Alcaligenes

Infect Immun, 1994 Sep, 62(9), 4075 - 80
Shuttle mutagenesis of Legionella pneumophila: identification of a gene associated with host cell cytopathicity; Arroyo J et al.; We performed shuttle mutagenesis of Legionella pneumophila . Mutants were screened for reduced cellular infectivity . Approximately 10% of the mutants had decreased cytopathicity . The DNA sequence of one locus was determined; the inferred amino acid sequence revealed homology with transport proteins including Escherichia coli TolC, Bordetella pertussis CyaE, and Alcaligenes eutrophus CzcC and CnrC.

Undersea Hyperb Med, 1994 Sep, 21(3), 265 - 75
Hydrogen gas is not oxidized by mammalian tissues under hyperbaric conditions; Kayar SR et al.; Mammalian tissues, including heart, lung, liver, kidney, spleen, and skeletal muscle of guinea pig, rat, or pig, were exposed to tritium (T2) and high pressures of H2 . Incorporation of the tritium label was measured to test for a latent capacity by mammalian tissues to oxidize H2 under conditions such as those experienced by deep divers breathing H2 . Tissues were removed aseptically, and either minced, homogenized, or prepared as live cell cultures . The tissues were placed in a chamber to which 8 mCi T2, 1 MPa He, and either 1 or 5 MPa H2 were added . After 1 h the chamber was decompressed . The tissues were spun briefly in a vortex mixer to facilitate elimination of T2 in the gas phase . Samples were analyzed by scintillation counting for tritium incorporation in the liquid phase or in the tissues . Saline and distilled water were used as negative controls . Palladium (Pd) beads immersed in water, and cultures of the H2-metabolizing bacterium Alcaligenes eutrophus were used as positive controls . The tissues incorporated on the order of 10 nCi T2.ml-1, which implied a H2 incorporation of 10-50 nmol H2.g-1.min-1 . However this incorporation was not different from that found in the water controls and was attributed to radioisotope effects . The Pd and bacterial samples incorporated over 1,000-fold more T2 than the mammalian tissues . We concluded that the mammalian tissues did not oxidize H2 under hyperbaric conditions, with a limit of detection of 100 nmol H2.g-1.min-1.

Biochemistry, 1994 Aug 9, 33(31), 9311 - 20
Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity; Gerngross TU et al.; Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E . coli UT5600 (ompT-) . Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein . The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1 . The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers . The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms . Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA . Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process . A mechanistic model has been proposed for the polymerization process {Griebel, R., Smith, Z., & Merrick, J . (1968) Biochemistry 7, 3676-3681} in which two cysteines are required for catalysis . This model is based on the well-characterized enzymes involved in fatty acid biosynthesis . To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared . The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein . CD and antibody studies suggest that the mutant proteins are properly folded . The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well . This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E . coli SJ16, requiring beta-alanine for growth . Growth of SJ16/pKAS4 on {3H}-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein . These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7573 - 7
Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions; Lonetto MA et al.; sigma E, an RNA polymerase sigma factor of apparent M(r) 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarose gene (dagA) of Streptomyces coelicolor . A degenerate oligonucleotide probe, designed from the N-terminal sequence of purified sigma E, was used to isolate the sigma E gene (sigE) . The predicted sequence of sigma E shows greatest similarity to sequences of seven other proteins: Myxococcus xanthus CarQ, Pseudomonas aeruginosa AlgU, Pseudomonas syringae HrpL, Escherichia coli sigma E, Alcaligenes eutrophus CnrH, E . coli FecI, and Bacillus subtilis SigX, a protein of unknown function . These eight proteins define a subfamily of eubacterial RNA polymerase factors sufficiently different from other sigma s that, in many cases, they are not identified by standard similarity searching methods . Available information suggests that all of them regulate extracytoplasmic functions and that they function as effector molecules responding to extracytoplasmic stimuli . A . eutrophus CnrH appears to be a plasmid-encoded factor.

