Alcaligenes

FEBS Lett, 1998 Nov 6, 438(3), 231 - 5
Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus; Pierik AJ et al.; A third hydrogenase was recently identified in the proteobacterium Alcaligenes eutrophus as a constituent of a novel H2-sensing multicomponent regulatory system . This regulatory hydrogenase (RH) has been overexpressed in cells deficient in both the NAD+-reducing {NiFe}-hydrogenase and the membrane-bound {NiFe}-hydrogenase . EPR, FTIR and activity studies of membrane-free extracts revealed that the RH has an active site much like that of standard {NiFe}-hydrogenases, i.e . a Ni-Fe site with two CN- groups and one CO molecule . Its catalytic power is low, but the RH is always active, insensitive to oxygen, and occurs in only two redox states.

J Mol Biol, 1998 Dec 4, 284(3), 751 - 60
Basis for monomer stabilization in Rhodopseudomonas palustris cytochrome c' derived from the crystal structure; Shibata N et al.; The crystal structure of an unusual monomeric cytochrome c' from Rhodopseudomonas palustris (RPCP) has been determined at 2.3 A resolution . RPCP has the four-helix (helices A, B, C and D) bundle structure similar to dimeric cytochromes c' . However the amino acid composition of the surface of helices A and B in RPCP is remarkably different from that of the dimeric cytochromes c' . This surface forms the dimer interface in the latter proteins . RPCP has seven charged residues on this surface contrary to the dimeric cytochromes c', which have only two or three charged groups on the corresponding surface . Moreover, hydrophobic residues on this surface of RPCP are two to three times fewer than in dimeric cytochromes c' . As a result of the difference in amino acid composition, the A-B surface of RPCP is rather hydrophilic compared with dimeric cytochromes c' . We thus suggest that RPCP is monomeric in solution because of the hydrophilic nature of the A-B surface . The amino acid composition of the A-B surface is similar to that of Rhodobacter capsulatus cytochrome c' (RCCP), which is an equilibrium admixture of monomer and dimer . The charge distribution of the A-B surface in RCCP, however, is considerably different from that of RPCP . Due to the difference, RCCP can form dimers by both ionic and hydrophobic interactions . These dimers are quite different from those in proteins which form strong dimers such as in Chromatium vinosum, Rhodospirillum rubrum, Rhodospirillum molischianum and Alcaligenes . Cytochrome c' can be classified into two types . Type 1 cytochromes c' have hydrophobic A-B surfaces and they are globular . The A-B surface of type 2 cytochromes c' is hydrophilic and they take a monomeric or flattened dimeric form .

J Biotechnol, 1998 Sep 17, 64(1), 23 - 38
The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production; Gerritse G et al.; Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations . The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids . In order to improve the lipase yield further, the phenotype enhancement method has been developed . This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P . alcaligenes strain with already high lipase productivity . One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes . These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa . Remarkably and dissimilar to P . aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present . For OrfX no equivalent can be found in the known protein data bases . On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK . Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons . A model is proposed for the regulation of the secretion system from P . alcaligenes.

J Biotechnol, 1998 Oct 8, 64(2-3), 177 - 86
Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha; Dennis D et al.; The acetoacetyl-CoA reductase and the polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (formerly Alcaligenes eutrophus) were expressed in Escherichia coli, Klebsiella aerogenes, and PHA-negative mutants of R . eutropha and Pseudomonas putida . While expression in E . coli strains resulted in the accumulation of poly(3-hydroxybutyrate) {PHB}, strains of R . eutropha, P . putida and K . aerogenes accumulated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) {poly(3HB-co-3HHx)} when even chain fatty acids were provided as carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {poly(3HB-co-3HV)} when odd chain fatty acids were provided as carbon source . This suggests that fatty acid degradation can be directly accessed employing only the acetoacetyl-CoA reductase and the PHA synthase . This is also the first proof that the PHA synthase from R . eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described.

J Biotechnol, 1998 Oct 8, 64(2-3), 125 - 35
Molecular cloning, sequencing and expression in Escherichia coli of the poly(3-hydroxyalkanoate) synthesis genes from Alcaligenes latus DSM1124; Genser KF et al.; Fragments of chromosomal DNA from Alcaligenes latus DSM1124 were cloned into Escherichia coli and transformants were screened for poly(D(-)-3-hydroxybutyrate) {P(3HB)} production during excess carbon supply . A plasmid harboring a 5.5-kb insert of A . latus DNA was isolated from a P(3HB)-producing bacterial colony . The insert was partially sequenced and three major open reading frames (ORFs) were found, representing the PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) genes . They show striking homology to the Ralstonia eutropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way . The only major difference is the replacement of 560 nucleotides by 160 non-homologous nucleotides in the 5' region of phaC in A . latus . The phaC ORF lacks 29 amino acids at the N-terminus, compared to that of R . eutropha, and starts with a GTG codon . The transcription start points of the operon were determined . P(3HB) production of recombinant E . coli strains harboring the pha operons of A . latus DSM1124 or R . eutropha H16 was investigated . Both operons gave rise to less than 5% P(3HB) formation during exponential growth . At the end of the growth phase, the P(3HB) content reached approximately 20% of cell dry mass . Under nitrogen-depleted conditions, the A . latus pha genes gave rise to 50-52% P(3HB), compared to 33-38% for the R . eutropha pha genes . No NADH oxidase activity was detectable in A . latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth.

Plasmid, 1998 Nov, 40(3), 203 - 13
Characterization of the Pac25I restriction-modification genes isolated from the endogenous pRA2 plasmid of Pseudomonas alcaligenes NCIB 9867; Yeo CC et al.; Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-modification (R-M) system, Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2 . The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp . The deduced amino acid sequence of the Pac25I methylase revealed significant similarity with the XcyI, XmaI, Cfr9I, and SmaI methylases . High sequence similarity was displayed between the Pac25I endonuclease and the XcyI, XmaI, and Cfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5'-C CCGGG-3') and are thus perfect isoschizomers . However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5'-CCCGGG-3') . Both the Pac25I methylase and endonuclease were expressed in Escherichia coli . An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of the Pac25I R-M operon . In addition, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes . The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria .

CLAO J, 1998 Oct, 24(4), 239 - 41
Alcaligenes xylosoxidans keratitis post penetrating keratoplasty in a rigid gas permeable lens wearer; Lin A et al.; PURPOSE: We report a case of Alcaligenes xylosoxidans keratitis following penetrating keratoplasty in a rigid gas permeable (RGP) lens wearer . METHODS: A 61 year old RGP lens wearer with a history of nonresponsive keratitis of the right eye which involved the graft margin was referred to us for treatment . Corneal cultures revealed growth of a gram-negative rod on the fifth day and the organism was subsequently identified as Alcaligenes xylosoxidans, which was resistant to most antibiotics and sensitive only to Bactrim, Timentin, and imipenem . RESULTS: Clinical improvement was observed within 24 hours after treatment with the use of topical i.v . Bactrim and topical i.v . Timentin 2% alternating every 30 minutes . Complete resolution of the infection with mild scarring was observed 6 weeks after treatment . CONCLUSIONS: Alcaligenes xylosoxidans is a potential cause of bacterial keratitis which should be considered in cases of nonresponsive gram-negative keratitis . The addition of topical Bactrim or Timentin may need to be considered in such cases.

Arch Microbiol, 1998 Nov, 170(6), 460 - 3
hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster; Buhrke T et al.; The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined . The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied . The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase . The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected . We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant . The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor . Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX.

Arch Microbiol, 1998 Nov, 170(6), 451 - 9
Duplication of hyp genes involved in maturation of {NiFe} hydrogenases in Alcaligenes eutrophus H16; Wolf I et al.; Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1 . Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A . eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies . Mutants with lesions in both copies showed clear alterations in hydrogenase activities . Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H2 . Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium . Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor . Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes . HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms . Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site.

J Biochem (Tokyo), 1998 Nov, 124(5), 876 - 9
Type 1 Cu structure of blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 at 2.05 A resolution: comparison of blue and green nitrite reductases; Inoue T et al.; The crystal structure of the blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) has been determined at 2.05 A resolution . AxgNIR contains both type 1 and 2 Cu sites, the geometry of the former being distorted tetrahedral . The superpositioning of the type 1 Cu sites in the blue enzyme and a green nitrite reductase revealed that the orientation of the Met150 side chain differed . The deviation of the Sdelta(Met150) atom from the axial position of the NNS plane formed by two Ndelta(His95 and His145) and one Sgamma(Cys136) atom caused the difference in the colors of the enzymes, i.e . blue and green.

FEBS Lett, 1998 Oct 2, 436(2), 239 - 42
The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase; Farver O et al.; The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO) . This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis . The reversible ET reaction proceeds with a rate constant kET = k(1-->2) + k(2-->1) of 450 +/- 30 s(-1) at pH 7.0 and 298 K . The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k(1-->2) = 185 +/- 12 s(-1) and k(2-->1) 265 +/- 18 s(-1) . The temperature dependence of K allowed us to determine the deltaH(o) value of the ET equilibrium to be 12.1 kJ mol(-1) . Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, deltaH* = 22.7 +/- 3.4 kJ mol(-1) and deltaS* = -126 +/- 11 J K(-1) mol(-1); backward reaction, deltaH* = 10.6 +/- 1.7 kJ mol(-1) and deltaS* = -164 +/- 15 J K(-1) mol(-1) . X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion . This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared . The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.

Tsitologiia, 1998, 40(6), 579 - 84
{Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction}; Ponomarev AG et al.; Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean . The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach . Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12474 - 9
A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus; Lenz O et al.; Oxidation of molecular hydrogen catalyzed by {NiFe} hydrogenases is a widespread mechanism of energy generation among prokaryotes . Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources . In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus . This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA . HoxB and HoxC share characteristic features of dimeric {NiFe} hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ* . A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2 . Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2 . Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain . Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation . The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.

Biotechnol Prog, 1998 Sep-Oct, 14(5), 680 - 8
Novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol; Leonard D et al.; The use of a membrane bioreactor with cell retention to achieve high biomass concentrations has been examined for phenol degradation by the bacteria Alcaligenes eutrophus . This process is particularly interesting for toxic substrates as the hydraulic dilution rate and the growth rate are independently controlled . In the case of a transitory excess of phenol, this potentially toxic situation can be overcome by modifying the substrate concentration or the dilution rate without any loss of cells . The injection of a gas phase at the filter inlet increased both the permeate flow rate (by a factor of 1 . 75) and the oxygen transfer capacity (by a factor of 1.5) . This has enabled the cell concentration to reach a maximal value of 60 g L-1 with a hydraulic dilution rate of 0.5 h-1 and a phenol feed concentration of 8 g L-1 . The volumetric productivity of this process corresponds to a phenol degradation rate approaching 100 kg m-3 day-1 . The on-line measurement of the characteristic yellow color of 2-hydroxymuconate semialdehyde, a metabolic intermediate of the phenol degradation pathway, in the permeate provides an interesting basis for process control of phenol supply into the reactor since the color intensity correlates directly to the specific rate of phenol degradation.

Lett Appl Microbiol, 1998 Aug, 27(2), 86 - 92
Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration; Andreoni V et al.; A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6,-TCP with stoichiometric release of Cl- . In cultures of Alc . eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation . Experiments conducted with soil microcosms gave evidence that: the degradative process had a biotic nature and was accompanied by microbial growth; the soil used presented an intrinsic degradative capacity versus 2,4,6-TCP; the specialized organism used as inoculum was effective in degrading 2,4,6-TCP in a short time . These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 253 - 60
Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes; Yeo CC et al.; Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563 . Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons . However, no other mer operon genes were found on Tn5563 . Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.

Can J Microbiol, 1998 Jun, 44(6), 554 - 64
The phbC (poly-beta-hydroxybutyrate synthase) gene of Rhizobium (Sinorhizobium) meliloti and characterization of phbC mutants; Willis LB et al.; Defined insertion mutations have been constructed in the Rhizobium (Sinorhizobium) meliloti phbC gene, which encodes poly-beta-hydroxybutyrate (PHB) synthase . The locus was isolated and subcloned from a genomic library of R . meliloti Rm1021 by complementation of phbC mutation of Alcaligenes eutrophus . PHB production was detected in wild-type R . meliloti under nutrient-limited conditions but not in rich medium . No PHB production was detected in the R . meliloti phbC mutants . The DNA sequence of the R . meliloti phbC gene was determined . The deduced polypeptide sequence is homologous to previously identified PhbCs from other bacteria . The R . meliloti phbC locus maps to pRmeSU47a, the smaller of the two megaplasmids in this strain.

Am J Nephrol, 1998, 18(5), 452 - 5
Peritoneal dialysis-associated peritonitis caused by Alcaligenes xylosoxidans; El-Shahawy MA et al.; Despite significant progress to decrease its incidence, peritonitis remains the main source of morbidity and treatment failure in patients on continuous ambulatory peritoneal dialysis (CAPD) . The majority of cases of peritonitis result from infection with aerobic gram-positive (Staphylococcus epidermidis and Staphylococcus aureus), or gram-negative organisms . Less common organisms that are also reported include anaerobic bacteria, fungi, and mycobacteria, which collectively account for less than 10% of isolates cultured . We report a case of peritoneal dialysis-associated peritonitis, and review the literature on peritonitis caused by Alcaligenes species . Alcaligenes xylosoxidans is a nonfermenting gram-negative rod and opportunistic pathogen that is motile with peritrichous flagella . The clinical features and microbiological data of our case, as well as the other previously reported cases of peritonitis caused by Alcaligenes species show no particular pattern of peritoneal dialysate cell count . However, the rate of recurrence of peritonitis is characteristically high . The cause of such a high rate of recurrence of peritonitis is probably a reflection of the predilection of Alcaligenes species to cause infection in the 'sicker' patients, and the almost universal resistance of this species to most antimicrobial agents . We, therefore, recommend that catheter removal be undertaken as early as the identification of the organism is made . Whether patients should be allowed to return to CAPD after recovery is a more difficult question . We suggest that a reevaluation of the patient's overall status be undertaken, including personal hygiene, exchange technique, presence of diabetes mellitus, malnutrition, and/or other factors that may render the patient more prone to infection with opportunistic pathogens.

Zentralbl Bakteriol, 1998 Jul, 288(1), 145 - 57
Epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans by antibacterial susceptibility testing, fatty acid analysis, PAGE of whole-cell protein and pulsed-field gel electrophoresis; Knippschild M et al.; Antibacterial susceptibility testing, fatty acid analysis, protein analysis and DNA analysis of Alcaligenes xylosoxidans subsp . xylosoxidans were compared to determine the efficiency of the methods available for strain typing . Thirty isolates were investigated: 20 clinical isolates from a nonsocomial outbreak in Essen (Germany), 9 clinical isolates from sporadic nosocomial cases in Paris (France) and reference strain ATCC 2402 . The highest microbiological discriminative power was exhibited by pulsed-field gel electrophoresis (PFGE) yielding nine types, followed by fatty acid methyl ester (FAME) analysis with six types, and antibacterial susceptibility testing and polyacrylamide gel electrophoresis with five types each . By combining the results of the four typing methods, 14 varieties could be differentiated . Protein analysis and fatty acid analysis failed to discriminate between isolates from Essen and Paris and the reference strain, while antibacterial susceptibility testing and DNA analysis clearly discriminated them . It is concluded that a combination of antibacterial susceptibility testing and PFGE typing is most suitable for epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans strains.

J Appl Microbiol, 1998 May, 84(5), 859 - 64
Change in bacterial community during biodegradation of aniline; Tani K et al.; The response of river water microbial communities to chemical compounds was monitored under laboratory conditions using aniline as a model . Bacteria were collected from unpolluted and polluted sites . Bacterial abundance (plate and total direct counting) and its relation to aniline biodegradation was examined . Colony hybridization with 16S rRNA oligonucleotide probes was used to study the changes in microbial community structure during biodegradation of aniline . The changes in bacterial abundance and community structure were related to biodegradation of aniline . Burkholderia-Pseudomonas (rRNA group III), an authentic Alcaligenes group became dominant despite the initial differences in the microbial communities, suggesting that these genera are the main aniline degraders in the aquatic environment.

Appl Environ Microbiol, 1998 Sep, 64(9), 3437 - 43
Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer; Sudesh K et al.; The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe . Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa) . Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A . eutrophus . The arrangement of PHA biosynthesis genes in C . acidovorans was also similar to that in A . eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa . The cloned fragment complemented a PHA-negative mutant of A . eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source . The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers . The PHA synthase of C . acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate . This leads to the suggestion that in C . acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.

J Clin Microbiol, 1998 Sep, 36(9), 2618 - 22
Phenotypic and genotypic characterization of clinical strains of CDC group IVc-2; Osterhout GJ et al.; CDC group IVc-2 is a gram-negative, oxidase-positive, nonfermentative bacillus that has been implicated in human infections, including septicemia and peritonitis . Biochemically it most closely resembles Bordetella bronchiseptica and Alcaligenes sp . Results of cellular fatty acid (CFA) and 16S rRNA gene analysis were combined with biochemical data to assist in identification and classification . The predominant CFAs were hexadecanoic acid (16:0), cis-9-hexadecanoic acid (16:1omega7c), cis-11-octadecanoic acid (18:1omega7c), and Delta-cis-9,10-methylenehexadecanoic acid (17:0cyc) . Small amounts (2 to 5%) of 3-hydroxytetradecanoic acid (3-OH-14:0), tetradecanoic acid (14:0), 2-hydroxyhexadecanoic acid (2-OH-16:0), and Delta-cis-11,12-methyleneoctadecanoic acid (19:0cyc) were also consistently present . The highest 16S rRNA gene similarity was with Ralstonia eutropha and Ralstonia solanacearum . The CFA and 16S rRNA gene sequence data support the inclusion of CDC group IVc-2 in the recently created genus Ralstonia, which includes R . eutropha, R . pickettii, and R . solanacearum.

Microbiology, 1998 Jul, 144 ( Pt 7), 1765 - 72
Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties; Hino S et al.; Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low Ks (the apparent half-saturation constant in Haldane's equation) and an extremely high KSI (the apparent inhibition constant) . To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl- Cat+) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614 . PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase . The cloned genes consist of eight complete ORFs, designated poxRABCDEFG . The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp . CF600, sharing 30-65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme . The kinetic constants for phenol-oxygenating activity of PAO1c(pROE217) were determined, and these were compared with those of strain E2 . The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined . A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines Ks and KSI values for the cellular phenol-oxygenating activity . It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2.

Kansenshogaku Zasshi, 1998 Jun, 72(6), 631 - 4
{Case report: subcutaneous abscess and thoracic empyema caused by Alcaligenes xylosoxidans}; Mizunoe S et al.; Alcaligenes xylosoxidans is a glucose-nonfermentative gram-negative rod which usually exists in the environment . This organism while causing pneumonia, sepsis, meningitis and urinary tract infection in the compromised host, rarely causes thoracic empyema . We report a case of thoracic empyema and subcutaneous abscess due to A . xylosoxidans . A 74-year-old male, who had undergone right total pneumonectomy for chronic necrotizing pulmonary aspergillosis a year ago, was admitted to our hospital because of fever . CT scans of the chest revealed a subcutaneous abscess and empyema . Empyema and subcutaneous pus were aspirated . Culture of materials produced A . xylosoxidans . There was no significant change on symptoms and examinations despite therapy with PIPC 4 g/day and thoracic drainage . Finally, surgical treatment was required and the patient was cured.

Appl Environ Microbiol, 1998 Aug, 64(8), 3014 - 22
Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments; Wagner-Dobler I et al.; A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities . Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals . After 6 months, stable population densities between 8 x 10(9) and 2 x 10(11) CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates . A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl . Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region . In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences . The R . opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used . In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives . Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R . opacus, independent of the pollution history of the original sample . R . opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.

Arch Microbiol, 1998 Sep, 170(3), 162 - 70
Synechocystis sp . PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria; Hein S et al.; During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp . PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight . Our analysis of the complete Synechocystis sp . PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene . The open reading frame slr1829 was therefore designated as phaE . The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii . The Synechocystis sp . PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus . Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A . eutrophus PHB-4 . These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp . PCC6803 PHA synthase . PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied . Western blot analysis of Synechocystis sp . PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A . eutrophus (PhaC) and of C . vinosum (PhaE and PhaC) revealed no immunoreaction.

Mol Microbiol, 1998 Jun, 28(6), 1059 - 66
The extracytoplasmic function sigma factors: role and regulation; Missiakas D et al.; Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes . One such class of sigma factors appears to control a variety of functions, including expression of heat-shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E . coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp . strain SS9 . Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors . They have also been characterized in Mycobacteria as well as gram-positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius . ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species . The promoter consensus sequences recognized by the ECF factors are also highly conserved . In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E . coli and in P . aeruginosa, to act as an anti-sigma activity . This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change . Presumably, an on-and-off switch of the anti-sigma activity leads to the release of the sigma factor and thereby to the co-ordinate transcription of the specific regulon it governs.

Clin Infect Dis, 1998 Jul, 27(1), 158 - 63
Microbiology of sputum from patients at cystic fibrosis centers in the United States; Burns JL et al.; During a phase III national collaborative study of aerosolized tobramycin (1 July 1995 through 30 September 1996), the microbiology of specimens from 595 patients at 69 cystic fibrosis (CF) centers was examined . Samples from three screening visits were processed in a single laboratory by means of standardized techniques for identification and susceptibility testing . From 1,753 pretreatment specimens, 5,128 pathogens were isolated (average, 2.9/specimen) . Of the 3,936 Pseudomonas aeruginosa isolates, 56.7% were mucoid . The specimens of 125 patients (21.0%) yielded tobramycin-resistant P . aeruginosa (213 isolates); 61 (10.3%), Stenotrophomonas maltophilia; and 52 (8.7%), Alcaligenes xylosoxidans . Isolation of Burkholderia cepacia was an exclusion criterion . Only visit 3 sputum samples were cultured for gram-positive organisms and fungi (n = 465 patients); samples from 201 patients (43.2%) yielded Staphylococcus aureus (18.8% of isolates were oxacillin-resistant), and those from 114 (24.5%) yielded an Aspergillus species . Compared with the Cystic Fibrosis Foundation Patient Registry, the current study identified many more patients colonized with S . maltophilia, A . xylosoxidans, Aspergillus species, and oxacillin-resistant S . aureus, suggesting the utility of standardized processing of CF specimens.

Biochim Biophys Acta, 1998 May 19, 1384(2), 197 - 203
Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus; Willis LB et al.; A screen for Rhizobium meliloti genes which improve the growth of Alcaligenes eutrophus on sucrose identified the first alcohol dehydrogenase gene (adhA) isolated from the Rhizobiaceae . R . meliloti adhA is constitutively expressed in A . eutrophus and has alcohol dehydrogenase activity . R . meliloti adhA mutants retain some alcohol dehydrogenase activity.

Appl Environ Microbiol, 1998 Jul, 64(7), 2644 - 51
Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain; Gerritse G et al.; Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions . A fed-batch fermentation process based on the secretion of the alkaline lipase from P . alcaligenes was developed . Due to the inability of P . alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively . The gene encoding the high-alkaline lipase from P . alcaligenes was isolated and characterized . Amplification of lipase gene copies in P . alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number . It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA . In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only . During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed . Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses . A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

Appl Environ Microbiol, 1998 Jul, 64(7), 2566 - 71
Aerobic mineralization of 2,6-dichlorophenol by Ralstonia sp . strain RK1; Steinle P et al.; A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France) . It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP)as the sole carbon and energy source at pH 7.5 and room temperature . The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations . At concentrations above 300 microM (50 mg.liter-1), 2,6-DCP increasingly inhibited its own degradation . The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes) . The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers . Substituents other than chlorine prevented degradation . The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors . The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 microM in continuous reactor culture supplied with H2O2 as an oxygen source . Lack of peroxide completely stopped further degradation of 2,6-DCP . Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity . A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 microM) with hydraulic residence times of 8 to 30 min.

Appl Environ Microbiol, 1998 Jul, 64(7), 2520 - 7
Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme; Peng X et al.; Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15) . The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway . A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA . Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA . An open reading frame encoding 334 amino acids was identified and designated ligZ . The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y . Noda, S . Nishikawa, K.-I . Shiozuka, H . Kadokura, H . Nakajima, K . Yano, Y . Katayama, N . Morohoshi, T . Haraguchi, and M . Yamasaki, J . Bacteriol . 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M . Kabisch and P . Fortnagel, Nucleic Acids Res . 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp . strain CA10 (S . I . Sato, N . Ouchiyama, T . Kimura, H . Nojiri, H . Yamane, and T . Omori, J . Bacteriol . 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E . L . Spence, M . Kawamukai, J . Sanvoisin, H . Braven, and T . D . H . Bugg, J . Bacteriol . 178:5249-5256, 1996) . The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage . Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase . LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion.

Appl Biochem Biotechnol, 1998 Spring, 70-72, 929 - 35
Accumulation of biodegradable copolyesters of 3-hydroxy-butyrate and 3-hydroxyvalerate in Alcaligenes eutrophus; Chua H et al.; Biodegradable copolyesters of 3-hydroxybutyrate-co-3-hydroxyvalerate (3HB-3HV) were produced by Alcaligenes eutrophus in a two-staged process, namely growth stage and nitrogen-deficient polyester-accumulation stage . When C5 was used as the sole carbon source, the copolyester contained 43 mol % of 3HV . A range of copolyesters with 0-43 mol % of 3HV could be produced by using a medium containing different concentration ratios of butyric acid C4 and C5 . Tm of PHB homopolymer was 177.6 degrees C and that of copolyester with highest 3HV mol fraction of 43% was 99.0 degrees C . C5 concentration in the medium could be an effective means to control the polymeric composition and mechanical properties of the copolyesters accumulated in A . eutrophus.

