expected to accumulate nitric oxide because of a lack of nitric oxide reductase activity, the norC and nirQ promoters showed significantly enhanced activity in promoting transcription relative to the parental strain, even at low nitrite concentrations . These results suggest that the nirQ and norC promoters are regulated by the concentration of endogenous nitric oxide rather than that of nitrite.

Eur J Biochem, 1998 Dec 1, 258(2), 559 - 66
The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans--implications for the interaction with cytochrome c peroxidase; Pettigrew GW et al.; The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated . The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21 . Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge . The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy . The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer . This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography . A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other . The resultant dipole moment of the dimer is therefore small . Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Appl Environ Microbiol, 1999 Jan, 65(1), 270 - 7
Phylogenetic affinity of a wide, vacuolate, nitrate-accumulating Beggiatoa sp . from Monterey Canyon, California, with Thioploca spp; Ahmad A et al.; Environmentally dominant members of the genus Beggiatoa and Thioploca spp . are united by unique morphological and physiological adaptations (S . C . McHatton, J . P . Barry, H . W . Jannasch, and D . C . Nelson, Appl . Environ . Microbiol . 62:954-958, 1996) . These adaptations include the presence of very wide filaments (width, 12 to 160 microm), the presence of a central vacuole comprising roughly 80% of the cellular biovolume, and the capacity to internally concentrate nitrate at levels ranging from 150 to 500 mM . Until recently, the genera Beggiatoa and Thioploca were recognized and differentiated on the basis of morphology alone; they were distinguished by the fact that numerous Thioploca filaments are contained within a common polysaccharide sheath, while Beggiatoa filaments occur singly . Vacuolate Beggiatoa or Thioploca spp . can dominate a variety of marine sediments, seeps, and vents, and it has been proposed (H . Fossing, V . A . Gallardo, B . B . Jorgensen, M . Huttel, L . P . Nielsen, H . Schulz, D . E . Canfield, S . Forster, R . N . Glud, J . K . Gundersen, J . Kuver, N . B . Ramsing, A . Teske, B . Thamdrup, and O . Ulloa, Nature {London} 374:713-715, 1995) that members of the genus Thioploca are responsible for a significant portion of total marine denitrification . In order to investigate the phylogeny of an environmentally dominant Beggiatoa sp., we analyzed complete 16S rRNA gene sequence data obtained from a natural population found in Monterey Canyon cold seeps . Restriction fragment length polymorphism analysis of a clone library revealed a dominant clone, which gave rise to a putative Monterey Beggiatoa 16S rRNA sequence . Fluorescent in situ hybridization with a sequence-specific probe confirmed that this sequence originated from wide Beggiatoa filaments (width, 65 to 85 microm) . A phylogenetic tree based on evolutionary distances indicated that the Monterey Beggiatoa sp . falls in the gamma subdivision of the class Proteobacteria and is most closely related to the genus Thioploca . This vacuolate Beggiatoa-Thioploca cluster and a more distantly related freshwater Beggiatoa species cluster form a distinct phylogenetic group.

J Bacteriol, 1999 Jan, 181(1), 161 - 6
Kinetics of nirS expression (cytochrome cd1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification: evidence for a denitrification-specific nitrate- and nitrite-responsive regulatory system; Hartig E et al.; After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis . nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd1 nitrite reductase involved in denitrification . Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates . We found that nirS, together with nirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon . In addition, we found a 2-kb monocistronic transcript . The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h . When the culture was subjected to a low oxygen tension, we observed a transient expression of nirS lasting for about 30 min . The continued transcription of the nirS operon required the presence of nitrate or nitrite . This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains . Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase . Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.

Protein Eng, 1998 Oct, 11(10), 917 - 23
Metal chelating properties of adenylate kinase from Paracoccus denitrificans; Perrier V et al.; Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp . The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms {Spurgin, P., Tomasselli, A.G., and Schiltz, E . (1989) Eur . J . Biochem., 179, 621-628} . However, adenylate kinase isolated from this bacterium was not reported to bind metal . These findings prompted us to clone the corresponding gene of P . denitrificans, and to characterize the enzyme overproduced in Escherichia coli . The deduced primary structure of adenylate kinase from P . denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly . The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one . The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding . The iron-chelated form of recombinant P . denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c . In conclusion, the absence of metal in the enzyme isolated from P . denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.

Biospectroscopy, 1998, 4(6), 365 - 77
Resonance Raman spectroscopic study of the caa3 oxidase from Thermus thermophilus; Gerscher S et al.; The terminal caa3 oxidase of Thermus thermophilus has been studied by resonance Raman spectroscopy . Using different excitation wavelengths in the Soret band region, it was possible to disentangle the resonance Raman spectra of the fully oxidized and fully reduced state in terms of the component spectra of the individual hemes a, a3, and c . For the heme a and a3 groups, the spectra reveal only minor differences compared to those of beef heart cytochrome c oxidase attributable to subtle modifications of the heme environment . These differences are not more pronounced than those between the oxidases from beef heart and Paracoccus denitrificans confirming the view that this oxidase of Th . thermophilus is a typical member of the aa3 oxidase superfamily . The heme c component spectra display far-reaching similarities with those of c-type cytochromes which serve as mobile electron carriers in the respiratory chain . These results imply that caa3 oxidase represents an integrated version of the noncovalent redox complex between cytochrome c and cytochrome c oxidase in higher organisms . On the other hand, the structural changes of cytochrome c in the noncovalent complex have no counterpart in the heme c component of the caa3 oxidase indicating a specific cytochrome c binding site for the mitochondrial enzyme.

Eur J Biochem, 1998 Nov 15, 258(1), 29 - 36
Characterization of cytochrome c-556 from the purple phototrophic bacterium Rhodobacter capsulatus as a cytochrome-c peroxidase; Hu W et al.; A cytochrome c-556 was purified from Rhodobacter capsulatus and the complete amino acid sequence was determined . It contains 328 amino acid residues and two typical heme-binding sites at cysteine residues 54 and 57 and at residues 200 and 203 . It is homologous to the family of bacterial cytochrome c peroxidases (BCCP) with 69% identity to Paracoccus denitrificans BCCP and 60% identity to Pseudomonas aeruginosa BCCP for which there is a three-dimensional structure . There is lesser similarity to the mauG gene products from methylotrophic bacteria which are thought to be involved in biosynthesis of the quinone cofactor of methylamine dehydrogenase . Translated genes from Escherichia coli and Helicobacter pylori are also related to the bacterial cytochrome c peroxidases . The divergence of this family of proteins is reflected in the fact that the reported sixth heme ligands are not conserved, except in Pseudomonas, Rhodobacter and Paracoccus . This suggests that homologs of BCCP may fold differently and/or may not have the same enzymatic activity as the prototypic protein from Ps . aeruginosa . We found that the Rb . capsulatus BCCP is active with both Rb . capsulatus cytochrome c2 and with horse cytochrome c as substrates (Km values 60 microm and 6 microm, respectively) . The turnover number was 40 s(-1) and the Km for peroxide was 33 microm . We have thus confirmed that the Rb . capsulatus protein is a cytochrome c peroxidase.

Rapid Commun Mass Spectrom, 1998, 12(23), 1967 - 71
Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry analysis of blue copper proteins . Azurin and mavicyanin; Fukuo T et al.; Two copper proteins azurin-1 and azurin-2 were isolated from denitrifying bacteria Alcaligenes xylosoxidans GIFU1051, and the mass spectrometric analysis of the proteins were carried out by both matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) . The mass spectrometric analysis was also carried out with the recombinant zucchini protein mavicyanin, which was obtained by expression in Escherichia coli . All the proteins were detected as positive ions with the copper atom being eliminated . The molecular weights were determined as 14,017.6 for azurin-1, 13,807.6 for azurin-2 and 11,808.8 for mavicyanin . The observed molecular weight of azurin-1 agrees within two daltons with that calculated from the amino acid composition . Azurin-2 was found to have one different amino acid residue when compared with the known azurin-2 isolated from A . xylosoxidans NCIB11015 . The measured molecular weight for the recombinant mavicyanin agrees within two daltons with that of calculated from the amino acid composition of the native protein; therefore, the recombinant mavicyanin is identical to the native protein.

Curr Microbiol, 1999 Jan, 38(1), 9 - 17
Polyphosphate-accumulating and denitrifying bacteria isolated from anaerobic-anoxic and anaerobic-aerobic sequencing batch reactors
Merzouki M, Delgenes JP, Bernet N, Moletta R, Benlemlih M.
In this study, phosphate-accumulating bacteria achieved complete phosphate removal in two different systems: an anaerobic-anoxic sequencing batch reactor and an anaerobic-aerobic sequencing batch reactor . This result shows that phosphate-accumulating bacteria in the A2 SBR can use nitrate as terminal electron acceptor instead of oxygen . Phosphate-accumulating bacteria accumulated phosphate with a rates between 30 and 70 mg P/L/h in the A/O SBR and between 15 and 32 mg P/L/h in the A2 SBR . Twenty denitrifying isolates were screened from A2 SBR and nine from A/O SBR . Identification of these isolates by the Biolog system and the API 20 NE identification kit revealed that the most active denitrifiers in both SBRs reactors were species of Ochrobactrum, Pseudomonas, Corynebacterium, Agrobacterium, Aquaspirillum, Haemophilus, Xanthomonas, Aeromonas, and Shewanella . The most active phosphate accumulating and denitrifying bacteria were identified as Agrobacterium tumefaciens B, Aquaspirillum dispar, and Agrobacterium radiobacter . This study showed that the active phosphate accumulating-bacteria were also the most efficient denitrifying bacteria in both reactors.

Microbiol Mol Biol Rev, 1998 Dec, 62(4), 1046 - 78
Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility; Baker SC et al.; Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P . denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common . Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration . Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains . The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled . There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P . denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified . In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus . While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.

Syst Appl Microbiol, 1998 Aug, 21(3), 365 - 73
Thauera linaloolentis sp . nov . and Thauera terpenica sp . nov., isolated on oxygen-containing monoterpenes (linalool, menthol, and eucalyptol) nitrate; Foss S et al.; The monoterpenes menthol, linalool, and eucalyptol were recently used as sole electron donor and carbon source for the isolation of three denitrifying bacterial strains 21Mol, 47Lol, and 58Eu . The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism . Monoterpenes were completely mineralised to carbon dioxide, nitrate was reduced to dinitrogen . Strain 47Lol utilised aliphatic monoterpenes, strain 21Mol oxygenated monocyclic monoterpenes, and strain 58Eu the bicyclic eucalyptol and monocyclic monoterpene alkenes . The fatty acid composition of the strains indicated an allocation to the rRNA group III of pseudomonads . Comparative 16S rRNA gene sequence analyses revealed that the new isolates can be assigned as members of the genus Thauera within the beta subclass of Proteobacteria . DNA-DNA hybridisation studies indicated a relateness of 68.5% between strains 21Mol and 58Eu which shared 36.0% and 40.6% DNA similarity with strain 47Lol . The strains are described as new species belonging to the genus Thauera, strain 47Lol (DSM 12138T) as T . linaloolentis sp . nov . and strains 21Mol and 58Eu as T . terpenica sp . nov . with strain 58Eu (DSM 12139T) as type strain.