FEMS Microbiol Rev, 1994 Aug, 14(4), 405 - 14
Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications; Collard JM et al.; Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals . It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO(4)2-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+ . Among the best characterized determinants are the cnr operon (resistance to Co, Ni) on pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn) . Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different . The resistance mechanism in both cases is based on efflux . The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane . Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell . This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents . The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR) . The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium . Metal crystals are eventually removed by the effluent stream and collected on a glass bead column . The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu . By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A . eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances . Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri . Bacterial luciferases produce light through the oxidation of fatty aldehydes . The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment.

J Biochem (Tokyo), 1994 Aug, 116(2), 229 - 35
Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins; Yamamoto K; Microbial endoglycosidases are useful for elucidating the structure and function of the oligosaccharide chains of glycoconjugates . Most of the microbial endo-beta-N-acetylglucosaminidases including Endo-H can preferentially act on high-mannose type chains of asparagine-linked oligosaccharides of various glycoproteins . Among them, Flavobacterium sp . enzyme is produced in large amounts by the inducing cultivation . Using this enzyme, the role of oligosaccharide chains in various microbial glycoenzymes such as Rhizopus glucoamylase, and yeast invertase was examined . The findings suggested that the oligosaccharide chains of them are essential participants in the stabilization of the enzyme and in the protection from proteolytic inactivation . Novel endo-beta-N-acetylglucosaminidases were also found in the culture broths of microorganisms . Unlike most microbial endo-beta-N-acetylglucosaminidases, Endo-M of Mucor hiemalis could act on a complex type oligosaccharide chains, which is similar to Endo-F2 in multiple form of Endo-F from Flavobacterium meningosepticum . The complete amino acid sequences of Endo-F1, -F2, -F3, -H, and Flavobacterium sp . enzyme were determined . All of them had two highly conserved regions common to a number of chitinases . Endo-alpha-N-acetylgalactosaminidase which hydrolyzes the O-glycosidic linkages in glycoproteins was found in the culture broth of only a few microorganisms . The production of Alcaligenes sp . enzyme was highly induced by the addition of porcine gastric mucin in the culture medium . There is some evidence that endo-alpha-N-acetylgalactosaminidases may recognize not only the glycon but also the aglycon amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiology, 1994 Jul, 140 ( Pt 7), 1713 - 22
3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.-) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp . strain O-1; Junker F et al.; Alcaligenes sp . strain O-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium-benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS) . The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage . The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate . Cells were harvested at this point from BS-, TS- and 2AS-salts medium . Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS . BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC) . These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS . We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells . A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC23O) were detected in 2AS-grown cells only . In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C23O-III), neither of which was present in 2AS-grown cells . The 3SC23O was purified to near homogeneity, found to be monomeric (M(r) 42,000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate . The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS . The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS . Strain O-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s) . One operon encodes at least the 2AS dioxygenase system and 3SC23O whereas the other encodes at least the BS/TS dioxygenase system and C23O-III.

J Bacteriol, 1994 Jul, 176(14), 4394 - 408
Biochemical and molecular characterization of the Alcaligenes eutrophus pyruvate dehydrogenase complex and identification of a new type of dihydrolipoamide dehydrogenase; Hein S et al.; Sequence analysis of a 6.3-kbp genomic EcoRI-fragment of Alcaligenes eutrophus, which was recently identified by using a dihydrolipoamide dehydrogenase-specific DNA probe (A . Pries, S . Hein, and A . Steinbuchel, FEMS Microbiol . Lett . 97:227-234, 1992), and of an adjacent 1.0-kbp EcoRI fragment revealed the structural genes of the A . eutrophus pyruvate dehydrogenase complex, pdhA (2,685 bp), pdhB (1,659 bp), and pdhL (1,782 bp), encoding the pyruvate dehydrogenase (E1), the dihydrolipoamide acetyltransferase (E2), and the dihydrolipoamide dehydrogenase (E3) components, respectively . Together with a 675-bp open reading frame (ORF3), the function of which remained unknown, these genes occur colinearly in one gene cluster in the order pdhA, pdhB, ORF3, and pdhL . The A . eutrophus pdhA, pdhB, and pdhL gene products exhibited significant homologies to the E1, E2, and E3 components, respectively, of the pyruvate dehydrogenase complexes of Escherichia coli and other organisms . Heterologous expression of pdhA, pdhB, and pdhL in E . coli K38(pGP1-2) and in the aceEF deletion mutant E . coli YYC202 was demonstrated by the occurrence of radiolabeled proteins in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively . A three-step procedure using chromatography on DEAE-Sephacel, chromatography on the triazine dye affinity medium Procion Blue H-ERD, and heat precipitation purified the E3 component of the A . eutrophus pyruvate dehydrogenase complex from the recombinant E . coli K38(pGP1-2, pT7-4SH7.3) 60-fold, recovering 41.5% of dihydrolipoamide dehydrogenase activity . Microsequencing of the purified E3 component revealed an amino acid sequence which corresponded to the N-terminal amino acid sequence deduced from the nucleotide sequence of pdhL . The N-terminal region of PdhL comprising amino acids 1 to 112 was distinguished from all other known dihydrolipoamide dehydrogenases . It resembled the N terminus of dihydrolipoamide acyltransferases, and it contained one single lipoyl domain which was separated by an adjacent hinge region from the C-terminal region of the protein that exhibited high homology to classical dihydrolipoamide dehydrogenases.