Appl Biochem Biotechnol, 1998 Spring, 70-72, 341 - 52
Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila; Umeda F et al.; Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus . H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P . acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA) . In this study, the pha genes of P . acidophila were cloned and a sequence analysis was carried out . A gene library was constructed using the cosmid vector pVK102 . A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16 . The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert . Subcloning was done . and the recombinant plasmid pEH74 was selected by hybridization with the A . eutrophus H16 pha genes . Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P . acidophila . The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined . The homologies of phaA, phaB, and phaC between P . acidophila and A . eutrophus H16 were 64.7, 76.1 and 56.6%, respectively.

J Bacteriol, 1998 Jun, 180(12), 3197 - 204
Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes; Schwartz E et al.; Alcaligenes eutrophus H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H2, supplying the organism with energy for autotrophic growth . The promoters of the structural genes for the SH and the MBH, PSH and PMBH, respectively, were identified by means of the primer extension technique . Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under condition which repressed hydrogenase synthesis . The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters . Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system . Both promoters were dependent on the minor sigma factor sigma 54 and on the hydrogenase regulator HoxA in vivo . PSH was stronger than PMBH under both heterotrophic and autotrophic growth conditions . The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures . The response regulator HoxA mediated low-level activation of PSH and PMBH in a heterologous system.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 791 - 6
Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335; Kang BS et al.; Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde . As our ongoing study to characterize biochemical and genetic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned . The C23O gene was localized in an 1.4-kb PstI fragment from A . eutrophus 335, and was expressed in E . coli HB101 . The C23O exhibited the highest aromatic ring-fission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol >> 4-methylcatechol > 3-methylcatechol, protocatechuate, 4-chlorocatechol > 3,4-dihydroxy-phenylacetate > 2,3-dihydroxybiphenyl . Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading frame (ORF) corresponding to the C23O gene was composed of 930 base pairs . A putative ribosome-binding sequence of AGGAG was found at about 10 nucleotides upstream the ORF which can encode a polypeptide of molecular weight 34 kDa consisting of 309 amino acid residues . The deduced amino acid sequence of C23O from A . eutrophus 335 exhibited the highest 59% identity with those of corresponding enzymes from Pseudomonas sp . CF600 (p VI150), P . putida HS1 (pDK1), and P . putida PpG7 (NAH7) . An alignment of amino acid sequences of extradiol-type dioxygenases including C23O from A . eutrophus 335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution.

Am J Infect Control, 1998 Apr, 26(2), 146 - 8
A pseudoepidemic of Alcaligenes xylosoxidans attributable to contaminated saline; Granowitz EV et al.; Alcaligenes xylosoxidans is an uncommon but serious cause of nosocomial epidemics . This report describes a cluster of two patients who underwent revision of hip arthroplasties and one patient who had a lumbar puncture . Cultures obtained during all three procedures showed A . xylosoxidans with similar antibiotic sensitivity patterns . An investigation found that saline used to process these specimens was contaminated with this organism.

Plasmid, 1998, 39(3), 187 - 95
IS1491 from Pseudomonas alcaligenes NCIB 9867: characterization and distribution among Pseudomonas species; Yeo CC et al.; A new insertion sequence, IS1491, has been cloned and sequenced . The 2489-bp IS1491 was isolated from a Pseudomonas alcaligenes NCIB 9867 (strain P25X) 4.8-kb PstI chromosomal fragment . IS1491 is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2 . Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21 family of insertion sequences, which include two IS elements previously isolated from P . alcaligenes P25X, IS1474, and IS1475 (Yeo, C . C., and Poh, C . L . (1997) . FEMS Microbiol . Lett . 149, 257-263) . Transposition assays showed that IS1491 transposed at a frequency of approximately 1.4 x 10(-6) . Transposition of IS1491 into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected . IS1491-like sequences were detected in total DNA of Pseudomonas putida NCIB 9869 (strain P35X), Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans, and Comomonas testosteroni by hybridization with IS1491 DNA .

Appl Microbiol Biotechnol, 1998 Mar, 49(3), 333 - 6
Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain; Fukui T et al.; The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated . When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A . eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase . In addition, a recombinant strain of A . eutrophus PHB-4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w) . The mole fraction of 3-hydroxyhexanoate units was 4-5 mol% whatever the structure of the triglycerides fed . The polyesters produced by the A . eutrophus strains from olive oil were 200-400 kDa (the number-average molecular mass) . The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A . eutrophus strains.

Appl Microbiol Biotechnol, 1998 Mar, 49(3), 258 - 66
In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum; Jossek R et al.; Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaECCv) was used to examine in vitro the specific synthase activity, turnover of R-(-)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions . The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase . Salts (MgCl2, CaCl2, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold . Specific PHA synthase activity was only partially affected by the added components . In general, a higher concentration of salt often inhibited the activity of PhaECCv without affecting the yield according to 3HB-CoA turnover . NAD+ and NADP+ (2 mM) inhibited PhaECCv completely, whereas NADH and NADPH did not . Macroscopic poly(3HB) granules were formed in vitro if PhaECCv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl2 was present . The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus . Scanning electron micrographs from the synthesized granules were obtained . The granules consisted of poly(3HB) that had a molar mass in the range (1-2) x 10(6) g/mol.

Mol Cells, 1998 Feb 28, 8(1), 62 - 7
Cloning of genes specifically expressed in rice embryogenic cells; Jung BK et al.; We have examined differences in gene expression pattern between embryogenic callus (EC) and nonembryogenic callus (NEC) derived from mature seed embryo of rice (Oryza sativa L . cv Donggin) . Three EC-specific transcripts were identified by differential display of amplified cDNAs . Specific expression of two partial cDNAs, designated as REC1 and REC2, respectively, was confirmed by a northern blot analysis . Partial nucleotide sequence of the clone REC1 showed no homology with any known genes, but partial amino acid sequence deduced from the clone REC2 exhibited 55-82% homology with nickel-cobalt-resistant proteins identified from a bacterium, Alcaligenes eutrophus CH34.

Biochemistry, 1998 Mar 3, 37(9), 3035 - 42
Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid; Saari RE et al.; The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 . This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate . TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine . In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation . The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA . The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases . Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D . Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate . The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions . On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D . The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events.

Biochem Mol Biol Int, 1998 Feb, 44(2), 235 - 43
Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes; Etchegaray A et al.; The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG . This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity . Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems . These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae . Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase.

Gene, 1998 Feb 27, 208(2), 243 - 51
Characterisation of the urease gene cluster in Bordetella bronchiseptica; McMillan DJ et al.; Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen . The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes . Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively . The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon . UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv . viciae, and may potentially be involved in nickel transport . A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon . BbuR shares homology with members of the LysR regulatory protein family . LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage . A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B . bronchiseptica . We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B . bronchiseptica from phagolysosomal damage . Comparison of the urease promoter regions of B . bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B . pertussis Tohama I revealed no differences in the ureD open reading frame between each species . A cluster of mutations in both B . pertussis and B . parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter . The inability of B . pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.

J Ind Microbiol Biotechnol, 1998 Jan, 20(1), 61 - 8
Effect of the siderophore alcaligin E on the bioavailability of Cd to Alcaligenes eutrophus CH34; Gilis A et al.; Alcaligin E, the siderophore of the heavy metal-resistant A . eutrophus strain CH34, was shown to interact with Cd and consequently affect its bioavailability and toxicity . The addition of alcaligin E markedly stimulated the growth in the presence of Cd of an alcaligin E-deficient CH34 derivative . Using bioluminescence assays, this effect could be assigned to a decrease in bioavailability of Cd in the presence of alcaligin E . However, Cd-uptake studies showed no influence of alcaligin E on the cellular concentration of Cd . Furthermore, by scanning electron microscopy, the morphology of precipitated Cd crystals was shown to be altered by alcaligin E . These data suggest that alcaligin E, besides its function in iron supply to the cell, provides a protection against heavy metal toxicity . A link between the A . eutrophus CH34 siderophore system and the czc-mediated Cd-efflux system is hypothesized.

Gene, 1998 Jan 19, 207(1), 9 - 18
Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10; Bergeron H et al.; The degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde (CAA), a toxic intermediate in the cells if it is not metabolized further by the NAD(+)-dependent CAA dehydrogenases . Here, we describe the cloning, sequence and expression in Escherichia coli of aldA, a plasmid-located CAA dehydrogenase-encoding gene of GJ10 as well as a chromosomal homolog, designated aldB . The DNA-predicted amino acid (aa) sequences of the two proteins (505 aa in AldA and 506 aa in AldB) are 84% identical . The cloned aldA and aldB genes were verified by their expression in the E . coli T7 polymerase/promoter and the pUC lac promoter systems . The expression level of AldA and its enzymatic activity towards CAA were both higher than those of AldB . In a hybrid construct, the 3'end of aldB was able to complement, although not completely, the corresponding portion of aldA to produce a functional gene . Both AldA and AldB proteins of GJ10 share the highest degree of sequence identity with an acetaldehyde dehydrogenase (ALDH) encoded by acoD of Alcaligenes eutrophus (77.3-78% identity) . Together with at least three other ALDHs of prokaryotic origin, these proteins apparently form a special class of ALDHs whose expressions are dependent on RpoN factors . By pulsed-field gel electrophoresis the 225-kb pXAU1 plasmid encoding aldA was shown to be linear.

Appl Environ Microbiol, 1998 Mar, 64(3), 930 - 9
Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds; Stoffels M et al.; This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100) . The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source . The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides . Two significant shifts in the bacterial community structure could be demonstrated . The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria . A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor . The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined . Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor . In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis . Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.

J Bacteriol, 1998 Mar, 180(5), 1023 - 9
Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus; Massanz C et al.; The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis . HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer . HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation . A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms . A deletion removing most of hoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide . While the hydrogenase dimer, produced by a strain deleted of hoxF and hoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH . Deletion of hoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity . Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.

Eur J Clin Microbiol Infect Dis, 1997 Dec, 16(12), 933 - 8
Bacteremia and respiratory involvement by Alcaligenes xylosoxidans in patients infected with the human immunodeficiency virus; Manfredi R et al.; Seven cases of Alcaligenes xylosoxidans bacteremia and/or respiratory disease in patients infected with the human immunodeficiency virus (HIV) are described . Reported only thrice previously in this setting, these bacterial complications occurred during different phases of HIV infection and were associated with leukopenia-neutropenia in four patients and a central vascular catheter in two . Although the majority of cases were diagnosed after day 3 of hospitalization, a distinct source of infection was never identified . In four patients with advanced underlying disease, a polymicrobial infection was present . In vitro resistance to aminoglycosides, first-generation cephalosporins, and aztreonam was identified, but treatment with fluoroquinolones, piperacillin, or an aminoglycoside in combination with either ceftazidime or pefloxacin was successful in all cases . The relevance of Alcaligenes xylosoxidans and related species of gram-negative non-glucose fermenting bacilli as opportunistic pathogens in the immunocompromised host and in the setting of HIV infection is briefly reviewed.

Arch Microbiol, 1998 Jan, 169(1), 52 - 60
The Alcaligenes eutrophus hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase, is required for heme biosynthesis during anaerobic growth; Lieb C et al.; The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite . When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis . With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment . Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa . In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified . The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN . HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX . Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A . eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector . Likewise, the A . eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth . Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A . eutrophus is nitrate-independent and repressed by oxygen.

Arch Microbiol, 1997 Dec, 168(6), 486 - 92
Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase; Henderson RA et al.; Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, DL-lactate or L-lactate . Unlike growing cultures, washed cells excreted significant amounts of pyruvate . The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production . The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA) . Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP . Citrate synthase was purified and shown to be a "large" form of the enzyme (Mr 227,000), comprising a single type of subunit (Mr 47,000) as found in several other gram-negative aerobes . The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed.

J Colloid Interface Sci, 1998 Jan 15, 197(2), 185 - 90
Biosorption of Heavy Metal Ions on Rhodobacter sphaeroides and Alcaligenes eutrophus H16
Seki H, Suzuki A, Mitsueda SI.
A fundamental study of the application of bacteria to the recovery of toxic heavy metals from aqueous environments was carried out . The biosorption characteristics of cadmium and lead ions were determined with purple nonsulfur bacteria, Rhodobacter sphaeroides and hydrogen bacteria, Alcaligenes eutrophus H16 that were inactivated by steam sterilization . A simplified version of the metal binding model proposed by Plette et al . was used for the description of metal binding data . The results showed that the biosorption of bivalent metal ions to whole cell bodies of the bacteria was due to monodentate binding to two different types of acidic sites: carboxilic and phosphatic-type sites . The number of metal binding sites of A . eutrophus was 2.4-fold larger than that of R . sphaeroides .

FEMS Microbiol Lett, 1998 Jan 15, 158(2), 159 - 65
Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region; Kwong SM et al.; The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined . An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length . The pRA2 replication region has two open reading frames (ORFs) . ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end . ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively . The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.

Appl Environ Microbiol, 1998 Feb, 64(2), 453 - 8
Alcaligenes eutrophus as a bacterial chromate sensor; Peitzsch N et al.; In Alcaligenes eutrophus CH34, determinants encoding inducible resistance to chromate (chr) and to cobalt and nickel (cnr) are located adjacent to each other on plasmid pMOL28 . To develop metal-sensing bacterial strains, a cloned part of plasmid pMOL28, which contains both determinants, was mutated with Tn5-lacZ . The chr::lacZ fusions were specifically induced by chromium; cnr was induced best by Ni2+ but was also induced by Co2+, Mn2+, chromate, Cu2+, Cd2+, and Zn2+ . The broad-host-range IncP1 plasmid pEBZ141, which contains a chr::lux fusion, was constructed . A . eutrophus AE104(pEBZ141), carrying a chr::lux transcriptional fusion, could be used as a biosensor for chromate when cultivated in glycerol as an optimal carbon source . Chromate and bichromate were the best inducers; induction by Cr3+ was 10 times lower, and other ions induced only a little or not at all . Interactions among induction of the chr resistance determinant, chromate reduction, chromate accumulation, and the sulfate concentration of the growth medium were demonstrated.

Appl Environ Microbiol, 1998 Jan, 64(1), 1 - 6
Repression of phenol catabolism by organic acids in Ralstonia eutropha; Ampe F et al.; During batch growth of Ralstonia eutropha (previously named Alcaligenes eutrophus) on phenol in the presence of acetate, acetate was found to be the preferred substrate; this organic acid was rapidly metabolized, and the specific rate of phenol consumption was considerably decreased, although phenol consumption was not abolished . This decrease corresponded to a drop in phenol hydroxylase and catechol-2,3-dioxygenase specific activities, and the synthesis of the latter was repressed at the transcriptional level . Studies with a mutant not able to consume acetate indicated that the organic acid itself triggers the repression . Other organic acids were also found to repress phenol degradation . One of these, benzoate, was found to completely block the catabolism of phenol (diauxic growth) . A mutant unable to metabolize benzoate was also unable to develop on benzoate-phenol mixtures, indicating that the organic acid rather than a metabolite involved in benzoate degradation was responsible for the repression observed.

Gene, 1997 Nov 20, 202(1-2), 103 - 14
Genetic characterization of insertion sequence ISJP4 on plasmid pJP4 from Ralstonia eutropha JMP134; Leveau JH et al.; Directly adjacent to the (tfdT-) tfdCDEF gene cluster for chlorocatechol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134, we identified a 0.9-kb DNA element, designated ISJP4, with the typical features of a bacterial insertion sequence . ISJP4 occurs as a single complete copy on plasmid pJP4 . About 9 kb away from this copy, in the tfdA-tfdS intergenic region, we found a 71-bp duplication of the ISJP4 right-hand extremity . In addition, we discovered a complete copy of ISJP4 on the chromosome of the R . eutropha JMP134 strain that we use routinely in our laboratory . We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R . eutropha JMP222 and JMP289, two previously pJP4-cured derivatives of JMP134 . By comparing both complete copies and their flanking regions, we could establish that element ISJP4 has a size of 915 bp and is bordered by 18-bp inverted repeats with one mismatch . Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of the IS4 family of bacterial insertion sequences, where it is mostly related to IS402 of Burkholderia cepacia . A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromosomal copy of ISJP4 . We could demonstrate that a piece of DNA that is flanked by two complete copies of ISJP4 can be transposed . Even more so, one complete ISJP4 plus its tfdA-tfdS intergenic remnant were sufficient to mediate transposition of intervening DNA . A possible role of ISJP4 in the formation of the tfd pathway genes will be discussed.

Appl Environ Microbiol, 1997 Dec, 63(12), 4765 - 9
Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli; Wang F et al.; Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium . One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium . By overexpressing an essential cell division protein, FtsZ, in recombinant E . coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium . A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E . coli in a defined medium . It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production . By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained . For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter . However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E . coli.

Mol Cells, 1997 Oct 31, 7(5), 620 - 9
Cloning and characterization of the regulatory genes phlR1 and phlR2 involved in phenol metabolism from Alcaligenes eutrophus JMP134; Kim Y et al.; One mutant (AEK201) of Alcaligenes eutrophus JMP134 deficient in phenol metabolism was isolated by transposon mutagenesis using pSUP2021, a suicide plasmid . The 14.5 kb EcoRI fragment containing Tn5 and flanking DNA was cloned from AEK201 and used to probe a gene bank of wild type by colony hybridization . All five positive cosmids isolated rendered AEK201 to grow on phenol . The data from subcloning revealed that a trans-acting factor encoded on the 2.3 kb SalI-HindIII fragment, which is common to all cosmids, allowed the mutant to restore three enzyme activities tested (phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase) . This fragment seems to act as a positive regulator on the entire phenol pathway . Another regulatory segment was subcloned from the 16.8 kb HindIII fragment on which phenol hydroxylase and catechol 2,3-dioxygenase activities were carried {Kim, Y., Ayoubi, P., and Harker, A . R . (1996) Appl . Environ . Microbiol . 62, 3227-3233} . The expression of phenol hydroxylase activity was entirely repressed in the presence of this segment in Pseudomonas aeruginosa PAO1c, but the enzyme activity was increased in A . eutrophus AEK301, suggesting that this trans-acting factor is both an activator and a repressor for phenol hydroxylase . Possible regulatory mechanisms for the phenol pathway in A . eutrophus JMP134 are discussed.

J Bacteriol, 1997 Nov, 179(22), 6871 - 9
New functions for the three subunits of the CzcCBA cation-proton antiporter; Rensing C et al.; The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport . The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit . The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation . Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter . Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins . Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein . Based on these data, a new working model for the function of the Czc system is discussed.

Microbiol Res, 1997 Sep, 152(3), 233 - 7
Rapid physiological characterization of microorganisms by biosensor technique; Riedel K et al.; Eleven microorganisms, Arxula adeninivorans LS3, Candida boidinii DSM 70034, Candida lactis-condensi DSM 70635, Pichia jadinii DSM 2361, Pichia minuta DSM 7018, Kluyveromyces lactis DSM 4394, Pseudomonas putida DSM 50026, Alcaligenes sp . DSM 30002, Arthrobacter nicotianae DSM 20123 as well as Issatchenkia orientalis DSM 70077 and Rhodococcus erythropolis DSM 311 were characterized by the sensor technique by injection of 30 different substrates and substrate mixtures . The obtained data which are based on the determination of respiratory rate of microorganisms are similar to physiological characteristics obtained with conventional methods . In comparison to these conventional methods the sensor technique works much more rapid and permits quantification of the data . Therefore, the described technique provides an alternative method for the characterization of microorganisms.

FEMS Microbiol Lett, 1997 Oct 15, 155(2), 179 - 84
Bacterial flavohaemoglobins: a consensus sequence and identification of a discrete enterobacterial group and of further bacterial globins; Membrillo-Hernandez J et al.; The amino acid sequences of haemoglobin-like proteins from the bacteria Alcaligenes eutrophus, Bacillus subtilis, Erwinia chrysanthemi, Escherichia coli, Vibrio parahaemolyticus, Vitreoscilla sp . and the yeast Saccharomyces cerevisiae were studied . Phylogenies based on distance and parsimony analysis showed that the eubacterial group can be easily distinguished from the other haemoglobin-like proteins . The construction of a consensus bacterial flavohaemoglobin based on the alignment of six bacterial and one yeast globins allowed the design of consensus primers to search for haemoglobin-like genes in other bacteria . PCR products of the expected size were found in Campylobacter jejuni, Salmonella typhimurium, Listeria monocytogenes, Rhizobium leguminosarum, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus.

Eur J Biochem, 1997 Sep 1, 248(2), 385 - 93
Isolation and characterization of D-threonine aldolase, a pyridoxal-5'-phosphate-dependent enzyme from Arthrobacter sp . DK-38; Kataoka M et al.; D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde . Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonas, and Pseudomonas, but not in yeasts or fungi . The enzyme was purified to homogeneity from one strain, Arthrobacter sp . DK-38 . The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa . This enzyme, as well as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme . Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity . The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation . A divalent cation was also essential for the thermal stability of the enzyme . The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity . The enzyme is strictly D-specific for the alpha-position, whereas it cannot distinguish between threo and erythro forms at the beta-position . Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different; the Km and Vmax values are 3.81 mM and 38.8 micromol x min(-1) x mg(-1) toward D-threonine, and 14.0 mM and 102 micromol x min(-1) x mg(-1) toward D-allothreonine . respectively . The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde . The enzyme also acts, in the same manner, on several other D-beta-hydroxy-alpha-amino acids, including D-beta-phenylserine, D-beta-hydroxy-alpha-aminovaleric acid, D-beta-3,4-dihydroxyphenylserine, and D-beta-3,4-methylenedioxyphenylserine.

Diagn Microbiol Infect Dis, 1997 Aug, 28(4), 173 - 8
Comparison of polymerase chain reaction and pulsed-field gel electrophoresis for the epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans in a burn unit; Lin YH et al.; Eighteen isolates of Alcaligenes xylosoxidans subsp . xylosoxidans were collected from clinical specimens of 15 patients in a burn unit and a plastic surgery ward over a 16-month period . Pulsed-field gel electrophoresis and polymerase chain reaction (PCR) were compared for the epidemiologic typing of these 18 isolates and fifteen epidemiologically unrelated strains . These 18 isolates demonstrated an identical fingerprint pattern and were easily distinguished from the 15 epidemiologically unrelated strains by pulsed-field gel electrophoresis typing and both enterobacterial repetitive intergenic concensus and repetitive extragenic palindrome-primed PCR fingerprinting . We conclude that pulsed-field gel electrophoresis analysis of XbaI-digested genomic DNA is a highly discriminatory and reproducible method for epidemiological typing of A . xylosoxidans subsp . xylosoxidans isolates . However, poor resolution due to frequent cutting in the smaller fragments (< 145.5 Kb) may lead to difficulty in interpretation . PCR is a rapid and highly discriminatory, but less reproducible, technique with occasional loss of major bands . The fingerprints produced by repetitive extragenic palindrome primed PCR had more intense bands and were easier to read than those produced by enterobacterial repetitive intergenic concensus-primed PCR in this study.

Biodegradation, 1997, 8(2), 113 - 24
Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site; Trajanovska S et al.; Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead . Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate . Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates . Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems . Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes . Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants . These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil . In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.

Plasmid, 1997, 38(2), 129 - 34
Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans; Chakraborty R et al.; Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans . The 1.4-kb fragment hybridized with IST2, an insertion sequence of T . ferrooxidans . The 1.2-kb fragment was cloned and sequenced . The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively . It displays 54.4% identity in 967 nucleotides of overlap with ISAE1 of Alcaligenes eutrophus . The IST445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T . ferrooxidans . In Southern blots of restricted genomic DNAs probed with IST445, each of the several strains of T . ferrooxidans gives a distinctive fingerprint . IST445 is present in the range of 10-20 copies per genome in the four strains studied.

J Biotechnol, 1997 Oct 2, 58(1), 33 - 8
Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose; Valentin HE et al.; A recombinant Escherichia coli strain has been developed that produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) when grown in complex medium containing glucose . This has been accomplished by introducing into E . coli DH5 alpha separate plasmids harboring the polyhydroxyalkanoate (PHA) biosynthesis genes from Ralstonia eutropha (formerly named Alcaligenes eutrophus) and the succinate degradation genes from Clostridium kluyveri, respectively . Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels reached 50% of the cell dry weight and contained up to 2.8 mol.% 4-hydroxybutyrate . The molecular weight of the polymer was 1.8 x 10(6).

Gene, 1997 Sep 1, 196(1-2), 209 - 18
The cis-diol dehydrogenase cbaC gene of Tn5271 is required for growth on 3-chlorobenzoate but not 3,4-dichlorobenzoate; Nakatsu CH et al.; The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined . The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III . Expression studies were carried out with Alcaligenes sp . strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60 . BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite . These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently . These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite . Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp . BR60 . The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains . o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins . These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).

Gene, 1997 Sep 1, 196(1-2), 69 - 74
Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications; Suarez A et al.; The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria . This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions . To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP . This modified gfp gene was also equipped with the strong translation signals of the atpE gene . Transfer of the mini-transposon into two different Pseudomonas spp . and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye . Individual GFP+ cells were readily observed by fluorescence microscopy.

Eur J Biochem, 1997 Aug 15, 248(1), 179 - 86
Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16; Bernhard M et al.; This study shows that the product of the hoxZ gene of Alcaligenes eutrophus H16 is a b-type cytochrome (cytochrome b(z)), which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H2-coupled respiration . The hoxZ product is not required for MBH translocation and H2-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride . The lack of cytochrome b(z) does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZdelta membranes . We show that the hoxZ product is a dihaem cytochrome b (haems with E(m7.0) of +10 mV and +166 mV) involved in H2-dependent electron transfer . We conclude that cytochrome b(z) of the A . eutrophus MBH complex is the link necessary for transfer of electrons from H2 to the ubiquinone pool and that it is required for attachment of MBH to the membrane.