Biochim Biophys Acta, 1998 Dec 1, 1409(2), 107 - 12
Electrochemically induced FT-IR difference spectra of the two- and four-subunit cytochrome c oxidase from P . denitrificans reveal identical conformational changes upon redox transitions; Hellwig P et al.; In order to study the role of subunits III and IV of the cytochrome c oxidase from P . denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared . These spectra reflect the alterations in the protein upon electron and proton transfer . Since the spectra are essentially identical, they clearly indicate that the additional subunits III and IV do not contribute to the FT-IR difference spectra of the four-subunit oxidase . Subunits III and IV are thus not involved in the reorganization of the polypeptide backbone and of single amino acids upon electron transfer and coupled proton transfer observed in the difference spectra in addition to heme contributions . The subtle differences between the FT-IR difference spectra that are attributed to the influence of protein-protein interactions between the subunits are discussed.

Biochim Biophys Acta, 1998 Dec 1, 1409(2), 99 - 105
FT-IR analysis of membranes of Rhodobacter sphaeroides 2.4.3 grown under microaerobic and denitrifying conditions; Mitchell DM et al.; Fourier transform infrared spectroscopic analysis of CO binding proteins in Rhodobacter sphaeroides reveals the presence of a membrane-bound nitric oxide reductase (Nor) . Nor has been clearly distinguished from the cytochrome oxidases by the temperature-dependence of relaxation following photodissociation of the CO complex at cryogenic temperatures . The center frequency and band shape, 1970 cm-1 and 20-30 cm-1 width at half-peak height, are similar to those reported for resonance Raman spectra of purified Paracoccus denitrificans Nor . Additional evidence is presented to indicate this enzyme is part of dissimilatory nitric oxide metabolism and that one of the genes in the nor operon required for production of an active Nor is not required for protein assembly or heme incorporation.

Biosci Biotechnol Biochem, 1998 Oct, 62(10), 1995 - 9
The role of the nirQOP genes in energy conservation during anaerobic growth of Pseudomonas aeruginosa; Arai H et al.; The nirQOP operon, which is located between the genes for nitrite reductase and nitric oxide reductase in Pseudomonas aeruginosa, encodes a putative ATP-binding protein and two putative transmembrane proteins . Phylogenetic analysis showed that NirO belongs to the family of subunit III of cytochrome oxidases but is distantly related to the other bacterial or mitochondrial proteins . P . aeruginosa strains that lacked the nirOP genes had all enzyme activities for denitrification and could grow under anaerobic conditions with nitrate or nitrite as an electron acceptor . However, the energy conservation efficiency of anaerobic respiration was lower in these strains than in strains harboring the nirOP gene.

FEMS Microbiol Lett, 1998 Nov 1, 168(1), 105 - 10
Denitrification by yeasts and occurrence of cytochrome P450nor in Trichosporon cutaneum; Tsuruta S et al.; Yeasts of various genera were screened for denitrifying activity, and several yeast strains such as Trichosporon cutaneum, Fellomyces fuzhouensis, and Candida sp . were found to exhibit distinct activities to convert nitrite to nitrous oxide . Dissimilatory nitrite reductase (Nir) or nitric oxide reductase (Nor) activities were detected in the cell-free extracts of these yeasts . Spectrophotometric as well as Western blot analyses showed that T . cutaneum contains Nor of the cytochrome P450 type . This is the first report that shows that denitrification is also distributed among yeasts although their systems are incomplete, only capable of reducing nitrite to nitrous oxide.

Microbiology, 1998 Sep, 144 ( Pt 9), 2427 - 39
Bacteria in post-glacial freshwater sediments; Miskin I et al.; Prokaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12,000 years of deposition, were examined for culturability, viability and community structure . The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria . Direct count procedures revealed 10(12) cells (g dry wt sediment)-1 in the surface sediments, which declined to approximately 10(9) cells (g dry wt sediment)-1 at 6 m depth of core (Representing approximately 10,000 years of deposition) . The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method . Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively . Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from 10(7) c.f.u . (g dry wt sediment)-1 to 10(1)-10(2) c.f.u . (g dry wt sediment)-1 below 3 m depth . Among culturable isolates, Gram-positives and Gram-negatives were found at all levels of the core, and spore-forming heterotrophs dominated . Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected at all depths . PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea . These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.

Biochemistry, 1998 Oct 20, 37(42), 14917 - 27
Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium; Spencer P et al.; The enzyme CbiL from the facultative anaerobe Salmonella typhimurium exhibits a high degree of homology to CobI from the aerobe Pseudomonas denitrificans (29% identity; 51% conservation obtained by a Blastp search of the ncbi database) . As CobI catalyzes the third methylation in the aerobic pathway to vitamin B12 it is proposed that CbiL catalyzes the analogous step in the anaerobic pathway . Potential metallo and metal-free substrates were characterized and their redox states defined by a combination of physicochemical techniques (MALDI-MS, NMR, UV/vis, IR, and EPR) and then used to investigate the function of CbiL . CbiL exhibited an absolute requirement for the presence of a metal ion (Co(II), Ni(II), or Zn(II)) within the tetrapyrrole substrate . CbiL had no preference for the redox state of its cobalt tetrapyrrole substrate, methylating both the reduced form, Co(II) 2, 7-dimethyl-dipyrrocorphin (Co(II)-precorrin-2), and the oxidized form, Co(III) 2,7-dimethyl-isobacterioclorin (Co(III)-factor-II) . In contrast CbiL had a marked preference for the oxidized Ni(II) and Zn(II)-2,7-dimethyl-isobacteriochlorin (Ni(II) and Zn(II)-factor-II) . Removal of the metal ion from a product of CbiL (Zn(II)-factor-III) allowed characterization by 13C NMR, identifying the tetrapyrrole as 2,7,20-trimethyl-isobacteriochlorin (factor-3), indicating that CbiL methylates at C20, the same site as that methylated by CobI . Competition experiments, utilizing isotopic labeling to distinguish otherwise identical mass substrates and products, revealed that oxidized Co(III) or Ni(II)-factor-II were equally good substrates, whereas Co(II)-precorrin-2 was much preferred over Ni(II)-precorrin-2 . Excess Ni(II)-precorrin-2 did not decrease CbiL methylation of Co(II)-precorrin-2, implying that CbiL has a low affinity for Ni(II)-precorrin-2 . These results are interpreted on the basis of tetrapyrrole ruffling occurring on the optimization of the metallo-N bond distances . The greater flexibility of the reduced precorrin-2 ring system allows greater deformation on accommodating the bound metal ion, the distortions imposed by bound Ni(II) or Zn(II) ions being larger than Co(II) . The resulting distortions imposed on the precorrin ring could then decrease catalysis by causing a departure from the optimal substrate conformation required for CbiL . On oxidation of the Ni(II) or Zn(II)-precorrin-2, the increased stiffness of the ring could then constrain the metallo-factor-II conformation toward that of the usual substrate, allowing greater methylation by CbiL . In contrast to its counterpart CobI in the aerobic pathway of B12 biosynthesis, which methylates the metal-free precorrin-2, these studies show CbiL to be the first methylase unique to the anaerobic pathway, methylating a metallo-precorrin-2 substrate . Implications of CbiL specificity for the mechanism of the anaerobic B12 pathway are discussed.

J Bacteriol, 1998 Oct, 180(20), 5454 - 7
Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product; Beller HR et al.; Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate . In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate . Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer {(R)-2-benzyl-3-carboxypropionic acid}, and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 347 - 54
Structure of azurin I from the denitrifying bacterium Alcaligenes xylosoxidans NCIMB 11015 at 2.45 A resolution; Li C et al.; Azurin I from Alcaligenes xylosoxidans NCIMB 11015 (AzN-I) was crystallized by using PEG 4000 as a precipitant . The crystals belong to the monoclinic crystal system and have a space group C2 with the unit-cell parameters of a = 130.67, b = 54.26, c = 74.55 A, and beta = 95.99 degrees . The structure of AzN-I has been solved by the molecular replacement method . Azurin II from the same bacterium (AzN-II) was chosen as the initial structural model . The final crystallographic R value is 17.3% and free R value is 23.6% for 10958 reflections at a resolution of 2.45 A . The root-mean-square deviations for main-chain atoms range between 0.19 and 0.26 A among the four independent molecules in the asymmetric unit . The Cu atom is coordinated to Ndelta of His46 and His117 at 2.0 (1) and 1.9 (1) A, and to Sgamma of Cys112 at 2.2 (1) A, while the carbonyl O atom of Gly45 and Sdelta of Met121 coordinate axially to Cu atom at 2.5 (1) and 3.1 (1) A, respectively . The Cu-N and Cu-S distances of AzN-I are quite similar to those of AzN-II, however, the Cu-SO (Gly45) bond length in AzN-I is 0.25 A shorter than the counterpart in AzN-II . The results have been used to discuss the differences in the spectra of these two proteins.

Acta Crystallogr D Biol Crystallogr, 1998 Mar 1, 54 ( Pt 2), 253 - 68
Crystallographic study of azurin from Pseudomonas putida; Chen ZW et al.; Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier . Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit . The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 A, beta = 107.79 degrees, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 A, beta = 103.12 degrees . The latter crystals were found to have four zinc ions incorporated into the crystal lattice . Both crystal structures were solved by the molecular-replacement method using the program MERLOT . The search model was the structure of azurin from Alcaligenes denitrificans . The crystallographic R factor for native azurin is 0.169 (Rfree = 0 . 257) from 8 to 1.92 A resolution, while that for zinc azurin is 0 . 181 (Rfree = 0.248) from 10 to 1.6 A resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 A . In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with Ndelta1 from His46 and His117, and one with the thiolate S atom of Cys112 . Two longer axial approaches are made by the Sgamma from Met121 and the carbonyl O atom from Gly45 . This results in a distorted trigonal bipyramidal co-ordination around the Cu atom . It further confirms the presence of a weak fifth bond to the copper in P . putida azurin, as with other azurin structures described at high resolution . The Ndelta1 atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans . In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain . In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules . The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site . The zinc ligands are Ndelta from His, carboxylate O atoms from Asp and Glu, Ogamma from Ser and water molecules . One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (-101) direction and is primarily responsible for the altered unit-cell parameters . Two of the other zinc ions bind to His83, one in each molecule.