J Bacteriol, 1994 Jul, 176(14), 4385 - 93
The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation; Lenz O et al.; Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16 . In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy . The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time) . A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth . We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases . The expression of both operons was lower in the mutant than in the wild-type strain . These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.

Plasmid, 1994 Jul, 32(1), 1 - 9
IS1139 from Streptococcus salivarius: identification and characterization of an insertion sequence-like element related to mobile DNA elements from gram-negative bacteria; Lortie LA et al.; An insertion sequence-like element, IS1139, was cloned and sequenced from Streptococcus salivarius ATCC 25975 chromosome . This insertion sequence-like element is 1168 bp long and is delimited by inverted repeats of 29 bp and by a duplicated sequence of 6 bp . This IS possesses an open reading frame that codes for a putative transposase of 339 amino acids which has, respectively, 94, 35, 33, and 30% amino-acid identity with the transposases of IS1161 from S . salivarius ATCC 25975, IS4351 from Bacteroides fragilis, IS30 from Escherichia coli, and IS1086 from Alcaligenes eutrophus . Sequence analysis revealed that these transposases may have evolved from a common ancestral gene . Southern hybridization of restriction endonuclease-digested genomic DNA from 21 strains of oral streptococci, using a probe specific to the transposase-encoding gene (tnpA), revealed that IS1139 is found in two strains of S . salivarius, ATCC 25975 and ATCC 13419, in eight and two copies, respectively.

J Bacteriol, 1994 Jun, 176(12), 3614 - 30
Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system; Kruger N et al.; E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells . The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively . Microsequencing of both proteins revealed identical amino acid sequences . By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H . Lorenzl, F.B . Oppermann, B . Schmidt, and A . Steinbuchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli . The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined . The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively . Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems . As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C . magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases . We found strong similarities between the AcoR and AcoX sequences and the A . eutrophus acoR gene product, which is a regulatory protein required for expression of the A . eutrophus aco genes, and the A . eutrophus acoX gene product, which has an unknown function, respectively . The aco genes of C . magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