FEMS Microbiol Lett, 1997 Sep 1, 154(1), 131 - 7
Monoclonal antibody against species-specific epitope of Pseudomonas aeruginosa Hsp60 protein cross-reacts with Pseudomonas stutzeri and other Pseudomonas species; Luneberg E et al.; In a previous study, we determined the epitope of the Pseudomonas aeruginosa Hsp60 heat shock protein which is recognized by the specific monoclonal antibody (MAb) 2528 . Subsequent investigations revealed a weak cross-reactivity of MAb 2528 with P . stutzeri, P . alcaligenes, P . mendocina and P . pseudoalcaligenes . To elucidate the molecular structure for these cross-reactions, we cloned the P . stutzeri hsp60 gene in Escherichia coli and determined the nucleotide sequence of the gene . In addition, the hsp60 gene of further Pseudomonas species was amplified and sequenced and amino acid substitutions within the epitope recognized by MAb 2528 were determined . The decapeptide QADIEARVLQ is unique to the P . aeruginosa Hsp60 protein, and cross-reaction of MAb 2528 reflects the phylogenetic relationship of Pseudomonas species as P . aeruginosa and all four cross-reacting species constitute a DNA homology group within the rRNA group I of the family Pseudomonadaceae, which belong to the gamma-subclass of the Proteobacteria.

J Biol Chem, 1997 Sep 12, 272(37), 23031 - 6
Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli; Eichhorn E et al.; The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation . The sequence relatedness of the TauD protein to the alpha-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an alpha-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J . R., Weiss, M . A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M . A., and Leisinger, T . (1996) J . Bacteriol . 178, 5438-5446) . TauD was overexpressed in E . coli to approximately 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure . The apparent Mr of 81,000 of the native protein and the subunit Mr of 37,400 were consistent with a homodimeric structure . The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine . The reaction also consumed equimolar amounts of oxygen and alpha-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction . The properties and amino acid sequence of this enzyme thus define it as a new member of the alpha-ketoglutarate-dependent dioxygenase family . The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at -20 degrees C for several weeks . Taurine (Km = 55 microM) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates . Among the cosubstrates tested, only alpha-ketoglutarate (Km = 11 microM) supported significant dioxygenase activity.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 411 - 8
Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli; Hein S et al.; The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli . Wild-type strains and other widely used non-recombinant strains of E . coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source . Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase . A 3.5-kb genomic SmaI/ApaI fragment from A . eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively . The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E . coli XL1-Blue . Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources . If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated . The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources . Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E . coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen . 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source . If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

Microbiology, 1997 Aug, 143 ( Pt 8), 2833 - 40
Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis; Yeo CC et al.; Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol . Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated Xin) which ranged in size from 2 kb to approximately 26 kb instability of DNA inserts in RP4::Xin hybrid plasmids was observed . The smallest insert present in a stable RP4::Xin6 hybrid plasmid, termed Xin6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA . Sequence analysis of Xin6 led to the discovery of an open reading frame with homology to the maturases of group II introns . The Xin6 insert also exhibited several features characteristic of a group II intron . These included the presence of the consensus sequence GUGYG at the 5' and and RAY at the 3' end of the intron . RNA secondary structure modelling of Xin6 also revealed the presence of perfectly conserved domains V and VI . Differences were detected in the Xin6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol . In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the Xin6 group II intron . This is the first reported group II intron isolated from Pseudomonas spp . and the first time that the mobility of a bacterial group II intron has been demonstrated.

J Bacteriol, 1997 Aug, 179(15), 4821 - 30
Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae; Fukui T et al.; A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) {P(3HB-co-3HHx)} from alkanoic acids or oils . The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) {i.e., the phaC gene from A . caviae}) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region . The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts . Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A . eutrophus mutant resulted in a decrease in the polyester content of the cells . Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A . caviae . The transconjugant of the A . eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.

J Biol Chem, 1997 Jul 4, 272(27), 17139 - 44
A Ni2+ binding motif is the basis of high affinity transport of the Alcaligenes eutrophus nickel permease; Eitinger T et al.; Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated . Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T . (1995) J . Bacteriol . 177, 1840-1843) . Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake . This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges . Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E . coli system . The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease . These mutations were reintroduced into the A . eutrophus wild-type strain . Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion.

Biotechnol Prog, 1997 Jul-Aug, 13(4), 347 - 54
Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures; Hahn JJ et al.; Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells . Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks . Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days) . Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots . However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins . Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture . These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable . A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation . Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA . The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium . This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.

Appl Environ Microbiol, 1997 Jul, 63(7), 2765 - 70
Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate; Ampe F et al.; During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (mu = 0.56 h-1) than when a single substrate was used (mu = 0.51 h-1 for benzoate alone and 0.44 h-1 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol) . Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol.g-1.h-1) . It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the beta-ketoadipate pathway . Indeed, the in vitro beta-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction.

Arch Microbiol, 1997 Jul, 168(1), 33 - 8
Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134; Prucha M et al.; An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134 and that exhibited muconolactone isomerizing activity was purified to homogeneity . The enzyme, involved in the isomerization of 3-methylmuconolactone had a high degree of sequence similarity with muconolactone isomerase of Alcaligenes eutrophus JMP 134 and other previously described muconolactone isomerases of the 3-oxoadipate pathway . Kinetic analysis showed that the enzyme has a substrate spectrum and a reaction mechanism similar to those of the muconolactone isomerase, but that it has distinct kinetic properties.

Biol Pharm Bull, 1997 Jun, 20(6), 667 - 9
Microbial contamination of antiseptic-soaked cotton balls; Oie S et al.; We investigated microbial contamination of in-use antiseptics at a hospital . No microbial contamination was observed in 70 samples of 0.02% benzalkonium chloride solution (500-ml volume), 70 samples of 1% titratable I2 povidone-iodine solution (250-ml volume), or 15 samples of 0.1% ethacridine lactate solution (500-ml volume) during use in reduced amounts . Nor was any microbial contamination observed in 70 samples of cotton balls soaked in 1% titratable I2 povidone-iodine solution in canisters or cotton gauze soaked in 70% (w/v) ethanol solution in canisters . However, among 70 samples of cotton balls soaked in 0.02% benzalkonium chloride solution in canisters, 6 (8.6%) were contaminated with 10(4) to 10(6) viable cells/ml . The microbial species detected were glucose non-fermentative bacilli such as Alcaligenes xylosoxidans and Pseudomonas putida . The contaminants obtained from cotton balls soaked in 0.02% benzalkonium chloride solution did not proliferate in that solution or in distilled water but showed rapid growth in the cotton balls soaked in either of these liquids . These findings suggested that benzalkonium chloride solution tends to become contaminated when cotton balls are immersed . Therefore, cotton balls soaked in benzalkonium chloride solution are not recommended as an antiseptic . When no other choice is available, the cotton balls should be soaked in benzalkonium chloride solution at the time of usage.

Biochem J, 1997 Jun 1, 324 ( Pt 2), 511 - 6
pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans; Abraham ZH et al.; The first quantitative characterization of the interaction of NO2(-) with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described . Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface . Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8 . Equilibrium gel filtration showed that binding of NO2(-) to the oxidized NiR was also pH-dependent . At pH 7.5, no binding was detectable, but binding was detectable at lower pH values . At pH 5.2, the concentration-dependence for binding of NO2(-) to the enzyme showed that approx . 4.1 NO2(-) ions bound per trimeric NiR molecule . Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2(-) ions per trimer . When corrected for this binding, a value of 3 NO2(-) ions bound per trimer of NiR, equivalent to the type-2 Cu content . The NO2(-)-induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity . Binding constants for NO2(-) at a single type of site, after allowing for the non-specifically bound NO2(-), were 350+/-35 microM (mean+/-S.E.M.) at pH 7.5 and <30 microM at pH 5.2 . The apparent Km for NO2(-) with saturating concentrations of dithionite as reductant was 35 microM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2(-) binds.

Appl Environ Microbiol, 1997 Jun, 63(6), 2266 - 72
Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria; Kamagata Y et al.; 2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure . By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D . Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria) . All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose {PTYG} or 0.1 x Luria broth {LB} medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders . Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth . PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene . This gene was transmissible to A . eutrophus JMP228 carrying a plasmid with a mutant tfdA gene . The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates . These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.

Microbiology, 1997 May, 143 ( Pt 5), 1691 - 9
Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad; Heiss G et al.; A new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity . A 1.6 kb DNA fragment was sequenced and an ORF of 954 bp identified . Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (MpcII) from Alcaligenes eutrophus JMP 222 . Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs . The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined . The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene . Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage . The enzyme required Fe2+ for maximal activity . In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.

Eur J Biochem, 1997 Apr 15, 245(2), 441 - 8
C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus; Massanz C et al.; Formation of enzymatically active {NiFe} hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits . Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Ni-containing subunit of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus H16 . Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis . This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme . In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon . Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni . Both mutant proteins contained only traces of H2-activating functions . H2-dependent reduction of NAD and benzylviologen, and D2/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained . These results allow the following conclusions: the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly.

FEMS Microbiol Lett, 1997 Apr 15, 149(2), 257 - 63
Characterization of IS1474, an insertion sequence of the IS21 family isolated from Pseudomonas alcaligenes NCIB 9867; Yeo CC et al.; A new insertion sequence (IS) designated IS1474 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) . IS1474 is a 2632 bp element which showed a characteristic IS structure with 12 bp inverted repeats (IRs) flanking a 2608 bp central region . IS1474 contained four open reading frames (ORF1-ORF4), two in each orientation . Similarities were detected between ORF1 and ORF2 and the putative transposases of the IS21 family . Sequences upstream from IS1474 were found to display up to 89% homology with IS53 from Pseudomonas syringae suggesting that IS1474 had inserted into another related IS element designated IS1475 . An open reading frame, ORF5, located at the junction of IS1474 and IS1475, showed similarities with the IstB protein of IS21 and could possibly be the transposase subunit of IS1475 . Transposition assays showed that IS1474 transposed at a relatively low frequency leading to cointegration with target plasmids . Hybridization studies showed that IS1474 is present in at least 13 copies in the chromosome of P25X and one copy on its endogenous plasmid.

Structure, 1997 Apr 15, 5(4), 497 - 507
Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp; Tarricone C et al.; BACKGROUND: The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species . The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties . Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function . This ability of VtHb to improve the growth properties of E . coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics . Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains . We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization . RESULTS: The VtHb three-dimensional structure conforms to the well known globin fold . Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7-E10 (defined according to the standard globin fold nomenclature) do not adopt the usual alpha-helical conformation, thus locating Gln53(E7) out of the heme pocket . Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8) . The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart . CONCLUSIONS: The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb . The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.

Microbiology, 1997 Apr, 143 ( Pt 4), 1271 - 86
Genes encoding the NAD-reducing hydrogenase of Rhodococcus opacus MR11; Grzeszik C et al.; The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far . The genes of the soluble hydrogenase were localized on a 7.4 kbp Asnl fragment of the linear plasmid pHG201 via heterologous hybridization . Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -W and ORF7 . The six latter ORFs belong to the gene cluster of the soluble hydrogenase . Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16 . The genes hoxF, -U, -Y and -H encode the subunits alpha, gamma, delta and beta, respectively . The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the beta subunit . Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known . ORF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria . Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R . opacus MR11 indicated that the hydrogenase genes are under control of a delta 70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level . Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes . Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E . coli, no active enzyme was detectable.

Int J Syst Bacteriol, 1997 Apr, 47(2), 522 - 8
Phylogeny of Thiobacillus cuprinus and other mixotrophic thiobacilli: proposal for Thiomonas gen . nov; Moreira D et al.; The complete 5S and 16S ribosomal DNA (rDNA) sequences of the facultatively chemolithotrophic bacterium Thiobacillus cuprinus and results of a comparison of these sequences with homologous sequences from several proteobacterial species supported affiliation of T . cuprinus with the beta 1 subgroup of the Proteobacteria . T . cuprinus, Thiobacillus intermedius, Thiobacillus perometabolis, and Thiobacillus thermosulfatus form a phylogenetic cluster that comprises some of the thiobacilli capable of mixotrophic growth . This cluster is related to some pseudomonads and Alcaligenes species belonging to the beta subclass . In addition, a low-frequency restriction fragment analysis (LFRFA) of some mixotrophic thiobacilli and some related species was carried out by using pulsed-field gel electrophoresis (PFGE) to determine the SpeI and XbaI macrorestriction patterns and genome sizes of these organisms . The correlation of the LFRFA results and the 16S rDNA analysis results and the usefulness of the two analyses are discussed . The PFGE fingerprints suggested that Thiobacillus sp . strain ATCC 27793 is related to T . intermedius rather than to T . perometabolis, as described previously . The distinctive characteristics of the mixotrophic species analyzed in this work, their phylogenetic relatedness, and their physiological differences from other groups belonging to the Proteobacteria, including other thiobacilli, suggest that these organisms should be transferred to a new genus, the genus Thiomonas gen . nov.

J Bacteriol, 1997 Apr, 179(8), 2595 - 607
Molecular characterization of genes of Pseudomonas sp . strain HR199 involved in bioconversion of vanillin to protocatechuate; Priefert H et al.; The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp . strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively . These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp . strain HR199 genomic library in the cosmid pVK100 . The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230 . The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively . The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh . The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp . strain ATCC 19151 (F . Brunel and J . Davison, J . Bacteriol . 170:4924-4930, 1988) . The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity to aldehyde dehydrogenases from different sources . The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases . Escherichia coli strains harboring fragment E230 cloned in pBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E . coli . High expression of vdh in E . coli was achieved with HE35 cloned in pBluescript SK- . The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture . Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.

Gene, 1997 Mar 25, 188(1), 91 - 4
A Bacillus subtilis locus encoding several gene products affecting transport of cations; Sturr MG et al.; A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K+ uptake-deficient Escherichia coli strain TK2420 . Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K+ . Insertional mutagenesis and subcloning identified a single gene responsible for the complementation . This gene coded for an apparent homolog of E . coli TrkA . Sequence analysis of the cloned region also revealed three additional open reading frames . These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na+ resistance in E . coli NM81 (delta nhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.

Arch Pediatr, 1997 Mar, 4(3), 260 - 2
{Neonatal meningitis caused by Alcaligenes xylosoxydans}; Bruel H et al.; BACKGROUND: Neonatal meningitis due to Alcaligenes xylosoxydans is exceptional; its diagnosis and treatment may be difficult . CASE REPORT: A neonate born at 42 weeks of GA to a mother who worked as a nurse in an intensive care unit was admitted on day 2 for a severe infection . Her cerebrospinal (CSF) contained 1,970 white cell/mm3, polymorphonuclear in majority: direct examination failed to show any germ but the CSF and blood cultures were positive for Alcaligenes xylosoxydans, a strain that was resistant to the initially given antibiotics . The patient was given piperacillin, 300 mg/kg/d for 21 days and completely cured with a follow-up of 6 months . CONCLUSIONS: This case shows that lombar puncture can be necessary in evaluating early neonatal sepsis; it also shows usefulness of piperacillin in some cases.

Appl Environ Microbiol, 1997 Mar, 63(3), 851 - 6
Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant; Morais PV et al.; Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant . These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system . The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains . One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P . fluorescens . The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System . Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile . This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P . aeruginosa or to any of the other type strains tested . Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P . alcaligenes . This study shows that characteristics similar to P . aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.

J Bacteriol, 1997 Mar, 179(5), 1655 - 63
A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species; Lenz O et al.; Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus . Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ . The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria . hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of {NiFe} hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU) . hoxJ encodes a protein with typical consensus motifs of histidine protein kinases . Introduction of the complete set of genes on a broad-host-range plasmid into A . eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2 . This repression was released by truncation of hoxJ . H2-dependent hydrogenase gene transcription is a typical feature of A . hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A . eutrophus H16 . Disruption of the A . hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source . RNA dot blot analysis showed that HoxJ functions on the transcriptional level . These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.

J Clin Microbiol, 1997 Mar, 35(3), 614 - 9
Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium; Henry DA et al.; We evaluated 819 isolates referred to us as "Burkholderia cepacia" from cystic fibrosis (CF) clinics and research laboratories from five countries; 28 (3.4%) were not B . cepacia . A further 12 (1.5%) organisms appeared to be other Burkholderia species, but identification could not be confirmed by conventional means . The most prevalently misidentified organisms were Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Comamonas acidovorans . Many of these organisms grew on oxidation-fermentation polymyxin-bacitracin-lactose (OFPBL) and Pseudomonas cepacia agars, selective media currently used for B . cepacia isolation . We developed a new medium, B . cepacia selective agar (BCSA), which is more enriched for the growth of B . cepacia yet which is more selective against other organisms than currently available selective agars . A total of 190 of 191 (99.5%) isolates of B . cepacia from patients with CF grew on BCSA without vancomycin, whereas 100% grew on OFPBL agar and 179 (94.2%) grew on P . cepacia agar . Of 189 other gram-negative and gram-positive organisms tested, 10 (5.3%) grew on BCSA without vancomycin . The addition of vancomycin to BCSA lowered the false positivity rate to 3.7% without further inhibition of B . cepacia . The false positivity rates for OFPBL and P . cepacia agars were 19.6 and 13.8%, respectively . Isolates of B . cepacia from CF patients grew most quickly on BCSA, with 201 of 205 (98.0%) being readily visible within 24 h, whereas 182 (88.8%) grew on OFPBL agar and 162 (79.0%) grew on P . cepacia agar within 24 h . We propose that the use of BCSA will allow investigators to overcome many of the difficulties associated with the identification of B . cepacia and should be considered for use as a primary isolation agar for specimens from patients with CF.

Infect Control Hosp Epidemiol, 1997 Feb, 18(2), 132 - 4
Evidence for the genetic unrelatedness of nosocomial Alcaligenes xylosoxidans strains in a pediatric hospital; Benaoudia F et al.; Restriction fragment-length polymorphism of ribosomal DNA and random amplified polymorphic DNA analysis were used to study the relationships among nine isolates of Alcaligenes xylosoxidans obtained from seven children in a pediatric hospital . All the children harbored only genotypically unrelated strains, thus excluding a common source of exposure or patient-to-patient strain transfer.

Can J Microbiol, 1997 Feb, 43(2), 202 - 5
The 2,4-dichlorophenol hydroxylase of Alcaligenes eutrophus JMP134 is a homotetramer; Farhana L et al.; 2,4-Dichlorophenol hydroxylase (DCP-hydroxylase) is a key enzyme in the pathway for degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in many bacteria . In Alcaligenes eutrophus JMP134, DCP-hydroxylase was reported to consist of two dissimilar types of subunit of 66 and 45 kDa, a structure which is different from that in other bacteria . Using a different procedure involving affinity purification and ion-exchange chromatography, we have purified active enzyme from JMP134 and show that it has a native molecular mass of approximately 245 kDa and consists of a single type of subunit of 66 kDa, similar to all other flavoprotein monooxygenase enzymes . A 45-kDa polypeptide, found in partially purified enzyme preparations, was not required for enzyme activity but had some serologic and N-terminal amino acid sequence similarity to the 66-kDa enzyme subunit.

Electrophoresis, 1997 Feb, 18(2), 202 - 4
Electrophoretic analysis of cyanide depletion by Pseudomonas alcaligenes; Zaugg SE et al.; Bacterial-facilitated depletion of cyanide is under development for remediation of heap leach operations in the gold mining industry . Capillary electrophoresis was found to be a powerful tool for quantifying cyanide depletion . Changes in cyanide concentration in aqueous suspensions of Pseudomonas alcaligenes bacteria and cyanide at elevated pH were easily monitored by capillary electrophoresis . The resulting data can be used to study rates of cyanide depletion by this strain of bacteria . Concentrations of these bacteria at 10(5) cells/mL were found to reduce cyanide from 100 ppm to less than 8 ppm in four days . In addition, other ions of interest in cyanide metabolism, such as formate, can be simultaneously analyzed . Direct UV detection of cyanide at 192 nm further simplifies the analytical method for these ions.

Appl Microbiol Biotechnol, 1997 Feb, 47(2), 140 - 3
Molecular mass of poly{(R)-3-hydroxybutyric acid} produced in a recombinant Escherichia coli; Kusaka S et al.; Poly{(R)-3-hydroxybutyric acid} (PHB) was produced at 37 degrees C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phb-CAB genes in Luria-Bertani media containing glucose at 10-30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied . PHB polymers accumulated within cells while glucose was present in the medium . The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E . coli, ranging from 0.5 MDa to 20 MDa . Under specific conditions (pH 6.0), E . coli produced PHB with an extremely high molecular mass (20 MDa) . It has been suggested that a chain-transfer agent is generated in E . coli cells during the accumulation of PHB.

Mol Microbiol, 1997 Feb, 23(3), 493 - 503
Two-component regulatory system involved in transcriptional control of heavy-metal homoeostasis in Alcaligenes eutrophus; van der Lelie D et al.; The czc determinant, which mediates resistance to Co2+, Zn2+ and Cd2+ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described) . Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR) . Transcription of czcCBA and of the URR was regulated by heavy-metal cations . DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR . Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion . Expression of both genes is also regulated by heavy metals . The genes of the URR yielded three mRNAs of approx . 1200, 500 and 200 nucleotides, respectively . The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.

Mol Gen Genet, 1997 Jan 27, 253(4), 499 - 506
Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds; Merlin C et al.; Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzoate derivatives . We constructed a cosmid library of Tn4371 DNA . The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon . Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp . KKS102, i.e . the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD . Consensus sequences for sigma54-associated RNA polymerase were found upstream of bphA1 and bphEGF . Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking sigma54 . In contrast to wild-type H16 exconjugants, the sigma54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371 bph gene expression on sigma54 . The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium . Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC.

Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 36 - 41
A novel transporter involved in cobalt uptake; Komeda H et al.; Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 . We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles . The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells . Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains . nhlF expression in R . rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni . The expression of both nhlF and nhlBA in R . rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression . These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.

Chin J Biotechnol, 1997, 13(1), 25 - 30
Studies on multiple forms of maltotetraose-forming amylase from Alcaligenes sp; Zhu J et al.; Zymograms of the cultural supernatant of Alcaligenes sp . showed three bands, the major one being G4A-1 and the minor two, G4A-2 and G4A-3 . Based on the electrophoretic homogeneity of the purified three bands and the enzymatic activities identified by a thin layer chromatography of the soluble starch hydrolysates, all the three bands were confirmed to be maltotetraose-forming amylase but in multiple forms . Neither glycosidase nor protease activities could be detected in the culture (only very weak protease activities were observed at 48 hours after cultivation), which indicate that the two enzymes were not involved in the amylase multiple-form formation . Only the relative amount of the two minor bands (but not the multiple-form pattern) was changed when the initial pH of the medium varied from 6.5-8.5 . An addition of 0.3% glucose raised the yield of G4A-2 and G4A-3.

Plasmid, 1997, 37(3), 189 - 98
Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain; Charnock C; The species type strain of Pseudomonas alcaligenes contains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively . Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related . pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C . This suggested that pECB3 is especially rich in these bases . Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1 . A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kb Pseudomonas-specific plasmid isolated from Pseudomonas syringae pv . savastonoi . The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced . Introduction of the putative replicon into the Escherichia coli plasmid pUC19 created a recombinant vector able to replicate in both E . coli and Pseudomonas spp . Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1--a finding supported by sequencing studies . Attempts at plasmid curing were unsuccessful . A phenotypic comparison with a non-plasmid-harboring strain of P . alcaligenes, based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth . Transformation of the non-harboring strain with pECB1-3, followed by selection on a minimal medium containing benzoate, gave no colonies . The advantages gained by possession of pECB1-3, if any, are at present unknown.

Mikrobiologiia, 1997 Jan-Feb, 66(1), 14 - 8
{Bacterial cell membrane ATPase in assessing heavy metal toxicity}; Gruzina TG et al.; Sensitivity of membrane ATPase to heavy metals was studied in bacterial stains Bacillus cereus ATCC 14579, Bacillus cereus B4368, and Alcaligenes eutrophus CH34 was studied . The inhibition effects of metals on membrane ATPase ranged in the same order (Au > Cu > Zn > Co > Mn) as their effects on the growth of cultures . It is suggested that membrane ATPase is one of the targets for the action of heavy metal ions within the microbial cell and, therefore, its activity may serve as an indicator of their toxicity.

Plasmid, 1997, 37(1), 22 - 34
Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality; Taghavi S et al.; We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb . To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20-30 kb inserted DNA . Subsequently, a cosmid walking strategy, combined with BamHI or EcoRI restriction analysis and hybridization, was used to construct the restriction maps for both enzymes . On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, accounting for a total size of approximately 180 kb . We also analyzed several rearranged derivatives of pMOL28 that were obtained after a process of temperature-induced mortality and mutagenesis (TIMM), which is characteristic for Alcaligenes eutrophus CH34 and related strains . The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivative of pMOL28 obtained after TIMM, were constructed . By comparing the pMOL28 and pMOL50 maps, at least two transposable elements were identified which participated in the formation of pMOL50 from pMOL28 during TIMM . These transposable elements were IS1086, which was recently sequenced, and a new element named IS1089, which is located on the 44-kb inserted DNA fragment in pMOL50 . Partial sequencing of IS1089 revealed similarity of this element with IS1071 of the chlorobenzoate catabolic transposon Tn5271 of Alcaligenes sp . BR60.