Biochemistry, 1998 Oct 6, 37(40), 13987 - 96
Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa; Nurizzo D et al.; Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO) . The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme . The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described {Fulop, V., et al . (1995) Cell 81, 369-377; Williams, P . A., et al . (1997) Nature 389, 406-412}, and we recently reported on the structure of oxidized NiR-Pa at 2.15 A {Nurizzo, D., et al . (1997) Structure 5, 1157-1171} . Although the domains carrying the d1 heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state . The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion . Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108 . Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd . Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d1 heme active site . Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 A away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form . The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd . Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse.

Appl Environ Microbiol, 1998 Oct, 64(10), 3769 - 75
Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples; Braker G et al.; A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR . Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis . Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed . Likewise, the method allowed a determination of the nir type of five laboratory strains . The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp . (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698 . For each of the two genes, at least one primer combination amplified successfully for all of the test strains . Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type . The specificity of the amplified products was confirmed by subsequent sequencing . These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples . This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

Biochim Biophys Acta, 1998 Jul 20, 1365(3), 393 - 403
Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes; Nicoletti F et al.; Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition . An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside . If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II . The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy . The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding . From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes . The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

Biochemistry, 1998 Sep 22, 37(38), 13102 - 9
The active site of the bacterial nitric oxide reductase is a dinuclear iron center; Hendriks J et al.; A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed . The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry . The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution . Similarly, it is dimeric in two-dimensional crystals . Images of these crystals have been processed at 8 A resolution in projection to the membrane . The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O . Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper . NorC is a membrane-anchored cytochrome c . Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases . Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4 . The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct . We conclude that the active site of NOR is a dinuclear iron center.

J Biol Chem, 1998 Oct 2, 273(40), 25703 - 12
Crystallographic and spectroscopic studies of native, aminoquinol, and monovalent cation-bound forms of methylamine dehydrogenase from Methylobacterium extorquens AM1; Labesse G et al.; Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH) . Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A) . This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported . We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors . Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions . The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution . These studies lead us to propose a model for the entrance and binding of the substrate in the active site.

Chemosphere, 1998 Jun, 36(13), 2721 - 30
Microbial hexachlorobenzene dechlorination under three reducing conditions; Chang BV et al.; The potential dechlorination of hexachlorobenzene (HCB) in medium by 1,2,3-trichlorobenzene (TCB)-adapted mixed culture under three reducing conditions was investigated . It was found that strongest to weakest HCB dechlorination occurred in the order of methanogenic conditions > sulfate-reducing conditions > denitrifying conditions . Under denitrifying conditions, no dechlorination was observed during the first 20 days of incubation . Biotransformation occurred in this order: HCB-->pentachlorobenzene (PCB)-->1,2,3,5-tetrachlorobenzene (TeCB)-->1,3,5-TCB + 1,2,4-TCB-->1,3-dichlorobenzene (DCB), HCB dechlorination was delayed following treatment with ferric chloride and manganese dioxide, but enhanced by the addition of lactate and pyruvate under methanogenic or sulfate-reducing conditions, the addition of acetate had no significant effect on HCB dechlorination under any of the three reducing conditions . Sequential dechlorination was observed at concentrations of 2-50 mg/L, but at a significantly slower rate at the highest concentrations.

FEBS Lett, 1998 Sep 4, 434(3), 322 - 4
Electron entry in a CuA mutant of cytochrome c oxidase from Paracoccus denitrificans . Conclusive evidence on the initial electron entry metal center; Malatesta F et al.; A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the CuA site was changed by site-directed mutagenesis to a mononuclear copper site {Zickermann, V., Wittershagen, A., Kolbesen, B.O . and Ludwig, B . Biochemistry 36 (1997) 3232-3236} was investigated by stopped-flow spectroscopy . Contrary to the behavior of the wild type enzyme, in this mutant cytochrome a cannot be reduced by excess cytochrome c in the millisecond time scale in which cytochrome c oxidation is observed . The results conclusively identify and establish CuA as the initial electron entry site in cytochrome c oxidase . Partial rapid reduction (ca . 20%) of the modified CuA site suggests that the mononuclear copper ion has a redox potential ca . 100 mV lower than the wild type, and that internal electron transfer to cytochrome a is > or = 10(3)-fold slower than with the wild type enzyme.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 313 - 21
Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides; Schwintner C et al.; Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp . denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes . Two large plasmids of 102 and 115 kb were found . The genes encoding the various reductases are not clustered on a single genetic unit . The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid . Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment . For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.

Biochem J, 1998 Oct 1, 335 ( Pt 1), 159 - 66
Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon; Raux E et al.; A 16 kb DNA fragment has been isolated from a Bacillus megaterium genomic library and fully sequenced . The fragment contains 15 open reading frames, 14 of which are thought to constitute a B . megaterium cobalamin biosynthetic (cob) operon . Within the operon, 11 genes display similarity to previously identified Salmonella typhimurium cobalamin biosynthetic genes (cbiH60, -J, -C, -D, -ET, -L, -F, -G, -A, cysGA and btuR), whereas three do not (cbiW, -X and -Y) . The genes of the B . megaterium cob operon were compared with the cobalamin biosynthetic genes of Pseudomonas denitrificans, Methanococcus jannaschii and Synechocystis sp . Taking into account the presence of cbiD and cbiG, the absence of a cobF, cobG and cobN, -S and -T, it was concluded that B . megaterium, M . jannaschii and Synechocystis sp., like S . typhimurium, synthesize cobalamin by an anaerobic pathway, in which cobalt is added at an early stage and molecular oxygen is not required.

FEMS Microbiol Lett, 1998 Sep 1, 166(1), 35 - 41
A two-component system involved in regulation of anaerobic toluene metabolism in Thauera aromatica; Leuthner B et al.; The genes for a two-component regulatory system of the denitrifying toluene-degrading bacterium Thauera aromatica were identified immediately upstream of the genes for benzylsuccinate synthase (bssDCAB), the first enzyme involved in anaerobic toluene metabolism . The genes apparently encode the regulators of toluene catabolic enzymes and were therefore termed tdiSR (for toluene degradation including sensor and regulator) . The tdiR gene product was overproduced in Escherichia coli and assayed for binding to a DNA fragment containing the 5' region of the bss operon . We observed specific DNA binding with cell extracts containing overproduced TdiR, but not with control extracts . The tdiSR genes are almost identical to two genes of Thauera strain T1, which have not been assigned a function so far . In addition, the derived gene products share similarity with regulators of toluene and styrene catabolic pathways in aerobic Pseudomonas species, and with the tutCB gene products of Thauera strain T1 . The latter have previously been implicated in regulating anaerobic toluene metabolism . Our data suggest that toluene catabolism under aerobic and anaerobic conditions is regulated by similar, but distinct two-component systems.

FEBS Lett, 1998 Aug 14, 433(1-2), 93 - 7
Intrinsic uncoupling of cytochrome c oxidase may cause the maternally inherited mitochondrial diseases MELAS and LHON; Mather MW et al.; Mutations in the human mtDNA gene encoding subunit III of cytochrome c oxidase (CO) have been reported to cause MELAS and LHON . Poracoccus denitrificans cells expressing substitutions homologous to these MELAS- and LHON-causing mutations had lower growth yield than wild type cells and lower efficiency of proton pumping by CO (e.g . lower H+/e ratio and lower deltapsi), but had similar CO activity . These results indicate that both substitutions (F263L > A212T) cause intrinsic uncoupling, which may be the direct cause of the diseases . These results also suggest that subunit III is involved in proton pumping.

Eur J Biochem, 1998 Aug 1, 255(3), 618 - 27
Cyclohexa-1,5-diene-1-carbonyl-CoA hydratase {corrected}, an enzyme involved in anaerobic metabolism of benzoyl-CoA in the denitrifying bacterium Thauera aromatica; Laempe D et al.; Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate . The central pathway of benzoyl-CoA metabolism is initiated by an ATP-driven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carbonyl-CoA . The 1,5-dienoyl-CoA intermediate is thought to be transformed to 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by a specific dienoyl-CoA hydratase catalyzing the formal addition of water to one of the double bonds . This dienoyl-CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate . Substrate and product were confirmed and a convenient spectrophotometric assay was developed . The equilibrium concentrations of substrate and product were almost equal . Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate . The enzyme of 28 kDa was purified from T . aromatica and was found to be highly specific for the cyclic 1,5-dienoyl-CoA . A second 29-kDa enoyl-CoA hydratase acted on crotonyl-CoA; this highly active enoyl-CoA hydratase also acted slowly on cyclohex-1-ene-1-carbonyl-CoA . The regulation of expression of dienoyl-CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pathway of anaerobic aromatic metabolism in T . aromatica . Extracts of Rhodopseudomonas palustris contained high activity of cyclohex-1-ene-1-carbonyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity . It appears that a variant of the benzoyl-CoA pathway is operating in R . palustris in which hydration of the 1,5-dienoyl-CoA does not take place . Rather, cyclohex-1-ene-1-carbonyl-CoA is hydrated to 2-hydroxycyclohexane-1-carbonyl-CoA {corrected}.

J Mol Biol, 1998 Sep 18, 282(2), 369 - 82
X-ray structure of a blue-copper nitrite reductase in two crystal forms . The nature of the copper sites, mode of substrate binding and recognition by redox partner; Dodd FE et al.; Denitrification is one of the main steps of the global nitrogen cycle that is sustained by prokaryotic organisms . Denitrifying bacteria use two entirely different enzymes in this process, one based on haem cd1 prosthetic groups and the other on type 1-type 2 Cu centres . Copper-containing nitrite reductases (NiRs) are sub-divided into blue and green NiRs, which are respectively thought to be redox partners of azurins and pseudo-azurins . Crystallographic structures of the blue nitrite reductase from Alcaligenes xylosoxidans (AxNiR) are presented in the oxidised hexagonal form and the substrate-bound orthorhombic form to 2.1 A and 2.8 A resolution, respectively . The complete amino acid sequence of AxNiR has been determined by conventional chemical analysis . A 3 A structure of AxNiR has been published where the modelling was based on the sequence of another blue NiR . The higher resolution of the hexagonal form together with the correct sequence allows a detailed comparison with the crystallographic structures of the green NiRs . There is a striking difference in the overall surface charge distribution between the two sub-groups, providing a neat structural explanation for their different reactivities to pseudoazurin or azurin and supporting the view that electron transfer proceeds via complex formation . A detailed examination of the type-1 Cu site, the site responsible for the colour, reveals several subtle differences, including a lateral displacement of 0.7 A for Smet . The structure of the type-2 Cu site, and changes that occur upon substrate binding are discussed in terms of the catalytic mechanism . The similarity of the type 2 Cu site to the catalytic Zn site in carbonic anhydrase and the catalytic Cu site of superoxide dismutase is re-examined in view of the high-resolution (2.1 A) structure .

Acta Microbiol Pol, 1998, 47(1), 65 - 75
Microbial degradation of phenol in denitrifying conditions; Blaszczyk M et al.; The biodegradation of phenpol in anaerobic conditions by mixed population of bacteria in batch cultures or continuous cultures in packed bed reactor in medium with phenol as sole carbon source was effective . Phenol in concentrations up to 500 mg/l was degraded by bacteria in batch cultures (incubation temperature 30 degrees C) with increasing maximal rate without lag phase and at higher concentrations (up to 1000 mg/l) the activity of the bacteria was preceded by a lag phase lasting from 9 to 15 days . Phenol was degraded in continuous cultures with maximum efficiency (about 2500 mg/l x day) in the following conditions: incubation temperature 30 degrees C, phenol concentration in the medium of 200 mg/l and retention time of about 2 hours . Lowering of the temperature of the culture to 13 degrees C and 20 degrees C resulted in 10 and 5-fold decrease in the efficiency of the process, expressed as mg/l X day, respectively . Analysis of the composition of the bacteria among the facultatively growing Gram-negative rods showed that the incubation temperature visibly affected the species composition and domination pattern of denitrifying bacteria although their percent participation remained the same.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 953 - 6
Azoarcus anaerobius sp . nov., a resorcinol-degrading, strictly anaerobic, denitrifying bacterium; Springer N et al.; A strictly anaerobic, nitrate-reducing bacterium, strain LuFRes1, was isolated using resorcinol as sole source of carbon and energy . The strain reduced nitrate to dinitrogen gas and was not able to use oxygen as an alternative electron acceptor . Cells were catalase-negative but superoxide-dismutase-positive . Resorcinol was completely oxidized to CO2 . 16S rRNA sequence analysis revealed a high similarity with sequences of Azoarcus evansii and Azoarcus tolulyticus . Strain LuFRes1T (= DSM 12081T) is described as a new species of the genus Azoarcus, Azoarcus anaerobius.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 889 - 94
Identification of denitrifier strain T1 as Thauera aromatica and proposal for emendation of the genus Thauera definition; Song B et al.; Bacterial strain, T1, originally isolated by P.J . Evans on the basis of its capacity for toluene degradation under denitrifying conditions, has been classified as Thauera aromatica . In a comprehensive study of strains of this species, it was found that the cells have a different type of flagellar insertion from that of cells of the type species of the genus, Thauera selenatis, suggesting the convenience of an emendation of the description of the genus Thauera . Further studies on a larger collection of strains with the above characteristics may serve in the future as the basis for the creation of a new generic designation.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 775 - 82
Microvirgula aerodenitrificans gen . nov., sp . nov., a new gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions; Patureau D et al.; A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge . The cells were Gram-negative, catalase- and oxidase-positive curved rods and very motile . They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors . The G&C content was 65 mol% . Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems . However, a phylogenetic analysis based on the 165 rRNA sequence showed that the aerobic denitrifier could not be assigned to any currently recognized genus . For these reasons a new genus and species, Microvirgula aerodenitrificans gen . nov., sp . nov., is proposed, for which SGLY2T is the type strain.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 635 - 9
Phylogenetic analysis and intrageneric structure of the genus Hyphomicrobium and the related genus Filomicrobium; Rainey FA et al.; Almost complete 16S rDNA sequences from the type strains of seven species of the genus Hyphomicrobium and of Filomicrobium fusiforme have been determined . The Hyphomicrobium species from two phylogenetic clusters that are only moderately related to each other . While cluster I contains the type species Hyphomicrobium vulgare, Hyphomicrobium aestuarii, Hyphomicrobium hollandicum and Hyphomicrobium zavarzinii, cluster II comprises Hyphomicrobium facilis, Hyphomicrobium denitrificans and Hyphomicrobium methylovorum . Within the two species cluster, the species are highly related . Phylogenetically, Filomicrobium fusiforme clusters moderately with Hyphomicrobium species . The lack of distinguishing phenotypical properties presently excludes the possibility of describing cluster II as a new genus.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 537 - 42
Roseobacter gallaeciensis sp . nov., a new marine bacterium isolated from rearings and collectors of the scallop Pecten maximus; Ruiz-Ponte C et al.; Four bacterial strains were isolated from larval cultures and collectors of the scallop Pecten maximus . They showed a high level of intragroup genomic relatedness (84-95%) as determined by DNA-DNA hybridization . The cells were Gram-negative, strictly aerobic, motile, ovoid rods . They grew at temperatures from 15 to 37 degrees C and from pH 7.0 to 10, but did not grow in the absence of NaCl and required growth factors . They had the ability to use a wide variety of compounds as sole carbon source: D-mannose, D-galactose, D-fructose, D-glucose, D-xylose, melibiose, trehalose, maltose, cellobiose, sucrose, mesoerythritol, D-mannitol, glycerol, D-sorbitol, meso-inositol, succinate, propionate, butyrate, gamma-aminobutyrate, DL-hydroxybutyrate, 2-ketoglutarate, pyruvate, fumarate, glycine, L-alpha-alanine, beta-alanine, L-glutamate, L-arginine, L-lysine, L-ornithine and L-proline . They exhibited oxidase and catalase activities but no denitrification activity . The isolates did not contain bacteriochlorophyll a . The G + C content ranged from 57.6 to 58 mol% . Phylogenetic analyses of the 16S rRNA sequence revealed that these isolates belong to the genus Roseobacter . On the basis of quantitative hybridization data, it is proposed that these isolates should be placed in a new species, Roseobacter gallaeciensis . The type strain is Roseobacter gallaeciensis BS107T (= CIP 105210T).

Biochim Biophys Acta, 1998 Aug 24, 1381(3), 347 - 50
The nirQ gene, which is required for denitrification of Pseudomonas aeruginosa, can activate the RubisCO from Pseudomonas hydrogenothermophila; Hayashi NR et al.; Two putative ATP-binding proteins encoded in the gene cluster for the Calvin cycle of Pseudomonas hydrogenothermophila (cbbQ) and for the denitrification of Pseudomonas aeruginosa (nirQ) have been found to be similar . The cbbQ gene has been shown to activate the RubisCO from P . hydrogenothermophila in E . coli . The nirQ was functionally substituted for cbbQ . The nirQ gene restored the anaerobic growth and the NOR activity of the nirQOP mutant of P . aeruginosa, while the cbbQ gene did not . Copyright Elsevier Science B.V.

J Bacteriol, 1998 Sep, 180(17), 4413 - 5
Denitrification by actinomycetes and purification of dissimilatory nitrite reductase and azurin from Streptomyces thioluteus; Shoun H et al.; Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N2O) . As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail . S . thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N2O, suggesting that the denitrification acts as anaerobic respiration . Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated . This is the first report to show that denitrification generally occurs among actinomycetes.

FEBS Lett, 1998 Aug 7, 432(3), 109 - 12
Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition; Schroter T et al.; The cyo operon coding for the membrane-bound bo3-type quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba3 quinol oxidase . Using the P . denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E . coli and the enzyme purified in a single step on a metal-chelating column . Whereas the activity of the isolated complex matches that of the oxidase purified directly from E . coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.

Biochemistry, 1998 Aug 25, 37(34), 11806 - 11
Tryptophan-136 in subunit II of cytochrome bo3 from Escherichia coli may participate in the binding of ubiquinol; Ma J et al.; In the cytochrome c oxidases, the role of subunit II is to provide the electron entry site into the enzyme . This subunit contains both the binding site for the substrate, cytochrome c, and the CuA redox center, which is initially reduced by cytochrome c . Cytochrome bo3 and other quinol oxidases that are members of the heme-copper oxidase superfamily have a homologous subunit II, but the CuA site is absent, as is the docking site for cytochrome c . Speculation that subunit II in the quinol oxidases may also be important as an electron entry site is supported by the demonstration several years ago that a photoreactive substrate analogue, azido-Q, covalently labeled subunit II in cytochrome bo3 . In the current work, a sequence alignment of subunit II of heme-copper quinol oxidases is used as a guide to select conserved residues that might be important for the binding of ubiquinol to cytochrome bo3 . Results are presented for point mutants in 24 different residue positions in subunit II . The membrane-bound enzymes were examined by optical spectroscopy and by determining the activity of ubiquinol-1 oxidase . In each case, the Km for ubiquinol-1 was determined as a measure of possible perturbation to a quinol binding site . The only mutant that had a noticeably altered Km for ubiquinol-1 was W136A, in which the Km was about sixfold increased . Thus, W136 may be at or close to a substrate (ubiquinol)-binding site in cytochrome bo3 . In the cytochrome c oxidases, the equivalent tryptophan (W121 in Paracoccus denitrificans) has been identified as the "electron entry site".

Biochemistry, 1998 Aug 25, 37(34), 11792 - 6
Analysis of the pathogenic human mitochondrial mutation ND1/3460, and mutations of strictly conserved residues in its vicinity, using the bacterium Paracoccus denitrificans; Zickermann V et al.; The human mitochondrial ND1/3460 mutation changes Ala52 to Thr in the ND1 subunit of Complex I, and causes Leber's hereditary optic neuropathy (LHON) {Huoponen et al . (1991) Am . J . Hum . Genet . 48, 1147} . We have used a bacterial counterpart of Complex I, NDH-1 from Paracoccus denitrificans, for studying the effect of mutations in the ND1 subunit on the enzymatic activity . The LHON mutation as well as several other mutations in strictly conserved amino acids in its vicinity were introduced into the NQO8 subunit of NDH-1, a bacterial homologue of ND1 . The enzymatic activity of the mutants in the presence of hexammineruthenium (rotenone-insensitive) and ubiquinone-1 (rotenone-sensitive) were assayed . In addition, the kinetics of the interaction of selected mutant enzymes with ubiquinone-1, ubiquinone-2, and decylubiquinone was studied . The results suggest that the mutated residues play an important role in ubiquinone reduction by Complex I.