Nat Biotechnol, 1997 Jan, 15(1), 63 - 7
PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo; Sim SJ et al.; A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site . Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E . coli DH5 alpha and analyzed for polyhydroxybutyrate production . The molecular weight of polymer isolated from recombinant E . coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E . coli containing the native A . eutrophus operon . A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon . Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Int J Syst Bacteriol, 1997 Jan, 47(1), 207 - 10
A phylogenetic analysis of aerobic polychlorinated biphenyl-degrading bacteria; Williams WA et al.; Several bacterial isolates were characterized based on their abilities to degrade specific polychlorinated biphenyls (PCBs) and their 16S rRNA gene sequences . The members of one group of bacteria consisting of Alcaligenes species, including the PCB-degrading bacterium Alcaligenes eutrophus H850, had strong abilities to degrade a broad range of PCBs but not the di-para-chlorine-substituted congeners . The members of another group, which included the PCB-degrading bacterium originally classified as Corynebacterium sp . strain MB1, had strong abilities to degrade di-para-chlorine-substituted PCBs . These bacteria were most likely different members of Rhodococcus species.

J Bacteriol, 1996 Dec, 178(23), 6824 - 32
The tfdR gene product can successfully take over the role of the insertion element-inactivated TfdT protein as a transcriptional activator of the tfdCDEF gene cluster, which encodes chlorocatechol degradation in Ralstonia eutropha JMP134(pJP4)
Leveau JH, van der Meer JR.
The tfdT gene is located upstream of and transcribed divergently from the tfdCDEF chlorocatechol-degradative operon on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 . It is 684 bp long and encodes a 25-kDa protein . On the basis of its predicted amino acid sequence, the TfdT protein could be classified as a LysR-type transcriptional regulator . It has the highest degree of similarity with the proteins TcbR, ClcR, and TfdR, which are involved in the regulation of chloroaromatic breakdown . Despite this homology, the TfdT protein failed to activate the expression of its presumed target operon, tfdCDEF . This failure could be attributed to the inability of TfdT to bind the tfdC promoter region, an absolute requirement for transcriptional activation . Sequence analysis downstream of the tfdT gene revealed the presence of an insertion element-like element . We postulate that this element disrupted the tfdT open reading frame, leading to a premature termination and the production of a truncated, disfunctional TfdT protein . As an alternative to the inactivated TfdT protein, we propose that the product of the tfdR gene (or its identical twin, tfdS), located elsewhere on plasmid pJP4, can successfully take over the regulation of tfdCDEF expression . The TfdR protein was capable of binding to the tfdC promoter region and activated tfdCDEF gene expression by a factor of 80 to 100 when provided in cis as a tfdR-tfdCDEF hybrid regulon . Although to a lesser extent, induction of tfdCDEF expression was also observed when no functional TfdR protein was provided, implying cross-activation by chromosomally encoded regulatory elements in R . eutropha JMP134(pJP4).

J Bacteriol, 1996 Dec, 178(23), 6714 - 9
Regulation of CO2 assimilation in Ralstonia eutropha: premature transcription termination within the cbb operon; Schaferjohann J et al.; In the facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), most genes required for CO2 assimilation via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectively, of strain H16 . These operons are subject to tight control exerted by a promoter upstream of the 5'-terminal cbbL gene that is regulated by the activator CbbR . The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK . Nevertheless, marked differential expression of the promoter-proximal ribulose-1,5-bisphosphate carboxylase-oxygenase genes cbbLS and the remaining distal genes occurs within the operons . Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression . Nuclease S1 mapping identified a major 3' end and a minor 3' end of the relatively stable cbbLS partial transcript just downstream of this structure . Moreover, operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional termination downstream of cbbS rather than increased the segmental stability of the cbbLS transcript . Premature transcription termination thus represents an important mechanism leading to differential gene expression within the cbb operons.

J Clin Microbiol, 1996 Dec, 34(12), 2909 - 13
PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams; Senda K et al.; We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods . A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime . Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan . These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse . The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S . marcescens AK9373 was also well conserved among blaIMP-positive strains . These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element . Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.

Mikrobiologiia, 1996 Nov-Dec, 65(6), 790 - 5
{Growth and substrate utilization by bacterial lawn on the agar surface: experiment and one-dimensional distributed model}; Belova SE et al.; Cell mass dynamics of the lawns formed by Pseudomonas fluorescens and Alcaligenes sp . and the distribution profiles of the residual substrate in the agar layer were monitored . After one or two days of culturing, the concentration of pyruvate in the top agar layer adjacent to the lawn dropped below the level of detection, and, from this moment, the substrate was supplied to the lawn by diffusion from underlying agar layers . Diffusion of pyruvate in noninoculated bilayered agar was found to follow Fick's equation with the diffusion coefficient of 0.042 cm2/h . A distributed mathematical model adequately describing the growth of bacterial lawn was developed based on the diffusion equation and the Monod-Herbert kinetic model . Notable distinctions between the two cultures studied were revealed: pseudomonads had higher growth and death rates than Alcaligenes sp . and exhibited a greater affinity for the substrate.

Eur J Clin Microbiol Infect Dis, 1996 Nov, 15(11), 876 - 9
Molecular epidemiology of Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans in a cystic fibrosis center; Vu-Thien H et al.; Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans have been isolated with increasing frequency from the sputum of patients with cystic fibrosis in a pediatric hospital . In 1994-95, 27 of 120 patients were persistently colonized, 17 with Burkholderia cepacia, eight with Alcaligenes xylosoxidans, and five with Stenotrophomonas maltophilia . Genotyping of 220 clinical isolates revealed that most of the Burkholderia cepacia strains were clonally related, suggesting either cross-infection or a common source of exposure . In contrast, neither cross-infection nor a common source of exposure appear to have occurred in the cases of Alcaligenes xylosoxidans or Stenotrophomonas maltophilia.

Microbiology, 1996 Nov, 142 ( Pt 11), 3283 - 93
RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway; Springael D et al.; Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid . The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids . Three Bph+ prime plasmids were transferred from A . eutrophus to Escherichia coli and back to A . eutrophus without modification of the phenotype . The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane . Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning . Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A . eutrophus and the catabolic genes were expressed in those hosts . Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp . B13 allowed the recipient to mineralize 3-chlorobiphenyl . Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources . 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.

Gene, 1996 Oct 10, 175(1-2), 109 - 13
IS1394 from Pseudomonas alcaligenes N.C.I.B . 9867: identification and characterization of a member of the IS30 family of insertion elements; Yeo CC et al.; A new insertion sequence designated IS1394 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) by entrapment in plasmid pUCD800 which carries the Bacillus subtilis sacB and sacR genes . The 1100-bp sequence contains 27-bp inverted repeats with 4 bp mismatch and has one long open reading frame, spanning 92.1% of the entire IS . The deduced 338 amino-acid sequence demonstrated homology (varying from 65% to 78% similarity and 36-67% identity) to transposases encoded by the IS30 family of IS elements . Comparison of four different IS-sacB junction sequences showed that IS1394 generated 3-bp direct repeats of target DNA upon insertion . IS1394 is present in at least 10 copies in the P25X genome but none was detected in its endogenous plasmid pRA2 . Hybridization experiments revealed that the distribution of IS1394 is limited to closely related strains, being present in three copies in Pseudomonas putida NCIB 9869 (P35X) and two copies in Pseudomonas alcaligenes ATCC type strain (ATCC 14904).

Biotechnol Appl Biochem, 1996 Oct, 24 ( Pt 2), 95 - 100
Hydrogenase encapsulation into red blood cells and regeneration of electron acceptor; Axley MJ et al.; Biochemical decompression has been proposed as a method for reducing the amount of time required for deep-sea divers to return to the surface . Divers breathing H2/O2 mixtures would be presented with hydrogenase enzyme, and decompression would be accelerated by means of the enzymic removal of excess H2 from the tissues . We have studied FAD as a hydrogenase electron acceptor that is capable of transferring electrons derived from H2 oxidation directly to O2 . Kinetic activity constants for the soluble hydrogenase from the bacterium Alcaligenes eutrophus H16 were determined with FAD, FMN and riboflavin as electron acceptors, and these values were compared with those obtained with the physiological electron acceptor NAD+ . The Michaelis constants (K(m)) were similar for FAD, FMN and NAD . However, the maximal catalytic-centre activity (Kcat) was much lower for the flavins, and the catalytic efficiency (Kcat/K(m)) with FAD was 1/20th the value for NAD+ . After enzyme-catalysed FAD reduction to FADH2, the FAD could be regenerated by addition of O2 and reduced again by the enzyme in the presence of H2 . Thus FAD served as a regenerable electron shuttle between H2 and O2 . H2O2, a by-product of FADH2 oxidation by O2, inhibited the enzyme . Much greater inhibition was observed with the reduced form of the enzyme . Active hydrogenase was efficiently encapsulated into human and pig red blood cells . Hydrogen consumption was seen with lysed carrier cells, but was demonstrated with unlysed carrier cells only when FAD was co-encapsulated along with enzyme . These results demonstrate that red blood cells encapsulating hydrogenase and FAD act as a system for continuous H2 consumption in a mammalian tissue without addition of exogenous factors, and such cells may provide a biotherapeutic method for reducing the risk and treatment of decompression sickness.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1042 - 55
Description of chlorophenol-degrading Pseudomonas sp . strains KF1T, KF3, and NKF1 as a new species of the genus Sphingomonas, Sphingomonas subarctica sp . nov; Nohynek LJ et al.; Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria . These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol% . The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid . These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied . In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40% . Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points . On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica . The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria . The xenobiotic compound degraders Alcaligenes sp . strain A175 and Pseudomonas sp . strain BN6 were identified as members of species of the genus Sphingomonas.

Arch Microbiol, 1996 Oct, 166(4), 245 - 51
Identification of cbbBc as an additional distal gene of the chromosomal cbb CO2 fixation operon from Ralstonia eutropha; Bommer D et al.; Ralstonia eutropha (formerly Alcaligenes eutrophus) strain H16 possesses two highly homologous cbb operons encoding most of the Calvin cycle enzymes . One copy of the operon is located on the chromosome, the other on the megaplasmid pHG1 of the organism . Sequence analysis of the region downstream of the presumptive 3'-terminal gene (cbbAc) of the chromosomal operon revealed the presence of an open reading frame comprising 2,274 bp . Evidence is presented that this open reading frame is an additional distal gene (designated cbbBc) of the operon . In contrast to the other genes of the operon, cbbBc is not duplicated in the plasmid-borne operon . The deduced amino acid sequence of the cbbBc product (757 residues, molecular mass 83.17 kDa) showed the highest similarity to the large catalytic subunits of various bacterial formate dehydrogenases (FDH), suggesting that cbbBc might represent a structural FDH gene of R . eutropha . However, the properties of a cbbBc mutant strain indicated that the potential gene product is not related to known FDH of the organism . Transcriptional analysis in the homologous host and heterologous expression in Escherichia coli demonstrated that cbbBc is an active gene, which apparently has no essential function in the autotrophic metabolism of R . eutropha . The gene is a novel member of cbb operons in autotrophic bacteria.

Mol Gen Genet, 1996 Sep 25, 252(4), 456 - 64
Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress; Juhnke H et al.; We have isolated several mutants of Saccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide . Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway . Allelism of the pos10 mutation (POS for peroxide sensitivity) to the zwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously . The second mutation, pos18, was complemented by transformation with a yeast genomic library . The open reading frame of the isolated gene encodes 238 amino acids . No detectable ribulose 5-phosphate epimerase activity was found in the pos18 mutant, suggesting that the corresponding structural gene is affected in this mutant . For that reason the gene was renamed RPE1 (for ribulose 5-phosphate epimerase) . RPE1 was localized to chromosome X . The predicted protein has a molecular mass of 25966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82 . Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases of Escherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus and Solanum tuberosum . We have characterized RPE1 by testing enzyme activities in rpe1 deletion mutants and in strains that overexpress RPE1, and compared the hydrogen peroxide sensitivity of rpe1 mutants to that of other mutants in the pentose phosphate pathway . Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.

Mol Gen Genet, 1996 Sep 13, 252(3), 237 - 48
The hydrogenase gene cluster of Rhizobium leguminosarum bv . viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity; Rey L et al.; Plasmid pAL618 contains the genetic determinants for H2 uptake (hup) from Rhizobium leguminosarum bv . viciae, including a cluster of 17 genes named hupSLCDEFGHIJK-hypABFCDE . A 1.7-kb segment of insert DNA located downstream of hypE has now been sequenced, thus completing the sequence of the 20441-bp insert DNA in plasmid pAL618 . An open reading frame (designated hypX) encoding a protein with a calculated M(r) of 62300 that exhibits extensive sequence similarity with HoxX from Alcaligenes eutrophus (52% identity) and Bradyrhizobium japonicum (57% identity) was identified 10 bp downstream of hypE . Nodule bacteroids produced by hypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 microM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity . These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein . The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by a hypX gene provided in trans . From expression analysis of hypX-lacZ fusion genes, it appears that hypX gene is transcribed from the FnrN-dependent hyp promoter, thus placing hypX in the hyp operon (hypBFCDEX) . Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis . Similarities were restricted to two distinct regions in the HypX/HoxX sequences . Region I, corresponding to a sequence conserved in N10-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C1), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase family, was located in the C-terminal half . These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C1 donor N10-formyltetrahydrofolate and transfer of the C1 to an unknown substrate, and catalysis of a reaction involving polarization of the C = O bond of an X-CO-SCoA substrate . These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.

Appl Microbiol Biotechnol, 1996 Sep, 46(2), 163 - 8
Cometabolic degradation of 4-chlorophenol by Alcaligenes eutrophus; Hill GA et al.; Alcaligenes eutrophus was grown in batch cultures using either phenol as a sole substrate or mixtures of phenol and 4-chlorophenol . Phenol was found to be the sole source for carbon and energy while 4-chlorophenol was utilized only as a cometabolite . Maximum growth rates on phenol reached only 0.26 h-1, significantly below the growth rates reported earlier with Pseudomonas putida . The cometabolite was found to decrease biomass yield and increase lag time before logarithmic growth occurred . Both phenol and 4-chlorophenol were found to inhibit the growth rate linearly with maximum concentrations of 1080 ppm and 69 ppm respectively, beyond which no growth occurred . The best-fit parameters are incorporated into a simple, dynamic (i.e . time-varying) model capable of predicting all the batch growth conditions presented here . It is shown that P . putida is capable of faster bioremediation when phenol is the sole carbon source or for mixed substrates with low concentrations of the cometabolite, but for high concentrations of 4-chlorophenol, A . eutrophus becomes superior because of the long lag times that occur in the Pseudomonas species.

J Clin Periodontol, 1996 Sep, 23(9), 853 - 60
The effect of cyclosporin-A on the oral microflora at gingival sulcus of the ferret; Fischer RG et al.; The effect of cyclosporin-A (CyA) on the dentogingival flora of ferrets with healthy and experimentally induced periodontal breakdown was studied . Five animals were given 10 mg/kg/d CyA . At the start of the experiments (day 0), ligatures were placed around 4 teeth in the right upper and lower jaws; corresponding contralateral teeth on the left side served as control . On days 0 and 28 (end of the experiment), microbiological samples were collected from the gingival sulcus of the experimental and the control teeth and from closely located gingival mucosa membrane . The samples were subjected to viable counts and to darkfield microscopic analyses . On day 0, facultative anaerobic rods, mainly Pasteurella spp, Alcaligenes spp, Corynebacterium spp . and Rothia spp dominated in the viable counts . No anaerobic bacteria were detected in the viable counts . On day 28 spirochetes increased in the experimental gingival sulcus samples and anaerobic bacteria appeared in most of the samples and constituted 40-60% of the total cultivable flora; Fusobacterium necrophorum and Eubacterium spp . predominated in the samples from the experimental sites . The results of the present study were compared with those of our previous investigation of ferrets not medicated with cyclosporin but also subject to experimental ligature periodontitis . Eubacterium spp . were absent in the animals not treated with cyclosporin, while this species was frequently present in the immunosuppressed ferrets . The results indicate that the presence of the large numbers of gram negative rods and of anaerobic bacteria may have enhanced the inflammatory process and further provoked the gingival overgrowth observed.

J Bacteriol, 1996 Sep, 178(18), 5499 - 507
Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor; Gilis A et al.; Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E . Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups . Alcaligin E promoted the growth of siderophore-deficient A . eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells . However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E . AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E . This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated . The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins . Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake . Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A . eutrophus CH34 . Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A . eutrophus CH34 chromosome in the proximity of the cys-232 locus.

Appl Environ Microbiol, 1996 Sep, 62(9), 3227 - 33
Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation; Kim Y et al.; Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A . R . Harker and Y . Kim, Appl . Environ . Microbiol . 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE . Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis . Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities . Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment . Complementation with a cosmid-based gene bank constructed from A . eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment . Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities . Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii . The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction . Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source . The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions.

J Bacteriol, 1996 Sep, 178(17), 5249 - 56
Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases; Spence EL et al.; The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139 . The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E . coli, and with human 3-hydroxyanthranilate dioxygenase . Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain . A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases . The A . eutrophus MpcI enzyme was expressed in E . coli, purified, and characterized as a protein with a subunit size of 33.8 kDa . Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.

Arch Biochem Biophys, 1996 Aug 15, 332(2), 248 - 54
Characterization of the gene encoding catechol 2,3-dioxygenase of Alcaligenes sp . KF711: overexpression, enzyme purification, and nucleotide sequencing; Moon J et al.; Catechol 2,3-dioxygenase (C23O) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of catechol to form 2-hydroxymuconic semialdehyde . The C23O gene of Alcaligenes sp . KF711 was overexpressed in Escherichia coli HB101 by using the lac promoter of pUC18, and its gene product was purified by using immunoaffinity chromatography . The purified C23O exhibited a 35-kDa single band on an SDS-polyacrylamide gel, and its ring-fission activity on dihydroxylated aromatics was 4-methylcatechol > 4-chlorocatechol > catechol > 3-methylcatechol >> 2,3-dihydroxybiphenyl . Nucleotide sequence analysis of the C23O gene revealed an open reading frame of 927 bp, which can encode a polypeptide of 308 amino acid residues . The predicted molecular mass of 35 kDa is in agreement with that of purified C23O on an SDS-polyacrylamide gel . The amino acid sequence of the C23O was compared with those of nine other extradiol-type dioxygenases, including 2,3-dihydroxybiphenyl dioxygenase (2,3-DHBD) and 1,2-dihydroxynaphthalene dioxygenase (1,2-DHND) . The C23O of Alcaligenes sp . KF711 exhibited 80 to 94% identity in amino acid sequence with other C23Os, and 20 to 25% identity with 1,2-DHND and 2,3-DHBDs . Furthermore, sequence comparison of 10 extradiol-type dioxygenases has led to identifying 19 evolutionarily conserved amino acid residues whose possible catalytic roles are proposed.

Protein Sci, 1996 Aug, 5(8), 1719 - 36
Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum; Zhu PP et al.; The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced . Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate . The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr . Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit . Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms . The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M . genitalium and numerous other organisms . An evolutionary tree places the M . capricolum odp2 gene product in close relationship to the corresponding proteins from A . laidlawii and M.genitalium . The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum . Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A . laidlawii and M . genitalium . The phosphotransacetylase from M . capricolum is related most closely to the corresponding protein from M . genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension . The acetate kinase from M . capricolum is related evolutionarily to the homologous enzyme from M . genitalium . Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M . capricolum, they are scattered in M . genitalium.

Int J Biol Macromol, 1996 Aug, 19(2), 121 - 30
Production of heteropolymeric polyhydroxyalkanoate in Escherichia coli from a single carbon source; Eschenlauer AC et al.; Poly{beta-hydroxybutyrate-co-beta-hydroxyvalerate} co-polymer, PHBV, is a polyhydroxyalkanoate (PHA) that has greater utility as a biodegradable thermoplastic polyester than poly-beta-hydroxybutyrate, PHB . In order to produce PHBV, a system of pathways is required to produce both hydroxybutyrate (HB) and hydroxyvalerate (HV) monomers from the sources of carbon . A working model for conversion of glucose to PHBV via acetyl- and propionyl-coenzyme A was constructed by expressing the PHA biosynthesis genes from Alcaligenes eutrophus in Escherichia coli strain K-12 under novel growth conditions . When 1 mM valine was added to 1% glucose medium, growth ceased and up to 2.5% of the incorporated monomers were HV; up to 4% were HV when 1 mM threonine was added as well . Threonine dehydratase (TD) converts threonine to alpha-ketobutyrate; TD is required for HV to be incorporated into PHA unless its transaminated reaction product, alpha-aminobutyrate, is added to the medium . Intracellular alpha-ketobutyrate accumulates when valine is added to the medium because valine, which cannot be metabolized to HV by E . coli strain K-12, stimulates TD and inhibits acetolactate synthase . In turn, alpha-ketobutyrate is converted to propionyl-CoA by the E . coli pyruvate dehydrogenase complex . This constitutes a defined system of pathways for synthesis of a heteropolymeric PHA from a single carbon source, which in the future could be transferred to other organisms including plants.

Arch Biochem Biophys, 1996 Aug 1, 332(1), 175 - 82
Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus; Schmitt U et al.; Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose . The enzyme is a homotetramer with a subunit molecular mass of 52 kDa . The absorption spectrum of the bacterial dihydropyrimidine dehydrogenase has maxima in the 300- and 400-nm region, suggesting a flavoprotein . The enzyme contains 4 mol FMN, about 24 mol iron and acidlabile sulfide per mole of protein, implying a flavoprotein with FeS centers . The bacterial dehydrogenase is NADPH dependent with B-side stereospecificity . The initial velocity patterns of the bacterial dehydrogenase together with isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium random kinetic mechanism, which is in contrast to results obtained for dihydropyrimidine dehydrogenase from pig liver . The pig liver enzyme adheres to a nonclassical two-site ping-pong kinetic mechanism {B . Podschun, P . F . Cook, and K . D . Schnackerz (1990) J . Biol . Chem . 265, 12966-12972}, whereas for the bovine enzyme a rapid equilibrium random kinetic mechanism was proposed based on steady-state kinetic data {D . J . T . Porter and T . Spector (1993) J . Biol . Chem . 268, 19321-19327}.

J Bacteriol, 1996 Aug, 178(15), 4522 - 9
The Alcaligenes eutrophus membrane-bound hydrogenase gene locus encodes functions involved in maturation and electron transport coupling; Bernhard M et al.; Alcaligenes eutrophus H16 produces two {NiFe} hydrogenases which catalyze the oxidation of hydrogen and enable the organism to utilize H2 as the sole energy source . The genes (hoxK and hoxG) for the heterodimeric, membrane-bound hydrogenase (MBH) are located adjacent to a series of eight accessory genes (hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, and hoxV) . In the present study, we generated a set of isogenic mutants with in-frame deletions in the two structural genes and in each of the eight accessory genes . The resulting mutants can be grouped into two classes on the basis of the H2-oxidizing activity of the MBH . Class I mutants (hoxKdelta, hoxGdelta, hoxMdelta, hoxOdelta, and hoxQdelta) were totally devoid of MBH-mediated, H2-oxidizing activity . The hoxM deletion strain was the only mutant in our collection which was completely blocked in carboxy-terminal processing of large subunit HoxG, indicating that hoxM encodes a specific protease . Class II mutants (hoxZdelta, hoxLdelta, hoxRdelta, hoxTdelta, and hoxVdelta) contained residual amounts of MBH activity in the membrane fraction of the extracts . Immunochemical analysis and 63Ni incorporation experiments revealed that the mutations affect various steps in MBH maturation . A lesion in hoxZ led to the production of a soluble MBH which was highly active with redox dye.

Biotechnol Prog, 1996 Jul-Aug, 12(4), 533 - 9
Bioreactor strategies for the treatment of growth-inhibitory waste: an analysis of thiodiglycol degradation, the main hydrolysis product of sulfur mustard; Lee T et al.; The microbial degradation of thiodiglycol, the primary hydrolysis product of sulfur mustard, by a pure culture of Alcaligenes xylosoxydans ssp . xylosoxydans (SH91) was accomplished in laboratory scale stirred tank reactors . This is a major component of the overall biodegradation process proposed for the complete mineralization of sulfur mustard . Several configurations were evaluated for degradation efficiency including batch, repeated batch, continuous stirred tank reactor (CSTR), and two-stage series CSTR . The repeated batch reactor provided the highest degradation rate of thiodiglycol . Further, this method degraded thiodiglycol in the liquid broth to below the detection limits (0.03 mM) . Both batch and repeated batch experiments were simulated by an unstructured mathematical model . Simulation results were in agreement with the experimental data, particularly at low TDG concentration (around 30 mM) . This study demonstrates the degradation of thiodiglycol using bioreactors and, more generally, is an experimental study of bioreactor designs for the degradation of growth-inhibitory substances.

Eur J Clin Microbiol Infect Dis, 1996 Jul, 15(7), 610 - 5
Bacteremia due to glucose non-fermenting gram-negative bacilli in patients with hematological neoplasias and solid tumors; Martino R et al.; Twenty-six patients with hematological or solid tumors who developed bacteremia caused by Stenotrophomonas maltophilia (n = 10), Pseudomonas putida (n = 6), Sphingomonas paucimobilis complex (n = 4) or Alcaligenes xylosoxidans (n = 6) in the period between 1993 and 1995 were studied . Seventeen patients were neutropenic during the infection, and 13 were undergoing bone marrow or peripheral blood stem cell transplantation . Twenty-three patients had catheter-related infections; only 3 of the 26 patients developed septic complications (all due to Stenotrophomonas maltophilia) . Twenty patients were cured following catheter removal, either as primary measure (n = 8) or salvage measure (n = 12) . Four responded to antibiotic therapy only, and two died of septic complications . Such infections in hematological and oncological patients have increased in this hospital from no cases in 1975 to 11 cases in 1995.