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9891 - 6
CuA and CuZ are variants of the electron transfer center in nitrous oxide reductase; Farrar JA et al.; Nitrous oxide reductase (N2OR) is a dimeric copper-dependent bacterial enzyme that catalyzes the reduction of N2O to N2 as part of the denitrification pathway . In the absence of an x-ray crystal structure, the current model of the nature of the copper sites within the enzyme is based on four copper atoms per monomer and assigns two copper atoms to an electron transfer center, CuA, a bis-thiolate-bridged dinuclear copper center found to date only in N2OR and cytochrome c oxidase, and two copper atoms to a second dinuclear center, CuZ, presumed to be the site of catalysis . Based on detailed analysis of the low temperature magnetic CD spectra of N2OR, this paper revises the current model and proposes that both CuA and CuZ are variants of an electron transfer center and hence that all of the observed optical features are due to this electron transfer center . It is proposed further that the presence of these different forms provides a mechanism for the delivery of two electrons to an active site comprising copper ions lacking thiolate coordination.

Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 301 - 8
Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain; Ohnishi T et al.; Our recent experimental data on iron-sulfur clusters and semiquinones in the complex I segment of the respiratory chain is presented, focusing on the Paracoccus (P.) denitrificans and bovine heart studies . The iron-sulfur cluster N2 has attracted the attention of investigators in the research field of complex I, since the mid-point redox potential of this cluster is the highest among all clusters in complex I, and is pH dependent (60 mV/pH) . It is known that this cluster is located either in the NQO6 (NuoB in E . coli/PSST in bovine heart nomenclature) or in the NQO9 (NuoI/TYKY) subunit in the amphipathic domain of complex I . Our preliminary data indicate that the cluster N2 is located in the NuoB rather than the long-advocated NuoI subunit, and may have a unique ligand structure . We previously reported spin-spin interactions between cluster N2 and two distinct species of semiquinone (designated SQNf and SQNs) associated with complex I . A parallel intensity change was observed between the SQNf (g = 2.00) signal and the N2 split g parallel signal, further supporting our proposed interaction between SQNf and N2 spins.

Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 53 - 9
Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria; Kotlyar AB et al.; The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented . They are compared to those observed in submitochondrial particles from bovine heart (SMP) . All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme . There were some prominent differences, though . The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet . The signal was hardly affected by the presence of gramicidin . The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s . The fast-relaxing signal vanished immediately upon anaerobiosis . The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme . Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C . The spin concentration of the NADH-reducible {2Fe-2S} cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Biochemistry, 1998 Jul 28, 37(30), 10555 - 62
The primary structure of soluble cytochrome c-551 from the phototrophic green sulfur bacterium Chlorobium limicola, strain Tassajara, reveals a novel c-type cytochrome; Klarskov K et al.; Chlorobium limicola, strain Tassajara, cytochrome c-551 is a soluble dimeric protein containing identical subunits of about 30 kDa . The amino acid sequence was determined by a combination of automated Edman degradation and mass analysis . There are 258 residues with a single heme binding site located at cysteine positions 172 and 175 . In addition, there is a disulfide bridge between Cys78 and Cys109, and a free cysteine at position 219 which was found to occur as cysteic acid . The only homologue of soluble cytochrome c-551 is the soxA protein which is part of the thiosulfate utilization operon of Paracoccus denitrificans . They are 32% identical with three small gaps . This is consistent with the observation that cytochrome c-551 is the electron acceptor for a thiosulfate-oxidizing enzyme . On the basis of the redox potential of 135 mV, the sixth heme ligand should be a methionine . Among the seven methionine residues that are present in c-551, only one is conserved, two residues ahead of the heme-binding site . The far-UV circular dichroism spectrum indicates 40% alpha helix and 25% beta secondary structure . No other known cytochrome c has such a mixed structure; they are either all helical or all beta . Thus, Chlorobium soluble cytochrome c-551 and soxA are likely to be representative of a new class of c-type cytochromes.

Eur J Biochem, 1998 Jul 1, 255(1), 125 - 32
31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states; Griffin KJ et al.; Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states . The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state . The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase . The wild-type human electron transfer apoprotein was reconstituted with {2,4a-13C2}FAD, {4,10a-13C2}FAD, or {U-15N4}FAD . The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states . The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature . In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD . The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD . The sp2 hybridisation of N(10) is small compared to other flavoproteins . In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD . The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR . The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states . The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.

Eur J Biochem, 1998 Jul 1, 255(1), 100 - 6
Mutational analysis of residues forming hydrogen bonds in the Rieske {2Fe-2S} cluster of the cytochrome bc1 complex in Paracoccus denitrificans; Schroter T et al.; Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and {2Fe-2S} cluster stability, the enzyme activity and the redox potential of the {2Fe-2S} cluster . In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the {2Fe-2S} cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma {Iwata, S., Saynovits, M., Link, T . A . & Michel, H . (1996) Structure 4, 567-579} . They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P . denitrificans . Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P . denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain . The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster . Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively . The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A) . Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.

Eur J Biochem, 1998 Jul 1, 255(1), 87 - 92
Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon; Brosche M et al.; Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon {Tybulewicz, V . L . J., Falk, G . & Walker, J . E . (1984) J . Mol . Biol . 179, 185-214} . A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb . blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum . Although the protein is present during the entire life cycle of a Rb . blasticus culture, it is most abundant early during the stationary phase . Plasmid constructs of the URF6 gene for overexpression in E . coli were made . These constructs were designed to obtain proteins both with and without His-tagging . In both cases, a protein product was visible in induced cells . The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography . By N-terminal sequencing of the His-tagged protein, its identity was confirmed.

Appl Environ Microbiol, 1998 Aug, 64(8), 3004 - 8
Effect of tungstate on nitrate reduction by the hyperthermophilic archaeon pyrobaculum aerophilum
Afshar S, Kim C, Monbouquette HG, Schroder I I.
Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor . Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen . This study demonstrates that P . aerophilum requires the metal oxyanion WO42- for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources . The addition of 1 &mgr;M MoO42- did not replace WO42- for the growth of P . aerophilum . However, cell growth was completely inhibited by the addition of 100 &mgr;M MoO42- to the culture medium . At lower tungstate concentrations (0.3 &mgr;M and less), nitrite was accumulated in the culture medium . The accumulation of nitrite was abolished at higher WO42- concentrations (<0.7 &mgr;M) . High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed . The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell . While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO42- levels of 0.7 &mgr;M or higher . The high specific activity of the nitrate reductase enzyme observed at low WO42- levels (0.3 &mgr;M or less) coincided with the accumulation of nitrite in the culture medium . This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon . We demonstrate here that nitrate reductase synthesis in P . aerophilum occurs in the presence of high concentrations of tungstate.

Microbiol Immunol, 1998, 42(6), 429 - 38
Emendation of genus Achromobacter and Achromobacter xylosoxidans (Yabuuchi and Yano) and proposal of Achromobacter ruhlandii (Packer and Vishniac) comb . nov., Achromobacter piechaudii (Kiredjian et al.) comb . nov., and Achromobacter xylosoxidans subsp . denitrificans (Rüger and Tan) comb . nov; Yabuuchi E et al.; Based on the results of GC content determination and 16S rRNA sequence analysis among the type strains of Achromobacter xylosoxidans, 4 Alcaligenes species, 5 Bordetella species, and 12 species of 4 other genera, the separation of genus Achromobacter Yabuuchi and Yano 1981, with the type species Achromobacter xylosoxidans, is confirmed . Alcaligenes ruhlandii (Packer and Vishniac) Aragno and Schlegel 1992 is a distinct species and not a senior synonym of Achromobacter xylosoxidans . Alcaligenes ruhlandii and Alcaligenes piechaudii Kiredjian et al 1986 are transferred to genus Achromobacter . Thus 2 new combinations, Achromobacter ruhlandii (Packer and Vishniac) and Achromobacter piechaudii (Kiredjian et al) are proposed; their type strains are ATCC 15749 and ATCC 43552, respectively . Alcaligenes denitrificans Ruger and Tan 1983 is also transferred to genus Achromobacter and ranked down to the subspecies of Achromobacter xylosoxidans . Thus a new subspecies name, Achromobacter xylosoxidans subsp . denitrificans (Ruger and Tan) is proposed . The type strain of the subspecies is ATCC 15173 . This proposal automatically creates type subspecies, Achromobacter xylosoxidans subsp . xylosoxidans, with type strain ATCC 27061 . An emended description of genus Achromobacter and of type species Achromobacter xylosoxidans are given.

Appl Environ Microbiol, 1998 Aug, 64(8), 3092 - 5
The requirement of RpoN (sigma factor sigma54) in denitrification by Pseudomonas stutzeri is indirect and restricted to the reduction of nitrite and nitric oxide; Hartig E et al.; The rpoN region of Pseudomonas stutzeri was cloned, and an rpoN null mutant was constructed . RpoN was not essential for denitrification in this bacterium but affected the expression levels and enzymatic activities of cytochrome cd1 nitrite reductase and nitric oxide reductase, whereas those of respiratory nitrate reductase and nitrous oxide reductase were comparable to wild-type levels . Since the transcription of the structural genes nirS and norCB, coding for nitrite reductase and the nitric oxide reductase complex, respectively, proceeded unabated, our data indicate a posttranslational process for the two key enzymes of denitrification depending on RpoN.

Appl Environ Microbiol, 1998 Aug, 64(8), 2925 - 30
Inhibition of anaerobic phosphate release by nitric oxide in activated sludge; Van Niel EW et al.; Activated sludge not containing significant numbers of denitrifying, polyphosphate {poly(P)}-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods . During the aerobic period, poly(P) accumulated up to 100 mg of P x g of (dry) weight . When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate . Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation . In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-beta-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished . In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO . The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase . Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO . It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms . The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO . The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.

Arch Microbiol, 1998 Aug, 170(2), 120 - 31
Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica; Heider J et al.; Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica . This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2 . Regulation was studied by three methods . 1 . Determination of protein patterns of cells grown on different substrates . This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways . 2 . Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates . The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates . 3 . Immunological detection of catabolic enzymes in cells grown on different substrates . Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity . However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity . This possibly indicates an additional level of regulation on protein level for these two reductases.

Extremophiles, 1997 Nov, 1(4), 207 - 11
A denitrifying bacterium from the deep sea at 11,000-m depth; Tamegai H et al.; The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench . The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively . However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land . From the results, it can be said that this organism is adapted to the environment of the deep sea . Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA . The cytochrome contents of the bacterium were similar to those of Ps . stutzeri in spectrophotometric studies.