Appl Environ Microbiol, 1996 Jul, 62(7), 2540 - 6
Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase; Williams MD et al.; A novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (Spodoptera frugiperda) by use of individual baculovirus vectors . This system includes expression of a dehydrase-domain mutant rat fatty acid synthase cDNA and the phbC gene encoding polyhydroxyalkanoate synthase from Alcaligenes eutrophus . The dehydrase-deficient fatty acid synthase provides de novo synthesis of R-(-)-3-hydroxybutyryl-coenzyme A as a premature termination product rather than palmityl-coenzyme A, the normal product of wild-type rat fatty acid synthase . High levels of this mutant multifunctional protein provide a suitable precursor pool of R-(-)-3-hydroxybutyryl-coenzyme A for conversion to poly-3-hydroxybutyrate in insect cells coexpressing the phbC gene product . This strategy for redesigning a poly-3-hydroxybutyrate biosynthetic pathway suggests a new method for generating structurally diverse polyhydroxyalkanoates by metabolic engineering.

Appl Environ Microbiol, 1996 Jul, 62(7), 2521 - 6
Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients; DiGiovanni GD et al.; This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients . The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance . Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D) . When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose . These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D . In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR . No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm . These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients . Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred . Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.

Appl Environ Microbiol, 1996 Jul, 62(7), 2470 - 6
Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation; Top EM et al.; The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway . The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF . Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB . To capture such plasmids from soil populations, we used a recipient strain of A . eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted . Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained . Among the transconjugants obtained were clones that contained a ca . 75-kb plasmid, pEMT8 . Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not . Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4 . Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp . strain RASC . Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF . Moreover, cell extracts from A . eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A . eutrophus(pEMT8)(pBH501aE) cultures did . These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.

Appl Environ Microbiol, 1996 Jul, 62(7), 2464 - 9
Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp . strain RASC; Suwa Y et al.; The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids . However, characterization of plasmid-cured strains of Burkholderia sp . strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain . Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5 . One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase . This is the first reported example of a chromosomally encoded tfdA . The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli . The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria . Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134 . The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.

Appl Environ Microbiol, 1996 Jul, 62(7), 2427 - 34
Isolation of Alcaligenes sp . strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols; Krooneman J et al.; Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route . The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail . Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways . Neither growth on nor respiration of catechol could be detected . Chloride production from 3CBA by L6 was strictly oxygen dependent . Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead . In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.

Microbiology, 1996 Jul, 142 ( Pt 7), 1807 - 17
Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations; Ampe F et al.; The growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated . In batch culture, growth was exponential and growth rate (mu) and yields (Y) were high {mu = 0.51 h-1 and Yx/benzoate = 0.56 mol carbon (mol carbon)-1} when low concentrations of benzoate (< 5 mM) were used . These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat {mu(max) = 0.55 h-1 and YX/benzoatemax = 0.57 mol carbon (mol carbon)-1} and the part of the energy for maintenance was limited (mATP = 4.3 +/- 2.2 mmol ATP g-1 h-1) . When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared . The specific rate of benzoate consumption was not altered, whereas growth was inhibited {Ki(benzoate) approximately 27 mM} . Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium . The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5.2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD+ intracellular content . The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism . Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.

J Bacteriol, 1996 Jul, 178(13), 3803 - 8
Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis; LaCelle M et al.; A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions . The hmp protein belongs to the family of two-domain flavohemoproteins, homologs of which have been isolated from various organisms such as Escherichia coli, Alcaligenes eutrophus, and Saccharomyces cerevisiae . These proteins consist of an amino-terminal hemoglobin domain and a carboxy-terminal redox active site domain with potential binding sites for NAD(P)H and flavin adenine dinucleotide . The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI . The requirement of FNR and NarGHJI for hmp expression is completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for transcriptional activation of narGHJI, which produces nitrite, leading to induction of hmp expression . In contrast, induction of hmp was still dependent on resDE in the presence of nitrite . A defect in hmp in B . subtilis has no significant effect on anaerobic growth.

Arch Microbiol, 1996 Jul, 166(1), 42 - 50
Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C . 1.13.11.1) from Alcaligenes eutrophus CH34; Sauret-Ignazi G et al.; Alcaligenes eutrophus CH34 used benzoate as a sole source of carbon and energy, degrading it through the 3-oxoadipate pathway . All the enzymes required for this degradation were shown to be encoded by chromosomal genes . Catechol 1,2-dioxygenase activity was induced by benzoate, catechol, 4-chlorocatechol, and muconate . The enzyme is most likely a homodimer, with an apparent molecular weight of 76,000 +/- 500 . According to several criteria, its properties are intermediate between those of catechol 1,2-dioxygenases (CatA) and chlorocatechol 1,2-dioxygenases (ClcA) . The determined Km for catechol is the lowest among known catechol and chlorocatechol dioxygenases . Similar Km values were found for para-substituted catechols, although the catalytic constants were much lower . The catechol 1,2-dioxygenase from strain CH34 is unique in its property to transform tetrachlorocatechol; however, excess substrate led to a marked reversible inhibition . Some meta- and multi-substituted catechols behaved similarly . The determined Km (or Ki) values for para- or meta-substituted catechols suggest that the presence of an electron-withdrawing substituent at one of these positions results in a higher affinity of the enzyme for the ligand . Results of studies of recognition by the enzyme of various nonmetabolised aromatic compounds are also discussed.

Protein Expr Purif, 1996 Jun, 7(4), 395 - 9
Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6 in Escherichia coli and its purification; Wakayama M et al.; We constructed the high-expression plasmid for D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6 . The appropriate Shine-Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3 . The resultant plasmid, which was named pKNSD2, showed a high D-aminoacylase activity in Escherichia coli JM109 cells transformed with it . The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg).

Arch Microbiol, 1996 Jun, 165(6), 418 - 20
Hydrogen oxidation by membranes from autotrophically grown Alcaligenes eutrophus H16: role of the cyanide-resistant pathway in energy transduction; Komen R et al.; Eighty percent of the ATP and proton electrochemical gradient (-ZDeltapH) formed during H2 oxidation in membranes from autotrophically grown exponential-phase cells of Alcaligenes eutrophus H16 was derived from a redox pathway that includes the membrane-bound hydrogenase complex and the cyanide-resistant (bo-type) oxidase . The H2/ubiquinone-1 oxidoreductase activity was coupled to energy transduction and was fully inhibited by the quinone along 2-n-hepthyl-4-hydroxyquinoline-N-oxide . We conclude that the cytochrome-c-containing pathway in exponential-phase A . eutrophus H16 cells plays a minor role in energy conservation.

Microbiology, 1996 May, 142 ( Pt 5), 1169 - 80
Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate; Leaf TA et al.; The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligenes eutrophus was used to construct a yeast plasmid which enabled expression of the functional synthase enzyme in Saccharomyces cerevisiae . Cells transformed with the synthase plasmid accumulated up to 0.5% of cell dry weight as PHB, with accumulation occurring in the stationary phase of batch growth . The identity of PHB in recombinant yeast cells was confirmed with 1H-NMR spectra of chloroform-extracted cell material . In addition, freeze-fracture electron microscopy revealed cytoplasmic granules exhibiting plastic deformations characteristic for PHB . GC results indicated a low background level of PHB in the wild-type strain, but intact polymer could not be detected by 1H-NMR . Formation of PHB in the recombinant strain implies the participation of native yeast enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA) . Inhibition studies with cerulenin indicated that the fatty acid synthesis pathway is not involved in PHB precursor formation . Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity {150-200 nmol min-1 (mg protein)-1} and acetoacetyl-CoA thiolase activity {10-20 nmol min-1 (mg protein)-1}, which together could synthesize monomer from acetyl-CoA . PHB accumulation was simultaneous with ethanol production, suggesting that PHB can act as an alternate electron sink in fermentative metabolism . We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native beta-oxidation protein possessing D-3-HB-CoA dehydrogenase activity and heterologous PHB synthase.

Lett Appl Microbiol, 1996 May, 22(5), 366 - 70
Growth and viability of Alcaligenes eutrophus JMP134 in seawater as affected by substrate and nutrient amendment; Nybroe O et al.; Growth and viability of Alcaligenes eutrophus JMP134 was studied in laboratory microcosms with 0.2 microns-filtered seawater prior to release in field-based mesocosms . In unamended systems JMP134 did not grow and viability, measured as direct viable counts combined with immunofluorescence microscopy, was 40-50% . Addition of a nitrogen + phosphorus nutrient mixture caused a greater growth response than amendment with a carbon substrate . Amendment with substrate and/or nutrients caused an increase in viability to ca 100% but only for a brief period coinciding with cell proliferation . Hence, Alc . eutrophus JMP134 has a limited survival potential in seawater unless it is supplied with additional nutrients.

Arch Microbiol, 1996 May, 165(5), 289 - 96
Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans; Fischer J et al.; Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air . Hydrogenase was located in the 140,000 x g supernatant in cell-free extracts . The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in a 85% pure preparation with a specific activity of 6.0 U (mg protein)-1 . With native PAGE, a mol . mass of 100 and 200 kDa was determined . With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed . Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD . The optimum of enzyme activity was at pH 9 and at 49 degrees C . Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme . The relationship of the T . ferrooxidans hydrogenase to other proteins was examined . A 9.5-kb EcoRI fragment of T . ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase . Antibodies against this enzyme did not react with the T . ferrooxidans hydrogenase in Western blot analysis . The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase . The N-terminal sequence of 20 amino acids of HoxG of T . ferrooxidans was 83.3% identical to that of the 60-kDa subunit . HupL, of the hydrogenase of Anabaena sp . Sequences of ten internal peptides of HoxG were 50-100% identical to the respective sequences of HupL of the Anabaena sp . hydrogenase.

Eur J Biochem, 1996 May 1, 237(3), 674 - 84
Alcaligenes eutrophus possesses a second pyruvate dehydrogenase (E1); Hein S et al.; Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase complex {Hein, S . & Steinbuchel, A . (1994) J . Bacteriol . 176, 4394-4408}, were identified on two nonrelated A . eutrophus chromosomal BamHI fragments by using a pdhA-specific DNA probe . These data indicated that A . eutrophus possesses, beside PdhA, two additional distinct pyruvate dehydrogenases (E1) . A 6.8-kbp genomic BamHI fragment of A . eutrophus was cloned, and sequence analysis of a 3.896-kbp region revealed the structural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (E1), which was not clustered with structural genes for other components of 2-oxo acid dehydrogenase complexes . The A . eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E1 components of the pyruvate dehydrogenase complexes of A . eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, which are also composed of only one type of subunit . Heterologous expression of pdhE in the aceEF deletion mutant E . coli YYC202 was demonstrated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy . These complementation studies indicated that the E1 component of the A . eutrophus pyruvate dehydrogenase complex can be replaced by a functionally active pdhE gene product.

Oncology, 1996 May-Jun, 53(3), 258 - 62
Infection by Alcaligenes xylosoxidans subsp . xylosoxidans in neutropenic patients; Knippschild M et al.; Alcaligenes xylosoxidans subsp . xylosoxidans (A . x . xylosoxidans) is a nonfermenting gram-negative peritrichous rod and opportunistic pathogen . The organism is frequently found in an aqueous environment . In the past few years, nosocomial infections caused by A . x . xylosoxidans have become more evident . The literature suggests that systemic infections are severe and often lethal and an optimal antibiotic therapy is not well established . This report describes nosocomial infections in 11 patients of a hematology ward over a 2-month period . Primary infection occurred during the neutropenic phase after cytotoxic chemotherapy . Reinfection spread from central venous catheters that had been implanted before the first infection . The bacteremia was successfully treated by imipenem . None of the 11 patients died from the bacteremia, but 3 died of their underlying diseases . Despite an intensive search for the source, the route of infection remained uncertain . Nosocomial infections by A . x . xylosoxidans are of growing importance in high-risk patients . Although the source of infection often remains unknown, infection seems to originate from contaminated solutions . Treatment with imipenem and the removal of central venous catheter systems successfully eliminated A . x . xylosoxidans, which adheres to plastic material.

Can J Microbiol, 1996 May, 42(5), 423 - 30
Isolation and characterization of resin acid degrading bacteria found in effluent from a bleached kraft pulp mill; Morgan CA et al.; Thirteen resin acid degrading bacteria enriched on abietic or dehydroabietic acids were isolated from waste water from the aerated stabilization basin of a bleached kraft pulp mill . Standard biochemical tests were used to characterize each isolate . Each isolate was tested for its ability to degrade six abietane- and pimarane-type resin acids . Resin acid concentrations were determined by high pressure liquid chromatography and UV absorbance . Cluster analysis based on phenotypic characteristics identified two distinct clusters of degraders that differed in their ability to utilize carbohydrates as carbon sources . Fatty acid methyl ester analysis of representative isolates from each cluster identified A19-6a and D11-13 as Comamonas and Alcaligenes species, respectively . To determine genotypic relatedness, enterobacterial repetitive intergenic consensus sequences were used to amplify genomic DNA fragments from 10 isolates . These results supported the phenotypic analysis for all isolates tested except A19-5 and A19-6b . These two organisms were clustered closely together based on phenotype but had distinctly different banding patterns, suggesting that they are not related genotypically . All isolates degraded a subset of the six resin acid congeners . Isolates A19-3, A19-6a, A19-6b, and D11-37 were the most effective at degrading all six congeners.

Infect Immun, 1996 May, 64(5), 1532 - 40
The Legionella pneumophila hel locus encodes intracellularly induced homologs of heavy-metal ion transporters of Alcaligenes spp; McClain MS et al.; We continued characterization of the Legionella pneumophila hel locus . Mutagenesis and DNA sequencing identified three genes similar to the czc and cnr loci of Alcaligenes eutrophus and the ncc locus of Alcaligenes xylosoxidans . On the basis of their similarity to these loci, we designated the L . pneumophila genes helC, helB, and helA . Mutations in the hel genes led to reduced cytopathicity towards U937 cells, although the mutant strains did not appear defective in other assays of virulence . Transcription of the hel locus was induced by the intracellular environment but was not induced by any of a variety of in vitro stress conditions . The function of the hel gene products remains to be determined.

Eur J Biochem, 1996 Apr 15, 237(2), 357 - 66
Metabolism of 5-chlorosubstituted muconolactones; Prucha M et al.; The stereochemistry of the four stereoforms of 5-chloro-3-methylmuconolactones could be deduced from NMR and stability data, and from the comparison with authentic (4R, 5S)-5-chloromuconolactone . Muconolactone isomerase of Alcaligenes eutrophus JMP 134 was shown to catalyze syn-elimination of hydrogen chloride from (4R, 5R)-5-chloro-3-methylmuconolactone, (4R, 5S)-5-chloro-3 -methylmuconolactone and (4R, 5S)-5-chloromuconolactone to form 3-methyl-trans-dienelactone, 3-methyl-cis-dienelactone and a 3:1 mixture of cis- and trans-dienelactone, respectively . 3-Methyl-trans-dienelactone was a substrate of pJP4-encoded dienelactone hydrolase of A . eutrophus JMP 134, whereas 3-methyl-cis-dienelactone transformation was negligible indicating a restricted substrate specificity of this enzyme . Both substrates were transformed into 3-methylmaleylacetate which in turn was a substrate for maleylacetate reductase . This compound was shown to possess a cyclic structure (4-hydroxy-3-methyl-muconolactone) under acidic conditions.

Eur J Biochem, 1996 Apr 15, 237(2), 350 - 6
Muconolactone isomerase of the 3-oxoadipate pathway catalyzes dechlorination of 5-chloro-substituted muconolactones; Prucha M et al.; An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)- and (4R,5S)-5-chloro-3-methyl- and (4R, 5S)-5-chloromuconolactone of principally 3-methyl-trans-, 3-methyl-cis-dienelactone and cis-dienelactone, respectively, was purified to homogeneity . The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases . Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100-kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida . Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)-5-chloro-3 -methylmuconolactone . (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone . The (4S, 5S)-structure was found to be an inhibitor of (4R, 5R)-5-chloro-3-methylmuconolactone transformation . Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 699 - 704
Cloning and sequence analysis of czc genes in Alcaligenes sp . strain CT14; Kunito T et al.; We have isolated 14 cadmium (Cd)-resistant, soil-borne bacteria . Among those, strain CT14, which was identified as an Alcaligenes sp., has a czc (cadmium, zinc, and cobalt divalent cation resistant determinant) system . Here we report the nucleotide sequence of 4 genes (czcCBAD) of the system . CzcCBA showed over 98% identity with those of A . eutrophus CH34, however, CzcD, the distal gene product, was 117 amino acids longer than that (199 amino acids) of A . eutrophus CH34, and had considerable similarity to the members of the CDF (cation diffusion facilitator) family proteins all over the region.

Bioseparation, 1996 Apr, 6(2), 125 - 32
Effects of heat shock on gram negative bacteria: use of lysis by sodium dodecyl sulphate as a probe for the integrity of DNA; Rees P et al.; Rheograms of Alcaligenes eutrophus (NCIMB 40529) and Escherichia coli (C90 NCIMB 10616) cells lysed by sodium dodecyl sulphate were compared before and after a variety of heat shock regimes . It was found that unheated cells produced a very characteristic shear thickening rheogram which could be destroyed by DNase treatment . Cells which had been subjected to heat shock produced rheograms very similar to DNase digested material . We thus suggest that the rheogram is largely due to the presence of intact DNA molecules . The extent and nature of the heat shock affected the shape of the rheogram of the SDS lysed material . Heat shock of cells after SDS lysis did not appear to significantly damage the DNA . Storage of the cells at 10 degrees C before heat shock considerably reduced the shear thinning effect of subsequent heat shock at 90 degrees C . We attribute the shear thinning effect of the heat shock to the action of nucleases which are activated and then depolymerise the DNA molecules.

Zhonghua Yi Xue Za Zhi (Taipei), 1996 Apr, 57(4), 301 - 4
Alcaligenes xylosoxidans neonatal meningitis: a case report; Pan CH et al.; Neonatal meningitis caused by Alcaligenes xylosoxidans is associated with a high mortality rate . The causative microorganism is resistant to most antimicrobials . Generally, once the organism has been isolated from the cerebrospinal fluid of the infected neonate, initial therapy with a third-generation cephalosporin and trimethoprim-sulfamethoxazole is recommended before determining its susceptibility to antimicrobials . There present this is the first of neonatal meningitis with transient diabetes insipidus cause by A . xylosoxidans . The patient was treated with dDAVP for seven days and a combination of imipenem plus trimethoprim-sulfamethoxazole for 28 days . The patient was discharged in a stable condition and the end of that time, but with sequelae of hydrocephalus and hearing impairment.

J Bacteriol, 1996 Apr, 178(8), 2368 - 74
Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product; Thiemermann S et al.; Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16 . While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen . Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme . Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel . Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence . Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW . These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.

Biochim Biophys Acta, 1996 Mar 7, 1293(1), 39 - 44
Hydroxylation of quinaldic acid: quinaldic acid 4-monooxygenase from Alcaligenes sp . F-2 versus quinaldic acid 4-oxidoreductases; Bubeck B et al.; The N-heterocycles quinaldic acid (quinoline 2-carboxylic acid), kynurenic acid (4-hydroxyquinoline 2-carboxylic acid), 2-oxo-1,2-dihydroquinoline, and xanthine are utilized by Alcaligenes sp . F-2 as sole source of carbon and energy . Although quinoline did not serve as growth substrate, 8-hydroxy-2-oxo-1,2-dihydroquinoline and 8-hydroxycoumarin, metabolites of the 'coumarin pathway' of quinoline catabolism, were isolated from the culture fluid during growth on 2-oxo-1,2-dihydroquinoline . Contrary to Serratia marcescens 2CC-1 and Pseudomonas sp . AK-2 (Sauter et al . (1993) Biol . Chem . Hoppe-Seyler 374, 1037-1046), which possess different molybdenum-containing hydroxylases catalysing the 4-hydroxylation of quinaldic acid to kynurenic acid with incorporation of oxygen derived from water and concomitant reduction of an electron acceptor, Alcaligenes sp . F-2 contains an inducible quinaldic acid 4-monooxygenase that catalyses the very same conversion in the presence of O2 and NADH . The activity of the monooxygenase was enhanced 1.5-fold by Fe2+ ions . The extremely thermolabile enzyme (apparent molecular mass: 155 kDa) exclusively accepted quinaldic acid as substrate . The 'pseudosubstrates' menadione, 8-hydroxyquinoline, and 8-hydroxy-2-oxo-1,2-dihydroquinoline effected consumption of NADH and oxygen without being hydroxylated . Quinaldic acid 4-monooxygenase was inhibited by sulfhydryl modifying and chelating agents, and by various divalent metal ions, whereas reducing agents did not affect enzymatic activity.

FEMS Microbiol Lett, 1996 Mar 1, 136(3), 231 - 8
Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex; Hein S et al.; Nucleotide sequence analysis of a 3.3-kb genomic EcoRI fragment and of relevant subfragments of a genomic 13.2-kb SmaI fragment of Alcaligenes eutrophus, which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region . The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative -35/-10 promoter in the order odhA, odhB, and odhL . In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique . Heterologous expression of the A . eutrophus odh genes in E . coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.

Protein Expr Purif, 1996 Mar, 7(2), 203 - 11
Expression and analysis of a bacterial poly(hydroxyalkanoate) synthase in insect cells using a baculovirus system; Williams MD et al.; An improved method for expression of poly-beta-hydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus has been developed using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) in BTI-TN-5B1-4 Trichoplusia ni cells which results in high level production of active PHA synthase . Confirmation of expression of authentic PHA synthase was obtained by Western analysis which also revealed the presence of several apparent proteolytic cleavage products . N-terminal sequence data were obtained from the 64-kDa protein which verified its identity . The PHA synthase produced in this system constitutes approximately 50% of total protein after 60 h of viral infection and is found approximately equally distributed in both soluble and membrane-associated fractions . The expression level allowed rapid purification of the soluble form of PHA synthase to approximately 90% homogeneity in a single liquid chromatography step on hydroxylapatite . Using a direct spectrophotometric assay, analyses show that the enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation between enzyme concentration and specific activity . Over 1000 units of soluble enzyme were obtained from a 250-ml culture of T . ni cells with an apparent initial specific activity of 12 mumol min-1 mg-1 . The amount of PHA synthase activity is significantly higher than previously obtained from much larger bacterial cultures . The method described here should provide a general approach for the expression of active PHA synthases from a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing proteins.

Mol Microbiol, 1996 Mar, 19(6), 1307 - 18
Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases; Reyes F et al.; The phototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen . In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment . A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb PstI fragment carrying the putative genes coding for the periplasmic nitrate reductase . In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon . These genes are required for nitrate reduction, as deduced by mutational and complementation studies . The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcaligenes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes . The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A . eutrophus . NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm . The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues . NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes . The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R . sphaeroides DSM 158 is a dimeric complex of a 90 kDa catalytic subunit (NAPA) and a 15 kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm . This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.

J Chem Technol Biotechnol, 1996 Feb, 65(2), 115 - 22
Survival of luxAB-marked Alcaligenes eutrophus H850 in PCB-contaminated soil and sediment; Van Dyke MI et al.; A rifampicin-resistant PCB-degrading Alcaligenes eutrophus H850 strain was marked with luxAB reporter genes and designated H850Lr . This strain was enumerated in soil by viable plating and counting of light-emitting colonies . The marked strain was also inoculated into soil and sediment microcosms contaminated with PCBs and treated with rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2Lr or inoculated with the P . aeruginosa UG2Lr strain . A . eutrophus H850Lr exhibited similar survival in sandy loam soil in the absence or presence of PCBs over 56 days . Survival of A . eutrophus H850Lr in PCB-contaminated sediment was less than in sandy soil under the same incubation conditions . Addition of P . aeruginosa UG2 rhamnolipids to soil increased the culturable indigenous heterotrophic population, and numbers of A . eutrophus H850Lr cells . P . aeruginosa UG2Lr cells did not affect survival of A . eutrophus H850, as cell enumerations after 2 months were the same as in microcosms containing only A . eutrophus H850 inoculum . P . aeruginosa UG2Lr survived in soils as demonstrated by the slight decrease in CFU from 1 x 10(8) to 2 x 10(6) CFU cm-3 after 2 months . Direct extraction of DNA from soil and purification for use in PCR amplification using primers specific for the bphC gene detected 8 x 10(2) A . eutrophus H850Lr CFU g-1 soil in PCB-contaminated soils . Colony lifts of bacteria isolated from microcosms containing PCB-contaminated soil did not hybridize with LB400 bphC probe . However, enrichment of PCB-contaminated soil with biphenyl, followed by DNA extraction and probing with bphC gene probe detected indigenous PCB-degrading bacteria containing a similar gene sequence in PCB-contaminated sediment . This study demonstrates the usefulness of using the lux reporter system in monitoring bacterial survival in PCB-contaminated soils and sediments.

Otolaryngol Head Neck Surg, 1996 Feb, 114(2), 332 - 4
Alcaligenes xylosoxidans subsp xylosoxidans in children with chronic otorrhea; Wintermeyer SM et al.; Because other known pathogens are frequently isolated with AXX, its clinical significance may be overlooked . AXX should not be considered a colonizer or contaminant, particularly in the presence of clinical signs and symptoms of infection . For a pediatric patient with AXX in the ear fluid, the choice of an antibiotic regimen should be based on in vitro activity of both AXX and other concurrent pathogens . Although the sensitivity of AXX may be highly variable, a number of antibiotics appear to be clinically useful.

J Antibiot (Tokyo), 1996 Feb, 49(2), 140 - 4
Kalimantacin A, B, and C, novel antibiotics produced by Alcaligenes sp . YL-02632S . II . Physico-chemical properties and structure elucidation; Tokunaga T et al.; Kalimantacin A, B and C are new antibiotics produced by Alcaligenes sp . YL-02632S . Their structures were elucidated to be novel long chain structure compounds containing O-carbamoyl, amide and carboxylic acid moieties based on various 2D NMR experiments and MS analysis.