Protein Eng, 1998 Apr, 11(4), 279 - 83
A novel, non-statistical method for predicting breaks in transmembrane helices; Nikiforovich GV; We have developed a novel, non-statistical procedure for predicting possible breaks in transmembrane helices based on energy calculations . The procedure consists of stepwise elongation of the 'core' helical fragment determined by consensus results of several available prediction procedures . At each step, we calculate the conformational energies corresponding to the regular 'frozen' helical conformer of the 'core' fragment elongated by two flanking residues, E(alpha), as well as those to several options for the fragment to enter or exit the helix by changing conformations of the flanking residues, Ei . The minimal values out of Ei - E(alpha), delta(k), can be viewed as a profile of relative energies, where each minimum of delta(k) is a signal to start or to stop transmembrane helix . We suggest that boundaries of the transmembrane helix would be determined by the signals closest to the 'core' sequence in the delta(k) profiles . Our procedure was applied to prediction of the N- and C-termini for 45 transmembrane helices from the photosynthetic reaction center from Rhodopseudomonas viridis, bacteriorhodopsin and the cytochrome c oxidase from Paracoccus denitrificans . The results clearly showed that it is significantly more probable that a prediction accuracy within an error of +/- 2 residues will be obtained by our procedure than by three different statistical approaches.

J Mol Biol, 1998 Jul 17, 280(3), 443 - 61
Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network; Okamoto A et al.; Aminotransferase reversibly catalyzes the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5'-phosphate (PLP) as a cofactor . Various kinds of aminotransferases developing into catalysts for particular substrates have been reported . Among the aminotransferases, aromatic amino acid aminotransferase (EC 2.6.1 . 57) catalyzes the transamination reaction with both acidic substrates and aromatic substrates . To elucidate the multiple substrate recognition mechanism, we determined the crystal structures of aromatic amino acid aminotransferase from Paracoccus denitrificans (pdAroAT): unliganded pdAroAT, pdAroAT in a complex with maleate as an acidic substrate analog, and pdAroAT in a complex with 3-phenylpropionate as an aromatic substrate analog at 2.33 A, 2 . 50 A and 2.30 A resolution, respectively.The pdAroAT molecule is a homo-dimer . Each subunit has 394 amino acids and one PLP and is divided into small and large domains . The overall structure of pdAroAT is essentially identical to that of aspartate aminotransferase (AspAT) which catalyzes the transamination reaction with only an acidic amino acid . On binding the acidic substrate analog, arginine 292 and 386 form end-on salt bridges with carboxylates of the analog . Furthermore, binding of the substrate induces the domain movement to close the active site . The recognition mechanism for the acidic substrate analog in pdAroAT is identical to that observed in AspAT . Binding of the aromatic substrate analog causes reorientation of the side-chain of the residues, lysine 16, asparagine 142, arginine 292* and serine 296*, and changes in the position of water molecules in the active site to form a new hydrogen bond network in contrast to the active site structure of pdAroAT in the complex with an acidic substrate analog . Consequently, the rearrangement of the hydrogen bond network can form recognition sites for both acidic and aromatic side-chains of the substrate without a conformational change in the backbone structure in pdAroAT .

J Bacteriol, 1998 Jul, 180(14), 3644 - 9
Evidence of two oxidative reaction steps initiating anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by the denitrifying bacterium Azoarcus anaerobius; Philipp B et al.; The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy . The anaerobic degradation of this compound was investigated in cell extracts . Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism . Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K3Fe(CN)6 as the electron acceptor . HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy . Average specific activities were 60 mU mg of protein-1 for resorcinol hydroxylation and 150 mU mg of protein-1 for HHQ dehydrogenation . Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation . These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.

FEBS Lett, 1998 Jun 12, 429(2), 216 - 20
Role of copper during carbon monoxide binding to terminal oxidases; Muntyan MS et al.; Under fully reduced conditions, reassociation kinetics of CO were studied in several terminal oxidases containing copper in their binuclear center . The purified Paracoccus denitrificans ba3-type quinol oxidase was found to recombine with CO monophasically (tau 25-30 ms) like oxidases of the bo type from Escherichia coli, the caa3 type from Bacillus halodurans FTU, and the bo type from Methylobacillus flagellatum KT . Oxidase of the aa3 type from bovine heart recombined with CO monophasically at a higher rate (tau 16-19 ms) than the studied copper-containing bacterial oxidases . After prolonged incubation in the presence of CO, oxidases of the ba3 and aa3 types changed their CO-binding properties . The contribution of the slow component was diminished while new fast components arose . Measurement of the metal content in the oxidases indicated that during the incubation, the enzymes lost their copper, the process being accompanied by the appearance of a fast CO recombination rate resembling that of the non-copper oxidases of the bd type from E . coli and the bb type from Bacillus halodurans FTU . This points to a role of copper in CO binding by terminal oxidases.

Appl Microbiol Biotechnol, 1998 May, 49(5), 618 - 23
Biodegradation of atrazine under denitrifying conditions; Crawford JJ et al.; Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification . The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated . The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems . Results of HPLC and TLC radiochromatography suggest that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation . Ring cleavage was demonstrated by 14CO2 evolution . Denitrification was confirmed by detection of 15N2 in headspace samples of K15NO3-amended anaerobic liquid cultures . In aquatic sediments, mineralization of uniformly ring-labeled {14C}atrazine occurred in both M91-3-inoculated and uninoculated sediment . Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation . Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2533 - 8
Kinetic Parameters of Denitrification in a River Continuum; Garcia-Ruiz R et al.; Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R . Wiske . The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve . The concentration of nitrate for half-maximal activity (Kmap) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 microM in the main river, but it was highest (640 microM) in the Wiske . The apparent maximal rate (Vmaxap) ranged between 35.8 and 324 micromol of N m-2 h-1 in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 micromol N m-2 h-1) . A study of nitrous oxide (N2O) production at the same time showed that rates ranged from below the detection limit (0.05 micromol of N2O-N m-2 h-1) at the headwater site to 27 micromol of N2O-N m-2 h-1 at the downstream site . In the Wiske the rate was up to 570 micromol of N2O-N m-2 h-1, accounting for up to 80% of total N gas production.

Appl Environ Microbiol, 1998 Jul, 64(7), 2432 - 8
Isolation and characterization of phenol-degrading denitrifying bacteria; van Schie PM et al.; Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds . Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date . In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized . Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica . The three strains were shown to be different from each other based on physiologic and metabolic properties . Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of Azoarcus species . Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus Azoarcus . The results of this study add three new members to the genus Azoarcus, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.

J Biochem (Tokyo), 1998 Jul, 124(1), 51 - 4
Characterization of a multi-copper enzyme, nitrous oxide reductase, from Rhodobacter sphaeroides f . sp . denitrificans; Sato K et al.; The nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f . sp . denitrificans, has been purified . The enzyme is composed of two identical subunits of 66 kDa, and contains four copper atoms per subunit . Copper supplementation of the medium resulted in a 3.5-fold increase in the enzyme yield with doubly enhanced specific activity . The activity of the purified nitrous oxide reductase was completely inhibited by 100 microM zinc ions.

Arch Microbiol, 1998 Jul, 170(1), 27 - 37
Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase; Matsson M et al.; Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain . Carboxin is a potent inhibitor of the enzyme of certain organisms . The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3'-methyl carboxin were isolated . Membranes of the mutants showed resistant succinate:quinone reductase activity . The mutation conferring carboxin resistance was identified in four mutants . They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex . The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide . P . denitrificans strains that overproduced wild-type or mutant enzymes were constructed . Enzymic properties of the purified enzymes were analyzed . The apparent Km for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme . Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds . A previously reported His to Leu replacement close to the {3Fe-4S} cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone . The Asp to Gly replacement in the P . denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments . It is likely that this loop is located in the neighborhood of the {3Fe-4S} cluster.

Zentralbl Hyg Umweltmed, 1997 Aug, 200(2-3), 152 - 62
Occurrence of gram-negative bacteria in drinking water undergoing softening treatment; Romano G et al.; A study was carried out on the presence of Gram-negative bacteria in the municipal waters of Bologna (Italy) undergoing softening using domestic ion exchangers with an automatic disinfection mechanism . The softening process was seen to cause a 15 fold increase in 22 degrees C and 36 degrees C heterotrophic plate counts . There was a 30 fold increase in Gram-negative bacteria and their number correlated directly with temperature and inversely with active residual chlorine . Organic matter had no effect on bacterial growth . The most commonly found bacteria were various species of Pseudomonas (87.6%) (Ps . acidovorans, Ps . denitrificans, Ps . fluorescens and Ps . testosteroni) followed by Aeromonas hydrophila (5.6%) and Stenotrophomonas (Xantomonas) maltophilia (3.8% in outgoing water) . Pseudomonas aeruginosa (present in 5.6% of incoming water samples and 0.4% of outgoing water) and Yersinia enterocolitica (present in 4.3% of incoming water samples and 1.1% of outgoing water) did not find favorable conditions for growth on the ion exchange resins.

Biochemistry, 1998 Jun 23, 37(25), 8839 - 47
Site-directed mutagenesis of the conserved threonine (Thr243) of the distal helix of fungal cytochrome P450nor; Okamoto N et al.; Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduction in denitrifying fungi . Threonine 243 (Thr243) of P450nor, which corresponds to the conserved threonine of monooxygenase cytochrome P450s, was replaced by 18 different amino acids via site-directed mutagenesis . The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligation, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural changes in the heme environment on Thr243 mutation . However, the NO reduction activity was dramatically altered by Thr243 mutation, depending on the properties of the replaced amino acids . The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retained on substitution of Asn, Ser, and Gly for Thr243, while it was profoundly decreased or lost on substitution with other amino acids . Kinetic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainly results from a decrease in the rate of reduction of the ferric-NO complex with NADH . On the basis of these enzymatic, kinetic, and spectroscopic results, as well as on the basis of the crystal data for native P450nor {Park, S.-Y., et al . (1997) Nat . Struct . Biol . 4, 827-832}, the role of the conserved threonine at the 243 position in the NO reduction reaction by P450nor is discussed . We also discuss structural similarities or differences in the vicinity of the conserved threonine between P450nor and other monooxygenase P450s.

Biotechnology (N Y), 1995 Feb, 13(2), 155 - 60
Engineered Fv fragments as a tool for the one-step purification of integral multisubunit membrane protein complexes; Kleymann G et al.; The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently insufficient for structural studies . To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments . cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli . The Fv fragments were engineered to serve as bifunctional adaptor molecules . The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column . The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and subcomplexes or individual subunits thereof . These membrane proteins were purified simply by combining the crude P . denitrificans membrane preparation with the E . coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidin affinity chromatography . Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag . The affinity column could thus be reused under continuous operation for several months . Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.