J Antibiot (Tokyo), 1996 Feb, 49(2), 136 - 9
Kalimantacins A, B and C, novel antibiotics from Alcaligenes sp . YL-02632S . I . Taxonomy, fermentation, isolation and biological properties; Kamigiri K et al.; Novel antibacterial antibiotics, kalimantacins A, B and C, have been isolated from the fermentation broth of Alcaligenes sp . YL-02632S . In this paper, the taxonomy of the producing strain, fermentation, isolation and biological activities of kalimantacins are reported . Kalimantacins inhibit the growth of Staphylococcus aureus and S . epidermidis including multiple-drug resistant strains.

Mol Biol Evol, 1996 Feb, 13(2), 324 - 33
Globins in nonvertebrate species: dispersal by horizontal gene transfer and evolution of the structure-function relationships; Moens L et al.; Using a new template based on an alignment of 145 nonvertebrate globins we examined several recently determined sequences of putative globins and globin-like hemeproteins . We propose that all globins have evolved from a family of ancestral, approx . 17-kDa hemeproteins, which displayed the globin fold and functioned as redox proteins . Once atmospheric O2 became available the acquisition of oxygen-binding properties was initiated, culminating in the various highly specialized functions known as present . During this evolutionary process, we suggest that (1) high oxygen affinity may have been acquired repeatedly and (2) the formation of chimeric proteins containing both a globin and a flavin binding domain was an additional and distinct evolutionary trend . Furthermore, globin-like hemeproteins encompass hemeproteins produced through convergent evolution from nonglobin ancestral proteins to carry out O2-binding functions as well as hemeproteins whose sequences exhibit the loss of some or all of the structural determinants of the globin fold . We also propose that there occurred two cases of horizontal globin gene transfer, one from an ancestor common to the ciliates Paramecium and Tetrahymena and the green alga Chlamydomonas to a cyanobacterium ancestor and the other, from a eukaryote ancestor of the yeasts Saccharomyces and Candida to a bacterial ancestor of the proteobacterial genera Escherichia, Alcaligenes, and Vitreoscilla.

J Bacteriol, 1996 Feb, 178(3), 888 - 93
Primary structure and phylogeny of the Calvin cycle enzymes transketolase and fructosebisphosphate aldolase of Xanthobacter flavus; van den Bergh ER et al.; Xanthobacter flavus, a gram-negative facultatively autotrophic bacterium, employs the Calvin cycle for the fixation of carbon dioxide . Cells grown under autotrophic growth conditions possess an Fe(2+)-dependent fructosebisphosphate (FBP) aldolase (class II) in addition to a class I FBP aldolase . By nucleotide sequencing and heterologous expression in Escherichia coli, genes encoding transketolase (EC 2.2.1.1.; CbbT) and class II FBP aldolase (EC 4.1.2.13; CbbA) were identified . A partial open reading frame encoding a protein similar to pentose-5-phosphate 3-epimerase was identified downstream from cbbA . A phylogenetic tree of transketolase proteins displays a conventional branching order . However, the class II FBP aldolase protein from X . flavus is only distantly related to that of E . coli . The autotrophic FBP aldolase proteins from X . flavus, Alcaligenes eutrophus, and Rhodobacter sphaeroides form a tight cluster, with the proteins from gram-positive bacteria as the closest relatives.

Eur J Biochem, 1996 Jan 15, 235(1-2), 351 - 8
hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system; Dernedde J et al.; In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH) . hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB {Dernedde, J., Eitinger, M . & Friedrich, B . (1993) Arch . Microbiol . 159, 545-553} . Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes . Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain . The resulting mutants fall into two classes . Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism . Class I mutants were unable to grow on hydrogen under autotrophic conditions . The enzymatic activities of SH and MBH were disrupted in all three class I mutants . Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation . Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane . In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity . In class I mutants this maturation was blocked . 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants . Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A . eutrophus.

FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 333 - 6
Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the beta-subclass of Proteobacteria; Springer N et al.; An almost full length 16S rRNA gene of the obligate bacterial endosymbiont Polynucleobacter necessarius was amplified using the polymerase chain reaction in combination with site-specific primers . The amplified DNA was directly sequenced and compared with other bacterial 16S rRNA sequences . P . necessarius belongs to the beta-subclass of Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum, and B . pickettii . In Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum, and B . pickettii . In situ hybridization with a specific oligonucleotide probe corroborated the assignment of the retrieved sequence to P . necessarius.

Biodegradation, 1996-97, 7(6), 435 - 43
Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp . JB1: metabolites and enzymes; Commandeur LC et al.; In contrast to the degradation of penta- and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp . JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays . Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded . Monochloro-benzoates and dichlorobenzoates were detected as metabolites . Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent . In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates . No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols . The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp . JB1 is unlikely.

New Microbiol, 1996 Jan, 19(1), 67 - 76
Heterotrophic bacteria in the Ross Sea (Terra Nova Bay, Antarctica); Maugeri TL et al.; Microbial research on temporal variation of bacterial densities was carried out on seawater samples collected from two field stations at different depths during the Antarctic summer (oceanographic campaign 1989/1990) . Bacterial densities evaluated on Marine Agar 2216 (Difco) and on TCBS Agar (Difco) after incubation at +4 degrees C for 21 days respectively ranged from 0 to 7.9 x 10(2) CFU/ml for heterotrophic bacteria and from 0 to 5.7 x 10(2) CFU/100ml for "presumptive vibrios" . During the period of observation, Vibrio densities showed a higher variability than those of total heterotrophic bacteria . A high percentage of gelatinolytic and chitinolytic vibrios was observed . The qualitative composition of heterotrophic bacterial communities was studied on 38 morphological and biochemical characteristics of 152 strains isolated from the stations . The data were subsequently used to determine the structure and metabolic potentialities of bacterial communities in the two sites . Almost all the heterotrophic, psychrotrophic isolates were non fermentative Gram-negative rods, belonging to the genera Pseudomonas/Alcaligenes, Flavobacterium/Cytophaga . The bacterial communities in the two coastal habitats investigated were clearly different.

Appl Biochem Biotechnol, 1996 Spring, 57-58, 779 - 89
Reactor comparisons for the biodegradation of thiodiglycol, a product of mustard gas hydrolysis; Pham MQ et al.; An environmentally benign method for the mineralization of sulfur mustard has been proposed involving chemical hydrolysis of sulfur mustard to thiodiglycol, and then the biological degradation of thiodiglycol to generate biomass and gaseous carbon dioxide . Alcaligenes xylosoxidans (SH91) was isolated based on its ability to utilize thiodiglycol as a sole carbon source . This article compares different biological reactor designs and experimentally assesses their relative effectiveness in degrading thiodiglycol using pure cultures of SH91 . The reactor configurations studied are batch, continuous stirred-tank reactor (CSTR), and CSTR with cell recycle . From the results, it is clear that the CSTR with cell recycle offers superior performance for a given residence time or volume . These pure culture data are necessary for accurate design of a pilot-scale system where mixed cultures will be employed because of a possible incomplete chemical hydrolysis step.

Genetika, 1996 Jan, 32(1), 146 - 9
{Introduction of mutator phage D3112 of Pseudomonas aeruginosa into Alcaligenes eutrophus var . metallotollerans (Strain CH34)}; Krylov VN; It is demonstrated that the intact genome of a D3112 tranposable phage (TP) of Pseudomonas aeruginosa, integrated into a recombinant plasmid RP4 :: D3112, can be transferred by means of conjugation from P . putida PpG1 (RP4npt :: D3112) donor cells into Alcaligenes eutrophus var . metallotollerans cells . P . aeruginosa strains are unacceptable as donors because they have a bactericidal effect on A . eutrophus . RP4npt :: D3112 plasmid is stably inherited by A . eutrophus with D3112 being expressed and successfully reproduced . However, TP loses the induction ability after UV irradiation or mitomycin C treatment . It is suggested that D3112 TP and its miniderivatives could be used in manipulations with A . eutrophus var . metallotolerans.

Int J Syst Bacteriol, 1996 Jan, 46(1), 252 - 8
Sutterella wadsworthensis gen . nov., sp . nov., bile-resistant microaerophilic Campylobacter gracilis-like clinical isolates; Wexler HM et al.; Campylobacter gracilis (formerly Bacteroides gracilis) is an asaccharolytic, nitrate-positive, urease-negative organism that requires formate and fumarate or hydrogen as a growth additive and may pit agar media . Clinical isolates that were obtained primarily from appendiceal and peritoneal fluid specimens and initially were identified in our laboratory as B . gracilis were later found to include "unusual" strains that could be distinguished by biochemical and genetic criteria . These unusual C . gracilis strains were bile resistant, could not reduce tetrazolium chloride under aerobic conditions if formate and fumarate were added to the medium, and could grow in the presence of 2 or 6% oxygen if no blood was added to the medium . C . gracilis, other campylobacters, and the unusual strains produced distinctive dehydrogenase patterns when gels were incubated anaerobically . A cellular fatty acid analysis revealed that the cluster formed by the unusual organisms was distinct from the (separate) clusters formed by C . gracilis, Bacteroides ureolyticus, and other Campylobacter species . 16S rRNA sequence data indicated that these organisms are not related phylogenetically to either C . gracilis or other Campylobacter species; the most closely related taxa as determined by rRNA sequence analysis were unrelated aerobes (members of the genera Bordetella, Alcaligenes, Rhodocyclus, and Comamonas) . DNA homology data confirmed that these taxa are separate groups . Our data indicate that the unusual organisms are members of a new genus and new species, for which we propose the name Sutterella wadsworthensis . The type strain of S . wadsworthensis is strain WAL 9799 (= ATCC 51579).

J Bacteriol, 1996 Jan, 178(1), 298 - 300
Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: comparison of catalytic properties; Muller D et al.; The maleylacetate reductases from Pseudomonas aeruginosa RHO1 and Alcaligenes eutrophus JMP134 were tested for activity and affinity to various maleylacetates as well as dechlorinating properties . The dechlorinating activity and the kcat/Km values revealed high-level similarity of these reductases to that of Pseudomonas sp . strain B13.

Sci Total Environ, 1995 Dec 15, 175(3), 275 - 85
Degradation of PCB in different soils by inoculated Alcaligenes xylosoxidans; Haluska L et al.; The degradation of PCB in soils by the biphenyl-utilising strain Alcaligenes xylosoxidans was studied in different soil types . In addition to the congener specificity, significant differences in the degradation of PCB by the strain in the different soil types were observed . Efficiency of degradation was generally better in sterilised soils, but the differences were not as significant as the differences observed between different soil types . These results indicate that the degradation of PCB is probably related not only to the capabilities of the strain employed and quality and amount of competitive species inhabiting the soils, but also to the soil sorption of the PCB congeners . Degradation is faster in the soils containing an intermediate amount of organic carbon with a high portion of total and aromatic carbon in humic acids.

EMBO J, 1995 Dec 15, 14(24), 6067 - 77
Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution; Ermler U et al.; The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6% . The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase . The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site . A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations . The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees . The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.

Plant Mol Biol, 1995 Dec, 29(6), 1279 - 91
Cloning of the amphibolic Calvin cycle/OPPP enzyme D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects; Nowitzki U et al.; Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D-ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach . Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E . coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source . The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum . A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E . coli genome . A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.

Appl Environ Microbiol, 1995 Dec, 61(12), 4500 - 4
Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100; Danganan CE et al.; Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy . The genes encoding the proteins involved in the first step (tftA and tftB {previously designated tftA1 and tftA2, respectively}) have been cloned and sequenced . The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays . The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group . These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134 . The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension . The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100 . The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters.

Vet Microbiol, 1995 Nov, 47(1-2), 61 - 70
Detection of Pseudomonas aeruginosa from ovine fleece washings by PCR amplification of 16S ribosomal RNA; Kingsford NM et al.; Two oligonucleotides were selected from the variable regions of the 16S rRNA gene of P . aeruginosa and used as PCR primers for the detection of P . aeruginosa . The specificity of the primers was tested against the following bacterial species; Pseudomonas putida, Pseudomonas cepacia, Xanthamonas maltophilia, Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas alcaligenes and Pseudomonas diminuta . These primers had a sensitivity of detection of 1 pg of chromosomal DNA or 1 x 10(5) cfu/microliters and were species-specific . The sensitivity of detection was increased to 1 fg or less than 10 cfu/microliters using a non-radioactively labelled probe . Using these PCR primers it was possible to detect the presence of P . aeruginosa from fleece washings collected from a flock of 100 sheep . Correlation between single PCR and bacteriological isolation showed agreement in 89% of fleece samples tested, 2% of the samples contained organic PCR inhibitors in the fleece washings, 3% were below the level of sensitivity of the test and the remaining 6% were culture negative for P . aeruginosa but PCR positive . Use of nested PCR did not increase the sensitivity of detection over single round PCR combined with the use of non-radioactively labelled probe.

Appl Microbiol Biotechnol, 1995 Nov, 43(6), 1039 - 43
Purification and characterization of N-carbamoyl-L-amino acid amidohydrolase with broad substrate specificity from Alcaligenes xylosoxidans; Ogawa J et al.; N-Carbamoyl-L-amino acid amidohydrolase was purified to homogeneity for the first time from Alcaligenes xylosoxidans . The enzyme showed high affinity toward N-carbamoyl-L-amino acids with long-chain aliphatic or aromatic substituents, and hydrolyzed those with short-chain substituents quite well . The enzyme hydrolyzed N-formyl- and N-acetylamino acids quickly and very slowly, respectively . The enzyme did not hydrolyze beta-ureidopropionate and ureidosuccinate . The relative molecular mass of the native enzyme was about 135,000 and the enzyme consisted of two identical polypeptide chains . The enzyme activity was significantly inhibited by sulfhydryl reagents and required the following divalent metal ions: Mn2+, Ni2+ and Co2+.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2115 - 9
Cloning and sequencing of a gene encoding D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6 and expression of the gene in Escherichia coli; Wakayama M et al.; The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified . The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues . The molecular weight of D-aminoacylase was calculated to be 51,918 . This value agreed well with the apparent molecular weight of 52,000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) . The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E . coli), with the exception of the removal of the N-terminal methionine processed after translation . The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6 . Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine and humans.

Appl Environ Microbiol, 1995 Nov, 61(11), 4061 - 8
Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp . under different culture conditions; Guerin WF et al.; The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments . The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp . (strain NP-Alk) under different batch culture conditions . Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 and NP-Alk, respectively . Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene . The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate . Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp . but remained at measurable levels in the pseudomonad even after 9 months . The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with 14C-labeled naphthalene as the substrate, was consistent with activity maintenance data . In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene . Adaptation in the uninduced Alcaligenes sp . occurred after many hours of exposure to naphthalene . In vivo labeling with 35S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results.

Appl Environ Microbiol, 1995 Nov, 61(11), 3788 - 95
Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements; Ogawa N et al.; Alcaligenes eutrophus NH9 was isolated from soil . This strain can utilize 3-chlorobenzoate (3-CB) as a sole source of carbon and energy . Most of the 3-CB-negative segregants had lost one of the plasmids present in the parent strain . The genes for catabolism of 3-CB were located within a 9.2-kb SacI fragment of this plasmid (pENH91) . The genes were found to hybridize with genes for components of the modified ortho cleavage pathway from Pseudomonas putida . In one of the 3-CB-negative segregants, the plasmid had undergone the deletion of a segment with a size of about 12.5 kb that covered the catabolic genes . The deletion event seemed to be the result of reciprocal recombination between two highly homologous sequences with sizes of 2.5 kb that were present as a direct repeat at the two ends of the region that included the catabolic genes . Nucleotide sequence analysis of homologous fragments revealed a structure that resembled an insertion sequence and relatedness to IS21 . During repeated subculturing of NH9 on liquid media with 3-CB, the culture was taken over by a derivative strain (designated NH9A) in which the degradative plasmid carried a duplicate copy of the 12.5-kb region that contained the catabolic genes . The duplication of these genes seemed again to have been mediated by recombination between the direct repeat sequences.

J Bacteriol, 1995 Nov, 177(22), 6568 - 74
Operator binding of the CbbR protein, which activates the duplicate cbb CO2 assimilation operons of Alcaligenes eutrophus; Kusian B et al.; The regulatory protein CbbR, which activates the transcription of the duplicate, chromosomally and megaplasmid pHG1-borne cbb CO2 assimilation operons of Alcaligenes eutrophus H16, was purified to homogeneity from Escherichia coli after heterologous expression of the cloned cbbR gene . The pure protein occurred as either a 63-kDa dimer at room temperature or a 125-kDa tetramer at 4 degrees C . CbbR bound to the 167-bp cbb control region separating the divergently oriented cbbR gene (defective copy on pHG1) from the cbb operon . DNase I footprinting revealed binding of the protein between position -29 and -74 relative to the transcriptional start point of the cbb operon, with a hypersensitive site at positions -47 and -48, suggesting potential DNA bending . Hydroxyl radical footprinting disclosed the same central binding region . The region was found to consist of two subsites to which the activator apparently bound in a cooperative manner . At higher CbbR concentrations, the binding region extended to position +13 . The overlapping arrangement of the operon promoter and CbbR-binding region (operator) suggests an interaction between CbbR and RNA polymerase to cause transcription activation . Transcriptional fusions with fragments carrying 1- or 2-bp insertions within the central region showed no operon promoter activity, although CbbR binding was not prevented by these mutations . Dissection of the central region enabled the differentiation of two apparently independent binding subsites . Strongly increased cbbR promoter activity originating from a fragment that contained only a part of the central region indicated negative autoregulation of cbbR transcription.

J Bacteriol, 1995 Nov, 177(21), 6309 - 12
Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm; Eismann K et al.; Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts . They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.

J Bacteriol, 1995 Oct, 177(20), 5826 - 33
Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase; Ampe F et al.; During batch growth of Alcaligenes eutrophus on benzoate-acetate mixtures, benzoate was the preferred substrate, with acetate consumption being delayed until the rate of benzoate consumption had diminished . This effect was attributed to a transcriptional control of the synthesis of acetyl coenzyme A (acetyl-CoA) synthetase, an enzyme necessary for the entry of acetate into the central metabolic pathways, rather than to a biochemical modulation of the activity of this enzyme . Analysis of a 2.4-kb mRNA transcript hybridizing with the A . eutrophus acoE gene confirmed this repression effect . In a benzoate-limited chemostat culture, derepression was observed, with no increase in the level of expression following an acetate pulse . Benzoate itself was not the signal triggering the repression of acetyl-CoA synthetase . This role was played by catechol, which transiently accumulated in the medium when high specific rates of benzoate consumption were reached . The lack of rapid inactivation of the functional acetyl-CoA synthetase after synthesis has been stopped enables A . eutrophus to retain the capacity to metabolize acetate for prolonged periods while conserving minimal protein expenditure.

Eur J Biochem, 1995 Oct 1, 233(1), 266 - 76
Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria; Schmitz O et al.; An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced . The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD(+)-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP(+)-dependent protein from Desulfovibrio fructosovorans . The cluster from A . variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A . eutrophus enzyme . In A . variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY, respectively), and a probably non-coding 0.9-kb segment in an unusual way . The hoxH partial sequence from Anabaena 7119 and Anacystis nidulans was amplified by PCR . Using the labeled segment from A . 7119 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A . 7119, Anabaena cylindrica, Anacystis nidulans and A . variabilis . The bidirectional hydrogenase from A . nidulans was purified and digests were sequenced . The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A . variabilis . Therefore the 8.9-kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria . Crude extracts from A . nidulans perform NAD(P)H-dependent H2 evolution corroborating the molecular biological demonstration of the NAD(P)(+)-dependent hydrogenase in cyanobacteria.

Mol Ecol, 1995 Oct, 4(5), 613 - 8
Gene transfer from a bacterium injected into an aquifer to an indigenous bacterium; Zhou JZ et al.; Two novel 3-chlorobenzoate-degrading bacteria were previously isolated from an aquifer in which no such bacteria could be enriched prior to the introduction of the 3-chlorobenzoate-degrading strain, Pseudomonas sp . B13 . To understand the origin of 3-chlorobenzoate-degrading genes in the two novel isolates, the 16S ribosomal RNA, clcD (dienelactone hydrolase) and clcA (chlorocatechol oxygenase) genes from these bacteria were amplified and sequenced . The partial 16S rRNA gene sequences and REP-PCR patterns showed that these two novel isolates were identical but differed from strain B13 . Phylogenetic analyses revealed that the novel isolates were closely related to Alcaligenes eutrophus in the beta subclass of the Proteobacteria, whereas strain B13 was related to Pseudomonas aeruginosa and P . mendocina in the gamma subclass of the Proteobacteria . In contrast, the clcD and clcA gene sequences were identical on strain B13 and these two isolates, indicating that the 3-chlorobenzoate-degrading genes were transferred from strain B13 to these isolates . What cannot be established is when this transfer occurred.

Mol Ecol, 1995 Oct, 4(5), 579 - 91
Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis; Knaebel DB et al.; We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils . Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P . oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone . Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure . This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively . The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes . Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene . Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products . For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results . This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA . A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils . Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil . However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.

Lett Appl Microbiol, 1995 Oct, 21(4), 246 - 8
Isolation and characterization of four polycyclic aromatic hydrocarbon degrading bacteria from soil near an oil refinery; Ashok BT et al.; Four bacterial strains (I-IV) capable of optimum growth on 0.1% naphthalene, anthracene or a mixture of naphthalene and phenanthrene were isolated from soil near an oil refinery . Two isolates (I and II) were identified as belonging to the genus Micrococcus, while strains III and IV were identified as Pseudomonas and Alcaligenes respectively . All the isolates were found to bear high molecular weight plasmid DNA (isolate I and IV 89%, II 67.5% and III 92.1% of lambda DNA), which is presumed to aid in the metabolism of polycyclic aromatic hydrocarbons . The strains also showed appreciable growth at high concentrations of NaCl (up to 7.5%).

Biol Chem Hoppe Seyler, 1995 Sep, 376(9), 561 - 8
Structural and immunological studies on the soluble formate dehydrogenase from Alcaligenes eutrophus; Friedebold J et al.; During growth with formate as the sole energy source the autotrophic bacterium Alcaligenes eutrophus synthesizes a cytoplasmic formate dehydrogenase . The enzyme is a molybdo-iron-sulfur-flavo protein and the major NADH-producing system under these growth conditions, although it was estimated to constitute only 0.65% of the soluble cell protein . An electron microscopic analysis of the purified enzyme revealed that the particle is made up of four nonidentical submasses, corroborating previous structural data . The NH2-terminal amino acid sequences of the enzyme subunits exhibited significant similarities to those of only one other heteromeric formate dehydrogenase, the enzyme from the methane-utilizing bacterium Methylosinus trichosporium . Metal analyses yielded 21.5 g-atom iron, 2.18 g-atom nickel, 0.76 g-atom molybdenum, and 0.59 g-atom zinc per mol of enzyme . Initial electron paramagnetic resonance spectroscopic studies showed at least three distinct signals which appeared upon reduction of the enzyme with NADH or formate . The corresponding spin systems could be attributed to iron-sulfur centers of the enzyme . Comparative immunostaining and activity-staining experiments using cell extracts from various bacteria established immunological similarities between the soluble formate dehydrogenase of A . eutrophus and the soluble enzymes from all tested facultative autotrophs as well as from M . trichosporium.

Appl Environ Microbiol, 1995 Sep, 61(9), 3274 - 81
2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes; Fulthorpe RR et al.; DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4) . The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA, -B, and -C . Most strains showed combinations of tfdA-, B-, and C-like elements that exhibited various degrees of homology to the gene probes . Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico . This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe . Members of the beta subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB . However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity . Members of the alpha subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB . The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria.

Biochem J, 1995 Aug 1, 309 ( Pt 3), 983 - 92
The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha; Berks BC et al.; The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced . The large and small subunits of the enzyme are coded by napA and napB . The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor . Cysteine-181 is proposed to ligate the molybdenum atom . It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases . A four-cysteine motif at the N-terminus of NapA binds a {4Fe-4S} cluster . The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation . napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family . NapC may be the direct electron donor to the NapAB complex . napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1489 - 92
Metal-characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp . strain 5f-1; Wakayama M et al.; N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp . 5f-1 was a zinc-metalloenzyme which contained 2.06-2.61 g.atom of Zn per mole of enzyme . The zinc atom was required for the catalytic activity and stability of the enzyme . The N-terminal amino acid sequence of Pseudomonas sp . 5f-1 D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp . xylosoxydans A-6.

J Bacteriol, 1995 Aug, 177(15), 4442 - 50
Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus; Kusian B et al.; Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism . An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons . The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced . Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons . The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli . For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells . The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters . The deficient cbbR gene located on pHG1 was transcribed as well . A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters . Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions . In contrast, the cbbR promoters displayed low constitutive activities . The data suggest that the chromosomal and plasmid-borne cbb promoters of A . eutrophus H16 are functionally equivalent despite minor structural differences.

Appl Environ Microbiol, 1995 Aug, 61(8), 3113 - 8
Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates); Jaeger KE et al.; The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P . fluorescens GK13, as well as that of an esterase purified from P . fluorescens GK 13, to various polyesters and to lipase substrates were analyzed . All lipases and the esterase of P . fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases . However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate) . The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P . aeruginosa lipase . Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.

Jpn J Antibiot, 1995 Aug, 48(8), 1009 - 25
{Antimicrobial activities of norfloxacin against clinical isolates from ocular infections}; Koguchi M et al.; In order to evaluate antimicrobial activity of norfloxacin (NFLX), minimum inhibitory concentration (MICs) of NFLX and control drugs were determined against clinical isolates from ocular infections that were obtained in our laboratory from July, 1993 to December, 1994 . The results are summarized as follows; 1 . Compared to MIC distributions of NFLX against clinical isolates from ocular infections studied in 1986 and 1987, the MIC80 of NFLX against Corynebacterium spp., Enterobacter spp., Serratia spp., Burkholderia cepacia, Flavobacterium spp., Alcaligenes spp . increased 8 times . Almost all of NFLX-resistant strains among them were ofloxacin (OFLX)-resistant, new quinolones resistant strains, and a part of them were aminoglycosides, beta-lactams-resistant as well, thus all of these strains were multiple drug resistant . 2 . MIC of NFLX against Pseudomonas aeruginosa were lower than that of OFLX . 3 . NFLX showed strong antimicrobial activities against so-called "particular bacteria" including Staphylococcus aureus subsp . aureus, Moraxella spp., Haemophilus spp., and P . aeruginosa from ocular infections . And MIC80 of NFLX against these bacteria was 0.05-1.56 microgram/ml . We observed that NFLX eye drops was administered so that concentrations above the MIC against these clinical isolates were maintained.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6279 - 83
Enzyme-catalyzed synthesis of poly{(R)-(-)-3-hydroxybutyrate}: formation of macroscopic granules in vitro; Gerngross TU et al.; A combined chemical and enzymatic procedure has been developed to synthesize macroscopic poly{(R)-(-)-3-hydroxybutyrate} (PHB) granules in vitro . The granules form in a matter of minutes when purified polyhydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus is exposed to synthetically prepared (R)-3-hydroxybutyryl coenzyme A, thereby establishing the minimal requirements for PHB granule formation . The artificial granules are spherical with diameters of up to 3 microns and significantly larger than their native counterparts (0.5 micron) . The isolated PHB was characterized by 1H and 13C NMR, gel-permeation chromatography, and chemical analysis . The in vitro polymerization system yields PHB with a molecular mass > 10 x 10(6) Da, exceeding by an order of magnitude the mass of PHAs typically extracted from microorganisms . We also demonstrate that the molecular mass of the polymer can be controlled by the initial PHA synthase concentration . Preliminary kinetic analysis of de novo granule formation confirms earlier findings of a lag time for the enzyme but suggests the involvement of an additional granule assembly step . Minimal requirements for substrate recognition were investigated . Since substrate analogs lacking the adenosine 3',5'-bisphosphate moiety of (R)-3-hydroxybutyryl coenzyme A were not accepted by the PHA synthase, we provide evidence that this structural element of the substrate is essential for catalysis.

J Biochem (Tokyo), 1995 Jul, 118(1), 204 - 9
Primary structure of N-acyl-D-glutamate amidohydrolase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6; Wakayama M et al.; The gene coding the N-acyl-D-glutamate amidohydrolase of Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete DNA sequence was determined . The N-acyl-D-glutamate amidohydrolase structural gene consists of 1,464 nucleotides and encodes 488 amino acid residues . The molecular weight of the enzyme was calculated to be 51,490 . This value is close to the apparent molecular weight of 59,000 determined for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) . The N-terminal amino acid sequence of the recombinant protein exactly matches the amino acid sequence derived from the DNA sequence and that determined from the Alcaligenes A-6 enzyme (NH2-MQEKLDLVIEGGWVIDGLGG) . The deduced amino acid sequence of the cloned N-acyl-D-glutamate amidohydrolase showed high sequence homology with those of N-acyl-D-aspartate amidohydrolase (46%) and D-aminoacylase (47%) from Alcaligenes A-6 . This fact strongly suggests that these three enzymes have evolved from a common ancestral gene.

Curr Microbiol, 1995 Jul, 31(1), 55 - 61
Analysis of the cbbF genes from Alcaligenes eutrophus that encode fructose-1,6-/sedoheptulose-1,7-bisphosphatase; Yoo JG et al.; The cbbF genes of the facultative chemoautotroph Alcaligenes eutrophus H16 are part of two highly homologous cbb operons . Both the chromosomal and the megaplasmid pHG1-borne copy of cbbF were cloned and sequenced . Subsequent analyses including comparison with known sequences from other organisms and heterologous expression in Escherichia coli revealed that each of the genes encodes fructose-1,6-bisphosphatase (FBPase) . A closely related activity likewise operating in the Calvin carbon reduction cycle, sedoheptulose-1,7-bisphosphatase, was also catalyzed by the two isoenzymes which were purified from autotrophically grown cells of A . eutrophus . Two-dimensional gel electrophoresis allowed the separation of the cbbF gene products . Preliminary physical evidence by Southern hybridization with a heterologous gene probe was obtained for the existence of a third FBPase gene, fbp, on the chromosome of the organism . Its product is probably involved in the heterotrophic carbon metabolism.

J Ind Microbiol, 1995 Jul, 15(1), 55 - 9
The effect of chemical pretreatment on the aerobic microbial degradation of PCB congeners in aqueous systems; Aronstein BN et al.; A series of experiments was conducted to examine the effects of chemical pretreatment on biodegradation of 14C-labeled PCB congeners in aqueous systems . Fenton's reagent was used to generate hydroxyl radicals (OH) which were successful in partially oxidizing/transforming otherwise recalcitrant molecules of tetrachlorinated PCB, but had little or no impact on the biodegradation of a monochlorinated congener . Application of Fenton's reagent (1% H2O2, 1 mM FeSO4) followed by inoculation with pure cultures Pseudomonas sp, strain LB 400 and Alcaligenes eutrophus, strain H850 resulted in the removal of approximately 38% of 2-chlorobiphenyl and 51% of 2,2',4,4'-tetrachlorobiphenyl in the form of 14CO2 . Comparison of the rate and extent of biodegradation of 2,2',4,4'-tetrachlorobiphenyl after the application of Fenton's reagent with the dynamic and final level of radioactivity in the aqueous phase of experimental system suggests two possible means of microbial utilization of tetrachlorinated PCB congener altered by chemical oxidation: (a) consumption of the partially oxidized chemical dissolved in the aqueous phase, and (b) direct microbial attack on the transformed compound, which may still be adhered to the solid surface.

Can J Microbiol, 1995 Jul, 41(7), 612 - 9
Characterization by arbitrary primer polymerase chain reaction of polychlorinated biphenyl (PCB)-degrading strains of Comamonas testosteroni isolated from PCB-contaminated soil; Joshi B et al.; In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York . Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site . Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined . Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians . Owing to the abundance of C . testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis . The PCB congener degradation pattern revealed a high degree of variability among the C . testosteroni isolates . The majority of the C . testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachlorinated biphenyl . Only four isolates could degrade more than 80% of hexachlorobiphenyl . All 12 isolates of C . testosteroni tested were able to attack 2,3,4,5,6,3',4'-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2',3',6'-heptachlorobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase . 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C . testosteroni isolates tested in this study . Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW 169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol).(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Jul, 61(7), 2487 - 92
Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli; Rhie HG et al.; Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} . We have used E . coli fadR (FadR is a negative regulator of fatty acid oxidation) and E . coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain . E . coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+ . Similarly, E . coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products . Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.

J Bacteriol, 1995 Jul, 177(14), 4157 - 61
Molecular characterization of a deletion-prone region of plasmid pAE1 of Alcaligenes eutrophus H1; Chow WY et al.; A 93-kb region (D region) of plasmid pAE1 of Alcaligenes eutrophus H1 has been found to have a high rate of spontaneous deletion . In this study, we constructed a restriction endonuclease map and carried out limited sequencing of an approximately 100-kb region from pAE1 which includes the D region (the deleted region) in order to detect and characterize repetitive sequences . Two types of repetitive sequences, the R1 and R2 sequences, were observed to flank the D region; within the D region are three copies of insertion element ISAE1 . The R1 and R2 sequences are arranged in direct and inverted orientations, respectively . Molecular analysis of the end product of the deletion is consistent with the hypothesis that the loss of the D-region DNA is the result of recombination between two copies of the R1 sequence . The R1 sequence encodes a 415-amino-acid protein which exhibits substantial sequence similarity to the lambda integrase family of site-specific recombinases . Its genetic function remains to be determined.

J Bacteriol, 1995 Jul, 177(13), 3885 - 9
Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway; Kasberg T et al.; The maleylacetate reductase from Pseudomonas sp . strain B13 functioning in the modified ortho pathway was purified and digested with trypsin . The polypeptides separated by high-performance liquid chromatography were sequenced . Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp . strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained . In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology . Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases . A NAD-binding motif in a beta alpha beta-element was detected.

Vet Med (Praha), 1995 Jul, 40(7), 217 - 20
{The NEFERM test in the identification of psychrotrophic bacteria isolated from milk and dairy products}; Pacova Z et al.; Sanitary and nutriti on quality of foods from animal sources, that means also of milk and dairy products, are significantly influenced by the presence of psychrotrophic bacteria, the lipolytic and proteolytic activity of which contributes to their nutritive and sensory changes . This applies particularly to psychrotrophic bacteria of the genus Pseudomonas, Shewanella, Alcaligenes and Flavobacterium (Muir and Phillips, 1984; Piton and Richard, 1985; Craven and Macauley, 1990) . These bacteria cause raw and pasteurized milk to turn bitter and gelatinize, and/or technological problems during processing . In the present paper the possible use of NEFERM-test and numerical identification system TNW was verified for identification psychrotrophic, nonfermentative bacteria isolated from milk and dairy products . This research is a continuation of our preceding work (Urbanova and Pacova, 1995) dealing with identification of these bacteria in poultry . The test comprised 35 psychrotrophic, gram-negative, oxidase-positive nonfermentative bacteria isolated from raw milk (30 strains) and hard cheeses (5 strains) . A commercial diagnostic kit NEFERM-test (Lachema a.s., Brno) and respective conventional methods were parallelly used for identification: the conventional methods were complemented by assays of oxidase (OXI) and beta-galactosidase (ONPG) using of commercial strips Lachema a.s . Additional tests were as follows: gelatin and Tween 80 hydrolysis, fluorescein production . The readings of yellow or orange pigment production were also applied . A numerical identification system TNW was used to process the results (Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Brno) . Correct identification of bacteria by means of commercial diagnostic kits depends particularly upon the high goodness of fit of the results obtained by conventional tests in relation to those obtained by commercial kits.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1995 Jul, 17(1), 57 - 67
A physical genome map of the Burkholderia cepacia type strain; Rodley PD et al.; Burkholderia cepacia (basonym Pseudomonas cepacia), the type species of the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis . A physical map of the 8.1 Mb genome of the B . cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques . Placed onto the macrorestriction map were 38 SpeI, 11 SwaI, 11 PacI, 11 PmeI and six I-CeuI sites, resulting in an average resolution of 105 kbp . Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size . The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid . Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B . cepacia . The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.

Biochem Biophys Res Commun, 1995 Jun 15, 211(2), 540 - 6
Purification and some of the properties of a novel secondary alcohol dehydrogenase from Alcaligenes eutrophus; Madyastha KM et al.; Alcaligenes eutrophus utilizing nerolidol, a sesquiterpene alcohol, as the sole source of carbon contains an inducible NAD(P)(+)-linked secondary alcohol dehydrogenase (SADH) . The enzyme was purified to homogeneity by a combination of salt precipitation, ion exchange and affinity matrix chromatographies . The apparent molecular mass of the enzyme was estimated to be 139 KDa with four identical subunits of 38.5 KDa . The enzyme carried out both oxidation and reduction reactions . At pH 5.5, enzyme catalyzed the stereospecific reduction of prochiral ketones to secondary alcohols . The pH optimum for the oxidation reaction was 9.5 . NADP+ and NADPH were respectively preferred over NAD+ and NADH for oxidation and reduction reactions . Some of the properties of this enzyme were found to be significantly different from those thus far described.

Mol Gen Genet, 1995 Jun 10, 247(5), 546 - 54
Characterization of three genes in the dam-containing operon of Escherichia coli; Lyngstadaas A et al.; The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS . We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa . Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode . The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle . In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe . Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph . The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions . Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division . We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph . Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.

Environ Health Perspect, 1995 Jun, 103 Suppl 5, 37 - 9
Characterization of the first enzyme in 2,4-dichlorophenoxyacetic acid metabolism; Hausinger RP et al.; This paper reviews the properties of the Alcaligenes eutrophus JMP134 tfdA gene product, the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation . The gene was overexpressed in Escherichia coli and several of its enzymatic properties were characterized . Although this enzyme catalyzes a hydroxylation reaction, it is not a monooxygenase . Rather, TfdA is an Fe(II) and alpha-ketoglutarate-dependent dioxygenase that metabolizes the latter cosubstrate to succinate and carbon dioxide . A variety of other phenoxyacetates and alpha-ketoacids can be used by the enzyme, but the greatest catalytic efficiencies were found using 2,4-D and alpha-ketoglutarate . The enzyme possesses multiple essential histidine residues, whereas catalytically essential cysteine and lysine groups do not appear to be present.

J Bacteriol, 1995 Jun, 177(11), 3166 - 75
aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp; Xu J et al.; Escherichia coli aldB was identified as a gene that is negatively regulated by Fis but positively regulated by RpoS . The complete DNA sequence determined in this study indicates that aldB encodes a 56.3-kDa protein which shares a high degree of homology with an acetaldehyde dehydrogenase encoded by acoD of Alcaligenes eutrophus and an aldehyde dehydrogenase encoded by aldA of Vibrio cholerae and significant homology with a group of other aldehyde dehydrogenases from prokaryotes and eukaryotes . Expression of aldB is maximally induced during the transition from exponential phase to stationary phase . Its message levels are elevated three- to fourfold by a fis mutation and abolished by an rpoS mutation . In addition, the expression of an aldB-lacZ fusion was decreased about 20-fold in the absence of crp . DNase I footprinting analysis showed that five Fis binding sites and one Crp binding site are located within the aldB promoter region, suggesting that Fis and Crp are acting directly to control aldB transcription . AldB expression is induced by ethanol, but in contrast to that of most of the RpoS-dependent genes, the expression of aldB is not altered by an increase in medium osmolarity.

J Bacteriol, 1995 May, 177(10), 2707 - 12
The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli; Nies DH; The function of the CzcABC protein complex, which mediates resistance to Co2+, Zn2+, and Cd2+ in Alcaligenes eutrophus by cation efflux, was investigated by using everted membrane vesicles of Escherichia coli and an acridine orange fluorescence quenching assay . Since metal cation uptake could not be measured with inside-out membrane vesicles prepared from A . eutrophus and since available E . coli strains did not express the Czc-mediated resistance to cobalt, zinc, and cadmium salts, mutants of E . coli which exhibited a Czc-dependent increase in heavy metal resistance were isolated . E . coli mutant strain EC351 constitutively accumulated Co2+, Zn2+, and Cd2+ . In the presence of Czc, net uptake of these heavy metal cations was reduced to the wild-type level . Inside-out vesicles prepared from E . coli EC351 cells displayed a Czc-dependent uptake of Co2+, Zn2+, and Cd2+ and a cation-triggered acridine orange fluorescence increase . The czc-encoded protein complex CzcABC was shown to be a zinc-proton antiporter.

J Bacteriol, 1995 May, 177(9), 2513 - 23
Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules; Pieper-Furst U et al.; The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus . The GA14 protein had no influence on the biosynthesis rate of PHB in E . coli XL1-Blue(pSKCO7), but this recombinant E . coli strain formed smaller PHB granules than were formed by an E . coli strain that expressed only the PHB operon . Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules . In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U . Pieper-Furst, M . H . Madkour, F . Mayer, and A . Steinbuchel, J . Bacteriol . 176:4328-4337, 1994) . This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules . Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A . eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.

J Bacteriol, 1995 May, 177(9), 2425 - 35
Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus; Wieczorek R et al.; A 5.0-kbp genomic EcoRI restriction fragment which complemented a third subclass of polyhydroxyalkanoic acid (PHA)-leaky mutants of A . eutrophus that accumulated PHA at a lower rate than the wild type was cloned from Alcaligenes eutrophus H16 . A 687-bp phaPAe gene on this fragment encoded a 24-kDa protein (M(r) = 23,963), which was referred to as the GA24 protein . The GA24 protein was solubilized from the granules and purified to electrophoretic homogeneity, and antibodies against the GA24 protein were obtained . The GA24 protein bound to the surface of PHA granules, as revealed by immunoelectron microscopy of whole cells and of artificial PHA granules . The GA24 protein contributed approximately 5% (wt/wt) of the total cellular protein, and it was the predominant protein present in the granules . It was synthesized only in cells accumulating PHA and only in amounts that could be bound to the granules; no soluble GA24 protein was detected . Tn5::mob-induced phaPAe mutants which were unable to synthesize intact GA24 protein formed only one large PHA granule per cell . The amino acid sequence of the GA24 protein revealed two closely related stretches consisting exclusively of nonhydrophilic amino acids at the C-terminal region, which are presumably involved in the binding of GA24 to the granules, as was recently proposed for a similar protein in Rhodococcus ruber . The GA24 protein seems to be a representative of phasins, which are a new class of protein that form a layer at the surface of PHA granules, like oleosins, which form a layer at the surface of triacylglycerol inclusions in oilseed plants.

J Bacteriol, 1995 May, 177(9), 2373 - 80
Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA; Zimmer D et al.; Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 54 . Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity . Mobility shift assays with DNA fragments containing the SH upstream region and purified beta-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A . eutrophus failed to detect specific binding . In contrast, A . eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA . This effect was not observed with comparable extracts prepared from hoxA mutants . A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift . Extracts prepared from a temperature-tolerant mutant of A . eutrophus gave a stronger retardation than did those from the wild type . Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33 degrees C because of a mutation in the regulatory gene hoxA . In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for temperature tolerance . The SH upstream region also contains sequence motifs resembling the E . coli integration host factor (IHF) binding site, and purified E . coli IHF protein shifted the corresponding indicator fragment.

Appl Environ Microbiol, 1995 May, 61(5), 1750 - 6
Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media; Gross MJ et al.; Seven chemicals, three buffers, and a salt solution known to affect bacterial attachment were tested to quantify their abilities to enhance the penetration of Alcaligenes paradoxus in porous media . Chemical treatments included Tween 20 (a nonionic surfactant that affects hydrophobic interactions), sodium dodecyl sulfate (an anionic surfactant), EDTA (a cell membrane permeabilizer that removes outer membrane lipopolysaccharides), sodium PPi (a surface charge modifier), sodium periodate (an oxidizer that cleaves surface polysaccharides), lysozyme (an enzyme that cleaves cell wall components), and proteinase K (a nonspecific protease that cleaves peptide bonds) . Buffers included MOPS {3-(N-morpholino)propanesulfonic acid}, Tris, phosphate, and an unbuffered solution containing only NaCl . Transport characteristics in the porous media were compared by using a sticking coefficient, alpha, defined as the rate at which particles stick to a grain of medium divided by the rate at which they strike the grain . Tween 20 reduced alpha by 2.5 orders of magnitude, to alpha = 0.0016, and was the most effective chemical treatment for decreasing bacterial attachment to glass beads in buffered solutions . Similar reductions in alpha were achieved in unbuffered solutions by reducing the solution ionic strength to 0.01 mM . EDTA, protease, and other treatments designed to alter cell structures did not reduce alpha by more than an order of magnitude . The number of bacteria retained by the porous media was decreased by treatments that made A . paradoxus more hydrophobic and less electrostatically charged, although alpha was poorly correlated with electrophoretic mobility and hydrophobicity index measurements at lower alpha values.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 May, 61(5), 1691 - 8
Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation; Top EM et al.; The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation . This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil . After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected . Two A . eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A . eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D) . By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients . However, when untreated control soil was used, no transconjugants were isolated . The various transconjugants had plasmids with seven different EcoRI restriction patterns . The corresponding plasmids are designated pEMT1 to pEMT7 . Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group . Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene . Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagn Microbiol Infect Dis, 1995 May-Jun, 22(1-2), 43 - 8
Antimicrobial activity of cefotaxime tested against infrequently isolated pathogenic species (unusual pathogens); Cormican MG et al.; The cefotaxime sodium spectrum of activity is very broad and includes many common species and a variety of less frequently isolated pathogens . We have reviewed the clinical microbiology literature (44 references) and the data base of the University of Iowa Hospitals and Clinics (Iowa City, IA) to collect data on the activity of cefotaxime against the less common species . Cefotaxime was consistently active against Actinobacillus actinomycetemcomitans, Capnocytophaga spp., Eikenella corrodens, Erysipelothrix rhusiopathiae, Pasteurella multocida, Plesiomonas shigelloides, and Fusobacterium nucleatum . The species Alcaligenes xylosoxidans, Flavobacterium spp., Stenotrophomonas (Xanthomonas) maltophilia, Bacillus cereus, Listeria monocytogenes, and Rhodococcus equi were uniformly cefotaxime resistant . For many other species there was considerable variation in reported minimum inhibitory concentrations . These data may be helpful in guiding therapy of unusual infections, particularly in the case of fastidious species, where the appropriate susceptibility testing methodology may not be immediately or routinely available.

Eur J Clin Microbiol Infect Dis, 1995 May, 14(5), 451 - 4
Isolation of a Bordetella avium-like organism from a human specimen; Dorittke C et al.; The isolation of a strain of Bordetella for which the species could not be determined but which most closely resembled Bordetella avium is reported . The strain was isolated in mixed culture from an ear swab of a patient suffering from chronic otitis media . The bacterium showed the typical biochemical reactions of Bordetella avium but differed in antimicrobial resistance pattern, protein and fatty acid composition, and DNA-DNA and DNA-rRNA hybridization . Further studies will clarify the taxonomic status of this strain within the Bordetella-Alcaligenes ribosomal RNA cluster.

J Bacteriol, 1995 Apr, 177(7), 1840 - 3
The Alcaligenes eutrophus protein HoxN mediates nickel transport in Escherichia coli; Wolfram L et al.; HoxN, an integral membrane protein with seven transmembrane helices and a molecular mass of 33.1 kDa, is involved in high-affinity nickel transport in Alcaligenes eutrophus H16 . From genetic analyses, it has been concluded that HoxN is a single-component ion carrier . To investigate this assumption, hoxN was introduced into Escherichia coli . The recombinant strain showed significantly enhanced nickel uptake in a short-interval assay . Likewise, growth in the presence of 63NiCl2 yielded a more than 15-fold-increased cellular nickel content . The HoxN-based nickel transport activity could also be demonstrated in a physiological assay: an E . coli strain coexpressing hoxN and the urease operon of Klebsiella aerogenes exhibited urease activity 10-fold greater than that in the strain lacking a functional hoxN . These results strongly suggest that HoxN is sufficient to operate as a nickel permease . Multiple sequence alignment of HoxN and four other bacterial membrane proteins implicated in nickel metabolism revealed two conserved signatures which may play a role in the nickel translocation process.

Nat Struct Biol, 1995 Apr, 2(4), 287 - 92
The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase; Strange RW et al.; Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding . X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre . The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu-histidine bond by approximately 0.08 A . Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

Appl Microbiol Biotechnol, 1995 Apr, 43(1), 171 - 7
Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids; Fava F et al.; The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBZOH) and 2,5-dichlorobenzoic acid (2,5-Cl2BZOH) by Pseudomonas sp . CPE2 strain, and of 3-chlorobenzoic acid (3-ClBZOH), 4-chlorobenzoic acid (4-ClBZOH) and 3,4-dichlorobenzoic acid (3,4-Cl2BZOH) by Alcaligenes sp . CPE3 strain were investigated . Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBZOH, 2,5-Cl2BZOH, 3,4-Cl2BZOH, 3-ClBZOH and 2-ClBZOH by about 75%, 70%, 55%, 7%, and 1%, respectively . However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase . When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBZOH dechlorination increased by 30%-35%, whereas the rate of 2,5-Cl2BZOH and 3,4-Cl2BZOH dechlorination increased by only 2%-10% . The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid . The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.

Arch Microbiol, 1995 Apr, 163(4), 291 - 9
Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus; Schaferjohann J et al.; In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1 . Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA . Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively . The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases . A phenotypic complementation analysis with a cbb operon mutant of A . eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome . No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon . Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.

Appl Environ Microbiol, 1995 Apr, 61(4), 1391 - 8
Regulated expression of the Alcaligenes eutrophus pha biosynthesis genes in Escherichia coli; Kidwell J et al.; A novel poly-beta-hydroxybutyrate (PHB) production system in which the expression and gene dosage of the Alcaligenes eutrophus pha biosynthetic operon were effectively regulated by cultivation temperature was constructed in Escherichia coli . The pha operon was fused to the negatively regulated tac promoter and cloned into a vector in which the copy number is temperature dependent . A two-phase process was employed to produce PHB during fed-batch growth . In the growth phase, the culture was maintained at a low temperature . Under this condition, the plasmid copy number was depressed and the number of LacI proteins was sufficient to repress tacupha transcription . The production phase was initiated by temperature upshift . At the elevated temperature, the number of plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tacupha transcription, synthesis of poly-beta-hydroxyalkanoate-specific proteins, and polymer synthesis . During the production phase, the PHB production rate was 1.07 g of PHB liter-1 h-1 under optimized conditions . This rate is comparable to that of bacteria which naturally produce this polymer.

Proteins, 1995 Apr, 21(4), 351 - 3
Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein; Ermler U et al.; A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant . The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2(1) with unit cell parameters of 52.2 A, 85.8 A, 103.9 A, and 81.8 degrees corresponding to two molecules per asymmetric unit . The crystals diffract at least to a resolution of 2.0 A and are suitable for an X-ray structure analysis.

Mol Microbiol, 1995 Apr, 16(2), 321 - 31
Genetic and molecular analysis of a regulatory region of the herbicide 2,4-dichlorophenoxyacetate catabolic plasmid pJP4; You IS et al.; In Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba) degradation plus mercury resistance . The plasmid genes specifying 2,4-D and 3-Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF . Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed . Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592 bp . Similar stem-loop structures were observed among other 2,4-D plasmids . The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32 kDa . The deduced amino acid sequence of tfdR indicates that it is a member of the LysR-type activators . Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4-D negative, but 3-Cba positive . In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4-D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.