J Appl Microbiol, 1998 Apr, 84(4), 607 - 18
Bacterial-fungal biofilms in flowing water photo-processing tanks; Elvers KT et al.; A complex seven species model community, including bacteria and fungi, was selected from organisms isolated from the walls of an industrial flowing water system . Growth rates of the species were determined in single and mixed batch culture growth . The rates were found to be significantly higher in mixed culture for Pseudomonas alcaligenes and Flavobacterium indologenes and higher in single culture for Xanthomonas maltophilia, Rhodotorula glutinis and Fusarium solani, whereas no significant difference was recorded for Alcaligenes denitrificans and Fusarium oxysporum . All species attached to PVC in single and mixed culture to form biofilms . Xanthomonas maltophilia, Alc . denitrificans, Ps . alcaligenes and F . solani biofilm cell densities cm-2 were significantly higher than attachment of the component species in mixed culture . Statistical analyses showed a significant difference in rate of colonization between single and mixed cultures for some species . No significant difference was noted between mixed culture cell densities cm-2 at laminar flows of Reynolds number 2.7 and 5.4.

Mol Microbiol, 1998 May, 28(3), 615 - 28
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism; Leuthner B et al.; Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica . The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate . We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties . The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres . The native molecular mass was 220 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels . The N-terminal sequences of the alpha- and alpha'-subunits were identical, suggesting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit . The composition of native enzyme therefore appears to be alpha2beta2gamma2 . A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced . The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry . Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site . The highest similarity was observed with pyruvate formate lyases and related proteins . The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the alpha'-subunit . The predicted cleavage site was verified using electrospray mass spectrometry . In addition, a gene coding for an activating protein catalysing glycyl radical formation was found . The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene . Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

Biochim Biophys Acta, 1998 May 6, 1364(2), 186 - 206
Iron-sulfur clusters/semiquinones in complex I; Ohnishi T; NADH-quinone 1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain . Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million . Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8 . These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I . From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned . Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I . Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron-sulfur clusters . Therefore, it has become possible to examine which subunits contain individual iron-sulfur clusters, their location within the enzyme and what their ligand residues are . The discovery of g=2.00 EPR signals arising from two distinct species of semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress . The intensity of semiquinone signals is sensitive to DeltamicroH+ and is diminished by specific inhibitors of Complex I . To date, semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems . This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron-sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts . Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit . The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin-spin interactions between cluster N2 and two distinct Complex I-associated species of semiquinone . Based on the splitting of the g1 signal of the cluster N2 and concomitant strong enhancement of the semiquinone spin relaxation, one semiquinone species was localized 8-11 A from the cluster N2 within the inner membrane on the matrix side (N-side) . Spin relaxation of the other semiquinone species is much less enhanced, and thus it was proposed to have a longer distance from the cluster N2, perhaps located closer to the other side (P-side) surface of the membrane . A brief introduction of EPR technique was also described in Appendix A of this mini-review .

Arch Microbiol, 1998 Jun, 169(6), 509 - 16
Phenylacetyl-CoA:acceptor oxidoreductase, a new alpha-oxidizing enzyme that produces phenylglyoxylate . Assay, membrane localization, and differential production in Thauera aromatica; Schneider S et al.; Anaerobic oxidation of phenylalanine and phenylacetate proceeds via alpha-oxidation of phenylacetyl-CoA to phenylglyoxylate . This four-electron oxidation system was studied in the denitrifying bacterium Thauera aromatica . It is membrane-bound and was solubilized with Triton X-100 . The system used dichlorophenolindophenol as an artificial electron acceptor; a spectrophotometric assay was developed . No other products besides phenylglyoxylate and coenzyme A were observed . The enzyme was quite oxygen-insensitive and was inactivated by low concentrations of cyanide . Enzyme activity was induced under denitrifying conditions with phenylalanine and phenylacetate, it was low in cells grown with phenylglyoxylate, and it was virtually absent in cells grown with benzoate and nitrate or after aerobic growth with phenylacetate.

Biochemistry, 1998 May 5, 37(18), 6436 - 45
Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones; Ohshima M et al.; A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures . Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes . The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I . Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III {He, D . Y., Gu, L . Q., Yu, L., and Yu, C . A . (1994) Biochemistry 33, 880-884}, this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates . In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1 . These observations indicated that bovine complex I recognizes the substrate structure with poor specificity . The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain . The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety . These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction . Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.

J Bioenerg Biomembr, 1998 Feb, 30(1), 89 - 97
Cytochrome c oxidase (heme aa3) from Paracoccus denitrificans: analysis of mutations in putative proton channels of subunit I; Pfitzner U et al.; One of the challenging features of energy-transducing terminal oxidases, like the aa3 cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen . As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis . The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.

J Bioenerg Biomembr, 1998 Feb, 30(1), 81 - 7
The role of electrostatic interactions for cytochrome c oxidase function; Kannt A et al.; In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins . Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers . The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein . A cluster of 18 titratable groups around the heme a3-CuB binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site . Predicted changes in net protonation were in reasonable agreement with experimentally determined values . The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.

J Bioenerg Biomembr, 1998 Feb, 30(1), 15 - 24
From NO to OO: nitric oxide and dioxygen in bacterial respiration; Hendriks J et al.; Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N-N bond (making N2O from two NO molecules) in the nitrogen cycle . It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC . It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron . NorC contains the c-type heme and NorB can be predicted to bind the other metal centers . NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity . Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane . Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter . This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.

Plant Cell Physiol, 1998 Apr, 39(4), 373 - 81
Molecular characterisation of the 76 kDa iron-sulphur protein subunit of potato mitochondrial complex I; Rasmusson AG et al.; Genes encoding subunits of complex I (EC 1.6.5.3) of the mitochondrial respiratory chain vary in their locations between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found on the plastid genome . In potato (Solanum tuberosum L.), the gene coding for the mitochondrial 76 kDa iron-sulphur protein is identified in the nuclear genome . The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves . The amino acid sequence is well conserved relative to the nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus and Neurospora crassa, respectively, and to the Paracoccus denitrificans homologue, most prominently in the region presumed to carry the iron-sulphur clusters . Polyclonal antibodies directed against the 78 kDa complex I subunit of N . crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75 kDa in solubilised stroma thylakoids from spinach chloroplasts . The 32 amino acid residues long presequence of the potato mitochondrial 76 kDa complex I subunit targets the precursor polypeptide into isolated potato mitochondria but not into isolated chloroplasts . These results suggest that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from, the mitochondrial subunit.

J Bacteriol, 1998 May, 180(10), 2718 - 22
Isolation of a periplasmic molecular chaperone-like protein of Rhodobacter sphaeroides f . sp . denitrificans that is homologous to the dipeptide transport protein DppA of Escherichia coli; Matsuzaki M et al.; A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f . sp . denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol . 37:333-339, 1996) . The protein was isolated from the periplasm of the phototroph . It had a molecular mass of 58 kDa and had no subunits . The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E . coli . The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein . The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution . The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.

Arch Microbiol, 1998 Apr, 169(4), 333 - 8
Anaerobic degradation of alpha-resorcylate by Thauera aromatica strain AR-1 proceeds via oxidation and decarboxylation to hydroxyhydroquinone; Gallus C et al.; Anaerobic degradation of alpha-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica . alpha-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway . Instead, alpha-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation . Nitrate, K3{Fe(CN)6}, dichlorophenol indophenol, and the NAD+ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD+ did not function as an electron acceptor . The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor . Oxidation of alpha-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of alpha-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with alpha-resorcylate under an air atmosphere . alpha-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps . The alpha-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0 . The primary oxidation product was not hydroxyhydroquinone . Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH.

Arch Microbiol, 1998 Apr, 169(4), 275 - 81
Genes coding for respiratory complexes map on all three chromosomes of the Paracoccus denitrificans genome; Winterstein C et al.; The genome of Paracoccus denitrificans (strain Pd1222) consists of three distinct DNA molecules when separated by standard pulsed-field gel electrophoresis with apparent molecular sizes of approximately 2, 1.1, and 0.64 Mb . When the separated chromosomes are digested by restriction enzymes and sizes of resulting fragments are summed up, the three chromosomes are composed of 1.83, 1.16, and 0.67 Mb . Since their migration behavior relative to size standards is largely independent of electrophoresis conditions, at least the two smaller chromosomes most likely represent linear molecules . The size analysis presented here allows an unequivocal distinction between groups of different strains of P . denitrificans and of Thiosphaera pantotropha, confirming an earlier cytochrome c analysis . When the genome was analyzed with different probes coding for respiratory enzymes, essential genes were found spread over all three chromosomes without any obvious clustering on any of the three forms.

Epidemiol Mikrobiol Imunol, 1998 Apr, 47(2), 47 - 51
{Identification of Pseudomonas stutzeri with the NEFERMtest, NEFERM test 24 and API 20NE commercial test kits}; Pacova Z et al.; A total of 35 strains of Pseudomonas stutzeri were tested by commercial diagnostic kits NEFERMtest, NEFERMtest 24 (both Lachema Co., Brno, Czech Republic) and API 20NE (bioMerieus sa, RCS Lyon, France) . The best identification efficacy was achieved by means of the commercial API 20NE (97.1%) . Only 60% strains were correctly identified by means of NEFERMtest . NEFERMtest 24 improved successful identification (80%) . Most misidentified strains were identified as Alcaligenes denitrificans and Al . xylosoxidans.

Biochemistry, 1998 May 19, 37(20), 7400 - 6
Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling; Behr J et al.; Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme . For this purpose, the hemA gene of the P . denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy . Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P . dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium . Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme . The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates . Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates . Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates . A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate . These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.

Biochemistry, 1998 May 19, 37(20), 7390 - 9
Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy; Hellwig P et al.; The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy . Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained . In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca . 1620-1680 cm-1) and amide II (ca . 1560-1540 cm-1) region in response to the redox transition of the cofactor(s) . In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes . A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme {Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mantele, W . (1996) FEBS Lett . 385, 53-57} . These signals were resolved into several components associated with the oxidation of different cofactors . For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl) . This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer . The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme . In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost . In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments . On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction . Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

J Biochem (Tokyo), 1998 Mar, 123(3), 369 - 75
Structure and function of bacterial cytochrome c oxidase; Iwata S; The crystal structure of cytochrome c oxidase from the soil bacterium Paracoccus denitrificans has been reported . This structure has provided a basis for understanding the mechanism of the redox-coupled transmembrane proton pump which is the key component of the respiratory chain in most aerobic organism . Over the past ten years, there have been many site-directed mutagenesis studies performed on bacterial oxidases . Structural features of Paracoccus oxidase have been summarized in the light of these mutagenesis studies and other structural works.

Biochemistry, 1998 Apr 28, 37(17), 6086 - 94
Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3; Olesen K et al.; We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides . Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper . Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide . The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods . The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored . The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity . The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center . His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper . The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

FEMS Microbiol Lett, 1998 May 1, 162(1), 61 - 8
Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments; Scala DJ et al.; Two PCR primer sets for the nitrous oxide reductase gene (nosZ) were developed . The initial primers were based on three sequences in GenBank and used to amplify nosZ from continental shelf sediments and from two denitrifiers in culture, Thiosphaera pantotropha and Pseudomonas denitrificans . Three unique marine sediment nosZ genes were identified and sequenced . The marine nosZ genes were most closely related to the nosZ genes of Paracoccus denitrificans or to Rhizobium meliloti . Alignment of all nosZ sequences currently available (n = 10) facilitated redesign of the PCR primers . Three new primer sets which amplify 1100 bp, 900 bp and 250 bp regions of the nosZ gene were designed and tested . The new primers robustly amplified nosZ fragments from samples in which the initial nosZ primers were only marginally successful.

Appl Environ Microbiol, 1998 May, 64(5), 1650 - 6
Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical; Coschigano PW et al.; The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source . Two mutants which have defects in this toluene utilization pathway have been characterized . A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated . The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da . The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da . Two additional small open reading frames have been identified, but their role is not known . The TutE protein has homology to pyruvate formate-lyase activating enzymes . The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme . Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3443 - 8
Rack-induced metal binding vs . flexibility: Met121His azurin crystal structures at different pH; Messerschmidt A et al.; The rack-induced bonding mechanism of metals to proteins is a useful concept for explaining the generation of metal sites in electron transfer proteins, such as the blue copper proteins, that are designed for rapid electron transfer . The trigonal pyramidal structure imposed by the protein with three strong equatorial ligands (one Cys and two His) provides a favorable geometry for both cuprous and cupric oxidation states . However, the crystal structures of the Met121His mutant of azurin from Alcaligenes denitrificans at pH 6.5 (1.89- and 1.91-A resolutions) and pH 3.5 (2.45-A resolution) show that the preformed metal binding cavity in the protein is more flexible than expected . At high pH (6.5), the Cu site retains the same three equatorial ligands as in the wild-type azurin and adds His121 as a fourth strong ligand, creating a tetrahedral copper site geometry with a green color referred to as 1.5 type . In the low pH (3.5) structure, the protonation of His121 causes a conformational change in residues 117-123, moving His121 away from the copper . The empty coordination site is occupied by an oxygen atom of a nitrate molecule of the buffer solution . This axial ligand is coordinated less strongly, generating a distorted tetrahedral copper geometry with a blue color and spectroscopic properties of a type-1 site . These crystal structures demonstrate that blue copper proteins are flexible enough to permit a range of movement of the Cu atom along the axial direction of the trigonal pyramid.

Arch Microbiol, 1998 Mar, 169(3), 179 - 87
New roles for CO2 in the microbial metabolism of aliphatic epoxides and ketones; Ensign SA et al.; Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically . In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria . One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2 . Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO2 in these processes . Both classes of compounds are metabolized by carboxylation reactions that produce beta-keto acids as products . The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases . Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD+ as cofactors . In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD+ undergoes reduction . Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation . Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions . ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha . These studies have identified new roles for CO2 as a cosubstrate in the metabolism of two classes of important xenobiotic compounds . In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO2 to organic substrates.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 121 - 9
Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis; Woodcock SC et al.; The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-l-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme . This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB . Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E . coli cysG deletion strain . Evidence is also presented to suggest that a gene within the S . typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

Biophys J, 1998 Feb, 74(2 Pt 1), 708 - 21
The coupling of electron transfer and proton translocation: electrostatic calculations on Paracoccus denitrificans cytochrome c oxidase; Kannt A et al.; We have calculated the electrostatic potential and interaction energies of ionizable groups and analyzed the response of the protein environment to redox changes in Paracoccus denitrificans cytochrome c oxidase by using a continuum dielectric model and finite difference technique . Subsequent Monte Carlo sampling of protonation states enabled us to calculate the titration curves of all protonatable groups in the enzyme complex . Inclusion of a model membrane allowed us to restrict the calculations to the functionally essential subunits I and II . Some residues were calculated to have complex titration curves, as a result of strong electrostatic coupling, desolvation, and dipolar interactions . Around the heme a3-CuB binuclear center, we have identified a cluster of 18 strongly interacting residues that account for most of the proton uptake linked to electron transfer . This was calculated to be between 0.7 and 1.1 H+ per electron, depending on the redox transition considered . A hydroxide ion bound to CuB was determined to become protonated to form water upon transfer of the first electron to the binuclear site . The bulk of the protonation changes linked to further reduction of the heme a3-CuB center was calculated to be due to proton uptake by the interacting cluster and Glu(II-78) . Upon formation of the three-electron reduced state (P1), His325, modeled in an alternative orientation away from CuB, was determined to become protonated . The agreement of these results with experiment and their relevance in the light of possible mechanisms of redox-coupled proton transfer are discussed.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 334 - 7
Trehalose as osmoprotectant in Rhodobacter sphaeroides f . sp . denitrificans IL106; Xu X et al.; The photosynthetic bacterium Rhodobacter sphaeroides f . sp . denitrificans IL106 can grow in high osmolarity . The accumulation of intracellular inorganic ions and organic solutes in cells grown in a synthetic medium containing different concentrations of NaCl was examined . Together with potassium ion, trehalose was the major organic osmoprotectant and its accumulation depended on the external salt concentration . Intracellular levels of glycine betaine and other osmoprotectants such as proline did not change when osmolarity increased.

Arch Microbiol, 1998 Feb, 169(2), 159 - 65
Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria; Benz M et al.; Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30 degrees C . Anaerobic nitrate-dependent ferrous iron oxidation was a biological process . One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate . Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30 degrees C in the presence of small amounts of acetate (between 0.2 and 1.1 mM) . The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply . The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria . Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments.

Gene, 1998 Feb 27, 208(2), 215 - 9
Rhizobium etli cycHJKL gene locus involved in c-type cytochrome biogenesis: sequence analysis and characterization of two cycH mutants; Tabche ML et al.; The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes . The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins . CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R . meliloti, and R . leguminosarum, respectively . CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B . japonicum, R . meliloti, R . leguminosarum, respectively . CycK of 666 aa shared 62, 73, and 90% homology with CycK from B . japonicum, R . meliloti, and R . leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% homology with CycL from the abovementioned species . The Tn5mob insertion present in the IFC01 strain was located in the cycH gene . This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis . A previously described R . etli cytochrome c-deficient MuD1lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH . Complementation analysis suggested that the MuD1lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn5mob insertion present in IFC01, which showed no polarity on cycJKL . Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.

Biodegradation, 1997, 8(4), 265 - 73
Implementation of natural attenuation at a JP-4 jet fuel release after active remediation; Cho JS et al.; After eighteen months of active remediation at a JP-4 jet-fuel spill, a residual of unremediated hydrocarbon remained . Further site characterization was conducted to evaluate the contribution of natural attenuation to control exposure to hazards associated with the residual contamination in the subsurface . Activities included the detailed characterization of ground-water flow through the spill; the distribution of fuel contaminants in groundwater; and the analysis of soluble electron acceptors moving into the spill from upgradient . These activities allowed a rigorous evaluation of the transport of contaminants from the spill to the receptor of groundwater, the Pasquotank River . The transport of dissolved contaminants of concern, that is benzene, toluene, ethyl benzene, xylene isomers (BTEX) and methyl-tertiary-butyl ether (MTBE), into the river from the source area was controlled by equilibrium dissolution from the fuel spill to the adjacent groundwater, diffusion in groundwater from the spill to permeable layers in the aquifer, and advective transport in the permeable layers . The estimated yearly loading of BTEX compounds and MTBE into the receptor was trivial even without considering biological degradation . The biodegradation of hydrocarbon dissolved in groundwater through aerobic respiration, denitrification, sulfate reduction, and iron reduction was estimated from changes in ground-water chemistry along the flow path . The concentrations of target components in permanent monitoring wells continue to decline over time . Long term monitoring will ensure that the plume is under control, and no further active remediation is required.

Biochemistry, 1998 Feb 24, 37(8), 2660 - 5
Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli; De Luca G et al.; Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively . The weak representativity of the enzyme among cell proteins has prevented its purification . This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli . The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol . EPR analysis of the reduced form of HndA indicates that it contains a single binuclear {2Fe-2S} cluster . This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2 . 000, respectively, and a midpoint redox potential of -395 mV . The UV-visible and EPR spectra of the {2Fe-2S} cluster indicate that HndA belongs to the {2Fe-2S} family typified by the Clostridium pasteurianum {2Fe-2S} ferredoxin . The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C . pasteurianum {2Fe-2S} ferredoxin . The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations . Evidence of a HndA organization based on two independent structural domains is discussed.

Biochemistry, 1998 Feb 24, 37(8), 2470 - 6
Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R . sphaeroides; Adelroth P et al.; In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I-362)) and threonine-359 (T(I-359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer . These residues have been proposed to be part of a proton-transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart . Mutation of K(I-362) and T(I-359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and approximately 35%, respectively, of those in the wild-type enzyme . The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of approximately 3 micros, not coupled to protonation reactions, is not affected in the mutant enzymes . However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of approximately 3 ms (at pH 9.0) is impaired in the KM(I-362) and TA(I-359) mutant enzymes . This is consistent with the slow reduction rate of heme a3 in the oxidized KM(I-362) enzyme because in the wild-type enzyme reduction of heme a3 is coupled to proton uptake . On the other hand, when reacting with O2, both the wild-type and mutant fully reduced enzymes become oxidized in approximately 5 ms, and proton uptake on this time scale is not affected . Hence, the results indicate that the KM(I-362) mutant enzyme is inactive because the proton-transfer pathway through K(I-362) and T(I-359) is involved in proton uptake during reduction of the oxidized binuclear center . Proton uptake during oxidation of the fully reduced enzyme takes place through a different pathway {through E(I-286) (Adelroth, P., et al . (1997) Biochemistry 36, 13824-13829)}.

J Biol Chem, 1998 Feb 27, 273(9), 5132 - 6
Tryptophan 121 of subunit II is the electron entry site to cytochrome-c oxidase in Paracoccus denitrificans . Involvement of a hydrophobic patch in the docking reaction; Witt H et al.; To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase . KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively . We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase . Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged . Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.

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