Int J Biol Macromol, 1995 Apr, 17(2), 86 - 92
Biosynthesis and characterization of hydroxybutyrate-hydroxycaproate copolymers; Caballero KP et al.; Most polyhydroxyalkanoates (PHAs) reported to date fall into one of two broad classes: either hydroxybutyrate-hydroxyvalerate copolymers (typified by the PHA produced by Alcaligenes eutrophus), or hydroxyoctanoate-rich heteropolymers (typified by the PHA produced by Pseudomonas oleovorans) . Few reports of copolymers rich in hydroxybutyrate (HB), but containing a minor proportion of a co-monomer with a higher carbon number than valerate, have appeared . Here we report on the biosynthesis and characterization of HB-rich polymers containing 2-4 mol% of hydroxycaproate (HC) units, as well as a terpolymer containing HC and hydroxyoctanoate (HO) units . These polymers were produced in good yield by Comomonas testosteroni, Bacillus cereus and an unidentified third organism when grown on caproate or octanoate . The minor co-monomers were found to be rejected from the PHB crystallites by X-ray analysis and by quantitative analysis of the melting point depression . The greatly reduced melting point, coupled with the retention of a high degree of crystallinity, could make these materials attractive as melt-processible thermoplastics.

Biochem Biophys Res Commun, 1995 Mar 28, 208(3), 943 - 9
Cloning and sequencing of the catechol 2,3-dioxygenase gene of Alcaligenes sp . KF711; Moon J et al.; The catechol 2,3-dioxygenase is an aromatic ring-fission enzyme catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde . A catechol 2,3-dioxygenase gene has been cloned from chromosomal DNA of Alcaligenes sp . KF711, and its sequence was determined . The catechol 2,3-dioxygenase gene was consisted of 927 nucleotides with ATG initiation codon and TGA termination codon, which can encode a polypeptide of molecular weight 35 kDa containing 308 amino acid residues . G+C content of the gene was 58 mol%, and a putative ribosome-binding sequence was identified at about 10 nucleotides upstream from the ATG initiation codon . The sequence of catechol 2,3-dioxygenase from Alcaligenes sp . KF711 exhibited 81-92% homology at nucleotide level and 84-92% homology at amino acid level with those of corresponding enzymes encoded in xylE of TOL plasmid, nahH of NAH7 plasmid, and dmpB of Pseudomonas CF600.

J Biol Chem, 1995 Mar 24, 270(12), 6991 - 6
Regulation of Saccharomyces cerevisiae flavohemoglobin gene expression; Crawford MJ et al.; The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function . It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species . We have studied its gene regulation in order to better understand its purpose in the cell . Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S . cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions . Transcription of the S . cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels . Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA . Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed . Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources . Physical and genetic mapping of the S . cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus . These data indicate that, despite the high degree of homology, the S . cerevisiae globin may have a function distinct from those proposed for bacterial globins.

Gene, 1995 Mar 21, 155(1), 95 - 100
Identification and characterization of the insertion element IS1070 from Leuconostoc lactis NZ6009; Vaughan EE et al.; A novel insertion sequence, designated IS1070, was identified on the lactose plasmid of Leuconostoc lactis NZ6009 by nucleotide sequence analysis . The 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame . The deduced 305-amino-acid sequence demonstrated homology to transposases of IS30 from Escherichia coli, IS4351 from Bacteroides fragilis, IS1086 from Alcaligenes eutrophus, IS1161 from Streptococcus salivarius, ISAS2 from Aeromonas salmonicida and a putative protein encoded by ORF3 of virus SpV1-R8A2 B from Spiroplasma citri . At least fifteen IS1070-like sequences were detected in the genome of the parent Lc . lactis strain and five of these were situated on plasmids . Analysis of the direct repeats of two of these copies with that of IS1070 revealed differences in the target duplication lengths.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 51 - 5
Degradation of trichlorophenols by Alcaligenes eutrophus JMP134; Clement P et al.; The degradation of chlorophenols by Alcaligenes eutrophus JMP134 (pJP4) was studied . The strain grew on 2,4,6-trichlorophenol or 2,4,6-tribromophenol as the sole carbon and energy source . Complete degradation of 2,4,6-trichlorophenol was confirmed by chloride release and gas chromatography analysis of supernatants from growth cultures . The 2,3,5-, 2,3,4-, 2,3,6- and 2,4,5-isomers of trichlorophenol did not support growth . However, up to 40% of 2,4,5-trichlorophenol was mineralized during growth of A . eutrophus on chemostats fed with either phenol (0.4 mM) or 2,4,6-trichlorophenol (0.4 mM) plus 2,4,5-trichlorophenol (0.1 mM) . Growth on 2,4,6-trihalophenols was also observed in A . eutrophus JMP222, the strain lacking pJP4, suggesting that this new degradative ability reported for A . eutrophus is not related to pJP4 encoded catabolic functions.

Arch Biochem Biophys, 1995 Mar 10, 317(2), 449 - 56
Kinetic mechanism studies of the soluble hydrogenase from Alcaligenes eutrophus H16; Keefe RG et al.; Purified soluble hydrogenase (H2:NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH . H2-dependent NAD+ reduction activities > or = 110 mumol NADH formed/min/mg protein at pH 8.0 and 30 degrees C were obtained which were stable for several hours at 4 degrees C . Kinetic studies were conducted anaerobically using activated enzyme for the purpose of evaluating the potential of using hydrogenase to enhance decompression of mammals breathing H2/O2 mixtures under hyperbaric conditions (i.e., at ambient pressures greater than 1 atm) . Using nonlinear curve fitting of the kinetic data, it was found that H2 and NAD+ bind hydrogenase via a ping pong bi bi mechanism with Km values (+/- SE) of 11 +/- 0.9 and 138 +/- 11 microM, respectively, at 30 degrees C and pH 8.0 . Sodium ions were found to reversibly inhibit hydrogenase via a dead-end type of inhibition in which two catalytic forms of the enzyme bind Na+ with dissociation constants calculated to be 8.3 +/- 1.2 and 49.8 +/- 11.5 mM . In the absence of NaCl, maximum NAD+ reduction activity was measured at pH 8.3 at 30 and 37 degrees C . In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values < or = 7.0, little or no difference was observed in activity in the presence or absence of 50 mM NaCl at a given temperature . Least squares analyses of the kinetic data indicated that substrate inhibition by H2 occurs at high substrate concentrations (Ki = 1.46 +/- 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H2.

Res Microbiol, 1995 Mar-Apr, 146(3), 245 - 50
Introduction of Pseudomonas aeruginosa mutator phage D3112 into Alcaligenes eutrophus strain CH34; Krylov V et al.; We have investigated the possibility of growing mutator phages from Pseudomonas aeruginosa on various isolates of Alcaligenes eutrophus . Although none out of 10 A . eutrophus strains were susceptible to infection with any of the phages tested, phage D3112 could be readily transferred in our model strain CH34 by means of an RP4::D3112 plasmid . CH34/RP4::D3112 lysogens were stable and produced phages . However, neither mitomycin C nor UV treatment increased the phage yield.

Am J Trop Med Hyg, 1995 Mar, 52(3), 231 - 5
Monoclonal antibodies to Pseudomonas pseudomallei and their potential for diagnosis of melioidosis; Rugdech P et al.; Monoclonal antibodies (MAbs) specific for Pseudomonas pseudomallei antigens were produced by immunizing BALB/c mice with a crude whole cell extract . Hybrids secreting MAbs specific for P . pseudomallei antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of crude whole cell extracts from P . pseudomallei, P . cepacia, P . aeruginosa, P . putida, P . alcaligenes, Xanthomonas maltophilia, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Salmonella typhi, S . krefeld, S . enteritidis, Proteus mirabilis, and Staphylococcus aureus . Of the six specific clones, clone 5F8, which was IgM-producing and which reacted with all 56 P . pseudomallei isolates, was selected for further characterization and evaluation of its possible diagnostic potential . Results obtained from the indirect ELISA against various P . pseudomallei antigens, from direct bacterial agglutination, and from immunofluorescence tests suggested that 5F8 reacted with the surface envelope, and probably specifically with an epitope of the lipopolysaccharide . The antibody could be readily used to identify P . pseudomallei in primary culture or in a simulated hemoculture . The antibody was also used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in patients with acute septicemic melioidosis.

Gene, 1995 Feb 27, 154(1), 81 - 5
Characterization of an acetyl-CoA C-acetyltransferase (thiolase) gene from Clostridium acetobutylicum ATCC 824; Stim-Herndon KP et al.; Thiolase (Thl) is an important enzyme at the junction in the pathway leading to the production of either acids (acetate or butyrate) or solvents (acetone, butanol or ethanol) during the growth of Clostridium acetobutylicum ATCC 824 . Cloning and expression of the Thl-encoding gene (thl) has been described {Petersen and Bennett, Appl . Environ . Microbiol . 57 (1991) 2735-2741}, as has the purification and properties of the enzyme {Wiesenborn et al., Appl . Environ . Microbiol . 54 (1988) 2717-2722} . Here, we present the complete nucleotide sequence (1.9 kb) of thl . The gene encodes a protein of 392 amino acids (aa) (41,237 Da), which mass is in agreement with previous findings using the purified protein . Primer extension analysis has defined the promoter region, and a stem-loop structure found at the end of thl indicates that it is not part of an operon . The aa sequence of Thl showed homology to those of four other beta-ketothiolases: (i) PhbC of Alcaligenes eutrophus, (ii) PhbA of Chromatium vinosum, (iii) PhbA of Thiocystis violacea and (iv) PhbA of Zoogloea ramigera . The C terminus of an open reading frame found upstream from the Thl sequence is similar to OrfX of Bacillus subtilis and to NfrC of Escherichia coli.

Int J Biol Macromol, 1995 Feb, 17(1), 7 - 12
Synthesis of high-molecular-weight poly({R}-(-)-3-hydroxybutyrate) in transgenic Arabidopsis thaliana plant cells; Poirier Y et al.; High-molecular-weight poly({R}-(-)-3-hydroxybutyrate) (PHB), a biodegradable thermoplastic, was produced from a suspension culture of transgenic Arabidopsis thaliana plant cells expressing two genes from the bacterium Alcaligenes eutrophus involved in the synthesis of PHB . The molecular structure of the plant-produced polymer was analysed by gas chromatography, mass spectrometry, proton nuclear magnetic resonance spectroscopy, infra-red spectroscopy, spectropolarimetry, differential scanning calorimetry, X-ray diffraction and size exclusion chromatography . The results indicate that the polymer from transgenic plants appears to have a chemical structure identical to that of PHB produced by bacteria . However, the molecular weight distribution of the plant-produced PHB was much broader than that of typical bacterial PHB.

J Ind Microbiol, 1995 Feb, 14(2), 186 - 99
Ion efflux systems involved in bacterial metal resistances; Nies DH et al.; Studying metal ion resistance gives us important insights into environmental processes and provides an understanding of basic living processes . This review concentrates on bacterial efflux systems for inorganic metal cations and anions, which have generally been found as resistance systems from bacteria isolated from metal-polluted environments . The protein products of the genes involved are sometimes prototypes of new families of proteins or of important new branches of known families . Sometimes, a group of related proteins (and presumedly the underlying physiological function) has still to be defined . For example, the efflux of the inorganic metal anion arsenite is mediated by a membrane protein which functions alone in Gram-positive bacteria, but which requires an additional ATPase subunit in some Gram-negative bacteria . Resistance to Cd2+ and Zn2+ in Gram-positive bacteria is the result of a P-type efflux ATPase which is related to the copper transport P-type ATPases of bacteria and humans (defective in the human hereditary diseases Menkes' syndrome and Wilson's disease) . In contrast, resistance to Zn2+, Ni2+, Co2+ and Cd2+ in Gram-negative bacteria is based on the action of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is called RND) . Another new protein family, named CDF for 'cation diffusion facilitator' has as prototype the protein CzcD, which is a regulatory component of a cobalt-zinc-cadmium resistance determinant in the Gram-negative bacterium Alcaligenes eutrophus . A family for the ChrA chromate resistance system in Gram-negative bacteria has still to be defined.

J Ind Microbiol, 1995 Feb, 14(2), 142 - 53
The czc operon of Alcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals; Diels L et al.; The plasmid-borne czc operon ensures for resistance to Cd2+, Zn2+ and Co2+ ions through a tricomponent export pathway and is associated to various conjugative plasmids of A . eutrophus strains isolated from metal-contaminated industrial areas . The czc region of pMOL30 was reassessed especially for the segments located upstream and downstream the structural genes czc CBA . In cultures grown with high concentrations of heavy metals, czc-mediated efflux of cations is followed by a process of metal bioprecipitation . These observations led to the development of bioreactors designed for the removal of heavy metals from polluted effluents.

Biofactors, 1995-96, 5(2), 87 - 92
FMN cofactor dissociation from the soluble hydrogenase of Alcaligenes eutrophus H16; Axley MJ et al.; The specific activity of purified soluble hydrogenase of Alcaligenes eutrophus H16 was found to vary with enzyme concentration . Specific activity as a function of concentration of purified enzyme could be fit to an equation describing the dissociation of a compound into two components . An association constant, kappa(a), was determined in this way to be 39.4 +/- 8.7 micrograms protein/ml . The concentration of the enzyme affected its kinetic parameters: a tenfold decrease in enzyme concentration caused by a reduction of the V(max) and Kappa(m) (NAD) values to 45% and 58%, respectively, of the values for undiluted (0.64 mg/ml) enzyme . Diaphorase (NAD-dependent reduction of benzyl viologen) specific activity of the hydrogenase was unaffected by dilution . The extent of dilution-induced activity loss was dependent on pH, with greater activity loss observed at higher pH values . The substrate NAD prevented loss of specific activity due to dilution, while the product NADH did not . Specific activity loss due to dilution as reversed with the addition of the cofactor FMN . Dilution of the hydrogenase caused an increase in the enzyme's specific flavin fluorescence . These results suggest that dilution of the soluble hydrogenase of Alcaligenes eutrophus causes dissociation of the cofactor FMN, and this activity loss should be taken into account as an important factor governing hydrogenase activity and kinetic properties.

Microbiol Immunol, 1995, 39(11), 897 - 904
Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen . Nov.: Proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb . Nov., Ralstonia solanacearum (Smith 1896) comb . Nov . and Ralstonia eutropha (Davis 1969) comb . Nov; Yabuuchi E et al.; Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phylogenetic analysis of 16S rDNA nucleotide sequences and rNA-DNA hybrization, Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia, and Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb . nov., Ralstonia solanacearum (Smith 1896) comb . nov., and R . eutropha (Davis 1969) comb . nov . are proposed . The type species of the new genus is R . pickettii . Type strain of R . pickettii is ATCC 27511T, of R . solanacearum is ATCC 10696T, and of R . eutropha is ATCC 17697T.

Appl Environ Microbiol, 1995 Jan, 61(1), 34 - 9
Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli; Hahn SK et al.; We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A . eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes . The amount of PHB degraded to a lower-molecular-weight compound in A . eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E . coli strain was negligible . However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform . To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance . The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E . coli strain were crystalline, while most of the PHB in A . eutrophus was in a mobile amorphous state . The stability of the native PHB in the recombinant E . coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology . In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E . coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction . The PHB preparations extracted from the two microorganisms had identical polymer properties.

Can J Microbiol, 1995, 41 Suppl 1, 216 - 21
High-level poly(beta-hydroxybutyrate) production in recombinant Escherichia coli in sugar-free, complex medium; Kalousek S et al.; The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively . To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E . coli transcription and translation signals for the PHB synthase gene (phbC) . The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain . The T7 gene 10 ribosome binding site is utilized for translational initiation . PHB production in E . coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence . In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E . coli when grown in Luria-Bertani medium without glucose supplement.

Can J Microbiol, 1995, 41 Suppl 1, 207 - 15
Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies; Lee SY et al.; A number of Escherichia coli strains including K12, B, W, XL1-Blue, DH5 alpha, HB101, JM109, and C600 were transformed with the stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid biosynthesis genes, and were subsequently compared for their ability to synthesize and accumulate poly(3-hydroxybutyric acid) (PHB) . The rate of PHB synthesis, the extent of PHB accumulation, and PHB yield from glucose varied considerably from one strain to another . Strains XL1-Blue and B harboring pSYL105 synthesized PHB at the highest rate to a final concentration of ca . 7 g/L in complex medium containing 20 g glucose/L . With an aim to reduce the cost of the medium, the effect on PHB accumulation of supplementing a defined medium with complex nitrogen sources was examined . A PHB concentration of 81 g/L could be obtained in 41 h from a pH-stat fed-batch culture of XL1-Blue(pSYL105) in a semidefined medium . When the availability of acetyl-CoA was increased by supplementing the medium with complex nitrogen sources, amino acids, or oleic acid, PHB synthesis by recombinant E . coli was enhanced.

Eur J Biochem, 1994 Dec 15, 226(3), 1053 - 61
Chlorocatechol 1,2-dioxygenase from Rhodococcus erythropolis 1CP . Kinetic and immunochemical comparison with analogous enzymes from gram-negative strains; Maltseva OV et al.; Chlorocatechol 1,2-dioxygenase from Rhodococcus erythropolis 1CP was purified to homogeneity . In contrast to chlorocatechol 1,2-dioxygenase from Gram-negative strains which have a very broad substrate tolerance, the Rhodococcus enzyme was relatively more specific and had a distinct preference for 4-substituted catechols . Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and manganese/64-kDa homodimer . The N-terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2-dioxygenase was determined and exhibited 15-22% identity to the published sequences of catechol 1,2-dioxygenases and other chlorocatechol 1,2-dioxygenases . Antiserum was raised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2-dioxygenase were affinity purified . Dot-blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocatechol 1,2-dioxygenases from Alcaligenes eutrophus JMP134 and Pseudomonas putida 87 . No reaction between these antibodies and above enzymes was observed using Western blotting . Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rhodococcus enzyme differs significantly from the known highly similar chlorocatechol 1,2-dioxygenases of Gram-negative strains and seems to be only distantly related to them.

FEMS Microbiol Lett, 1994 Dec 1, 124(2), 141 - 50
Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway; Huang M et al.; The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin . Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), apoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region . The amino acid sequences deduced from acoA, acoB, and acoC for E1 alpha (M(r) 34639), E1 beta (M(r) 37268), and E2 (M(r) 39613) of the P . putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A . eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria . Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M(r) 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases . Expression of the P . putida ADH in Escherichia coli was demonstrated . The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.

Biodegradation, 1994 Dec, 5(3-4), 301 - 21
Evolution of chlorocatechol catabolic pathways . Conclusions to be drawn from comparisons of lactone hydrolases; Schlomann M; The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates . They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modified ortho-cleavage pathway . Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity . In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone . While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant . They seem to share an alpha/beta hydrolase fold, but the sequences comprising the fold are quite dissimilar . Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway . Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains of Alcaligenes and Burkholderia (Pseudomonas) cepacia is investigated) . Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor . The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.

J Mol Biol, 1994 Nov 4, 243(4), 549 - 57
Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster; Du L et al.; Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome . Six accessory genes of the cluster, hupABYCDE, begin 4.8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional {NiFe} hydrogenase . The sequencing of the intervening 4.8 kb of hup-specific DNA has now been completed . This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT and hupV . These genes potentially encode polypeptides with predicted masses of 27.7, 22.3, 11.4, 16.2, 31.3, 8.1, 16.2 and 36.7 kDa, respectively . All eight genes are transcribed from the same strand as hupSL and hupABYCDE . A chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE . The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome . Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O2-dependent H2 oxidation . The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins . HupZ and HupR may both be involved in transferring electrons from hydrogenase to the electron transport chain . A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase . It is now clear that the genes controlling {NiFe} hydrogenase activity in many bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.

Appl Environ Microbiol, 1994 Nov, 60(11), 4053 - 8
Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil; Neilson JW et al.; Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment . Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms . The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site . Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation . A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil . Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4 . In addition, we confirmed the transfer through PCR amplifications of the tfdB gene . Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses . An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

J Bacteriol, 1994 Nov, 176(22), 7045 - 54
Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A; Schmidt T et al.; The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment . By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre . The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34 . ncc is not expressed in Escherichia coli . The nre locus causes low-level nickel resistance in both Alcaligenes and E . coli strains . The nucleotide sequence of the ncc locus revealed seven open reading frames designated nccYXHCBAN . The corresponding predicted proteins share strong similarities with proteins encoded by the metal resistance loci cnr (cnrYXHCBA) and czc (czcRCBAD) of A . eutrophus CH34 . When different DNA fragments carrying ncc genes were heterologously expressed under the control of the bacteriophage T7 promoter, five protein bands representing NccA (116 kDa), NccB (40 kDa), NccC (46 kDa), NccN (23.5 kDa), and NccX (16.5 kDa) were detected.

Appl Environ Microbiol, 1994 Oct, 60(10), 3585 - 91
Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments; Taghavi S et al.; Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Alcaligenes eutrophus CH34 . A 12.9-kb A . eutrophus-Escherichia coli shuttle vector, pMOL850, was constructed to optimize electroporation conditions . This vector is derived from the E . coli plasmid pSUP202 and contains the replication region of the A . eutrophus megaplasmid pMOL28 . Electroporation was used to transform A . eutrophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and transformants were selected for resistance to heavy metals . Electroporation was also performed with endonuclease-digested genomic DNA . Transformation of markers affecting lysine biosynthesis (lysA194) and biosynthesis of the siderophore alcaligin E were observed . Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination . However, during transformation of ale::Tn5-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers.

Curr Microbiol, 1994 Oct, 29(4), 229 - 35
Cloning and sequencing of a chromosomal fragment from Clostridium acetobutylicum strain ABKn8 conferring chemical-damaging agents and UV resistance to E . coli recA strains; Azeddoug H et al.; A 3.3-kb DNA fragment of Clostridium acetobutylicum conferred methyl methane sulfonate (MMS), mitomycin C (MC), and UV resistance to recA strains of E . coli when cloned on the pUC19 plasmid . Analysis of the nucleotide sequence of the total insert and results of in vitro transcription-translation experiments showed that the insert directed the synthesis of three polypeptides referred to as ORFa, ORFb, and ORFc of 23.6, 15.3, and 21 kDa, respectively . None of the polypeptides presented a relationship with the RecA protein of E . coli or products of genes involved in the SOS response . The deduced amino acid sequence of ORFb and ORFc are highly homologous to those deduced from two genes specifying resistance to tellurium salts present on plasmid pMER610 harbored by Alcaligenes sp.strains and to an AMP-binding protein (CABP1) found in Dictyostelium discoideum . The existence of these homologous proteins suggests that they may perform a similar key function in the three unrelated organisms.

J Bacteriol, 1994 Sep, 176(17), 5401 - 8
Identification of a novel gene, aut, involved in autotrophic growth of Alcaligenes eutrophus; Freter A et al.; The aerobic facultative chemoautotroph Alcaligenes eutrophus was found to possess a novel gene, designated aut, required for both lithoautotrophic (hydrogen plus carbon dioxide) and organoautotrophic (formate) growth (Aut+ phenotype) . Insertional mutagenesis by transposon Tn5-Mob localized the gene on a chromosomal 13-kbp EcoRI fragment . Physiological characterization of various Aut- mutants revealed pleiotropic effects caused by the transposon insertion . Heterotrophic growth of the mutants on substrates catabolized via the glycolytic pathway was slower than that of the parent strains, and the colony morphology of the mutants was altered when grown on nutrient agar . The heterotrophic derepression of the cbb operons encoding Calvin cycle enzymes was abolished, although their expression was still inducible in the presence of formate . Apparently, the mutation did not affect the cbb genes directly but impaired the autotrophic growth in a more general manner . The conjugally transferred wild-type EcoRI fragment allowed phenotypic in trans complementation of the mutants . Further subcloning and sequencing identified a single open reading frame (aut) of 495 bp that was sufficient for complementation . The monocistronic aut gene was constitutively transcribed into a 0.65-kb mRNA . However, its expression appeared to be low . Heterologous expression of aut was achieved in Escherichia coli, resulting in overproduction of an 18-kDa protein . Database searches yielded weak partial sequence similarities of the deduced Aut protein sequence to some cytidylyltransferases, but no indication for the exact function of the aut gene was obtained . Hybridizing DNA sequences that might be similar to the aut gene were detected by Southern hybridization in the genome of two other autotrophic bacteria.

J Bacteriol, 1994 Sep, 176(17), 5284 - 9
Integration and excision of a 2,4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer; Ka JO et al.; A self-transmissible 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmid, pKA2, has been identified in a new 2,4-D-degrading strain, Alcaligenes paradoxus 2811P, isolated from agricultural soil . pKA2 occurred as a 42.9-kb plasmid in strain 2811P . A derivative strain, 2811C, was isolated from a stock culture in which the entire pKA2 plasmid was apparently integrated into the host chromosome without loss of the 2,4-D+ phenotype . This interpretation is based on the disappearance of a free plasmid DNA band, a shift in the tfdA-hybridizing band to the chromosome, loss of transmissibility of the 2,4-D+ trait, and appropriate shifts in Southern hybridization bands of plasmid DNA compared with whole-cell DNA . The integrated plasmid of strain 2811C was excised either precisely or imprecisely after continued transfer on 2,4-D-containing medium . This suggests that a chromosome-free plasmid cycle may occur to optimize fitness under conditions of specific resource fluctuation . Another new 2,4-D-degrading strain, Pseudomonas pickettii 712, which was isolated from the same field plot but at a different time, was found to carry a plasmid that is nearly identical to pKA2 . The plasmid of this strain, pKA4, is 40.9 kb long and has features in common with pKA2, such as high self-transmissibility, hybridization only to the tfdA gene among the 2,4-D-metabolic genes of 2,4-D-degradative plasmid pJP4, and similar restriction endonuclease-generated fragments . Furthermore, the genetic homology between the two plasmids was high since all fragments of pKA2 hybridized to pKA4 . These results suggest that these two plasmids are closely related and thus their occurrence in two genera in nature is the result of natural horizontal gene transfer.

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