J Clin Microbiol, 1994 Jun, 32(6), 1547 - 9
Controlled clinical evaluation of Isolator and ESP aerobic blood culture systems for detection of bloodstream infections; Kirkley BA et al.; A controlled clinical evaluation comparing the Isolator system (Wampole Laboratories, Cranbury, N.J.) and the ESP 80A blood culture bottle in the automated ESP system (Difco Laboratories, Detroit, Mich.) was performed with 10,535 blood culture sets from patients with suspected septicemia . Of 1,150 positive cultures, 844 positive cultures from 285 patients with 394 septic episodes fulfilled the study criteria for minimum blood sample requirements in each system and clinical significance of isolates . The Isolator system detected statistically significantly more positive cultures of Staphylococcus aureus (P < 0.001), Enterococcus spp . (P = 0.007), Escherichia coli (P = 0.001), Alcaligenes xylosoxidans (P = 0.02), Xanthomonas maltophilia (P = 0.01), Candida albicans (P < 0.001), and Candida glabrata (P = 0.05) . The Isolator system detected significantly more septic episodes due to S . aureus (P < 0.001), X . maltophilia (P = 0.02), and C . albicans (P = 0.004) than did the ESP 80A bottle; however, the two systems did not otherwise significantly differ in their abilities to detect septic episodes due to other organisms.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 429 - 36
Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp . strain O-1 with subsequent meta ring cleavage and spontaneous desulphonation to 2-hydroxymuconic acid; Junker F et al.; 2-Aminobenzenesulphonic acid (2AS) is degraded by Alcaligenes sp . strain O-1 via a previously detected but unidentified intermediate . A mutant of strain O-1 was found to excrete this intermediate, which was isolated and identified by m.s., 1H- and 13C-n.m.r . as 3-sulphocatechol (3SC) . Proteins from cell extracts of strain O-1 were separated by anion-exchange chromatography . A multicomponent oxygenase was observed to convert 1 mol each of NADH, O2 and 2AS into 1 mol each of 3SC, NH3 and NAD+ . The enzyme presumably catalysed formation of the ring of a 2-amino-2,3-diol moiety, and elimination in the amino group led to a rearomatization . 3SC was further degraded via meta ring cleavage, which could be prevented by inactivation of the 3-sulphocatechol-2,3-dioxygenase (3SC23O) with 3-chlorocatechol . In Tris buffer, the separated 3SC23O catalysed the reaction of 1 mol each of 3SC and O2 involving a transient yellow intermediate, and release of 1 mol of sulphite and two organic products . The major product was identified by n.m.r . and by g.c./m.s . as 5-carboxypenta-2,4-dien-5-olide (CPDO), an indicator of formation of 2-hydroxymuconic acid (2HM) . The second product was identified as the Z,E isomer of 2HM by comparison with authentic material . When the CPDO in the product mixture was chemically hydrolysed to (Z,E)-2HM, 1 mol of (Z,E)-2HM/mol of 3SC was observed . If oxygenation of 3SC by 3SC23O was carried out in phosphate buffer, only a single product was detected, a keto form of 2HM . This dioate was also formed from authentic (Z,E)-2HM in phosphate buffer . Formation of the natural product (Z,E)-2HM from the xenobiotic, 3SC, seems to involve oxygenation to the unstable 2-hydroxy-6-sulphonomuconic acid semialdehyde, which hydrolyses spontaneously to 2HM . There would appear to be at least one spontaneous reaction per enzyme reaction in this pathway.

Mol Microbiol, 1994 Jun, 12(6), 1025 - 32
A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus; Eitinger T et al.; The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy-dependent uptake of nickel ions . Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis . To distinguish between these possibilities varying segments of the amino-terminal end of the transporter were fused to the Escherichia coli enzymes alkaline phosphatase (PhoA) or beta-galactosidase (LacZ) . The enzymatic activity of 16 HoxN-PhoA and 15 HoxN-LacZ fusions was determined . On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two-dimensional model for the nickel transporter was developed . This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices . The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.

Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5099 - 103
Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme; Fu C et al.; Nickel is an essential component of all H2-uptake hydrogenases . A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced . This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively . The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter . Based on its hydropathy profile, HupN is predicted to be a transmembrane protein . It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus . A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions . pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level . Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested . pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7 . The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein . Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells . The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.

FEMS Microbiol Lett, 1994 May 15, 118(3), 249 - 54
Stability and activity of hydrogenases of Methanobacterium thermoautotrophicum and Alcaligenes eutrophus in reversed micellar systems; Hoppert M et al.; In water-in-oil microemulsion the membrane-associated F420-hydrogenase of Methanobacterium thermoautotrophicum (strain Marburg) and the membrane-bound hydrogenase of Alcaligenes eutrophus H 16 (MBH) showed prolonged activity at elevated temperatures (measured as hydrogen production) as compared to aqueous buffer solution . The temperature optimum of the reactions was about 15 degrees C higher than in aqueous buffer solution . Activity of the almost completely inactivated F420-hydrogenase could be partially recovered by transfer into microemulsion.

Ann N Y Acad Sci, 1994 May 2, 721, 407 - 22
Molecular diagnostics for polychlorinated biphenyl degradation in contaminated soils; Layton AC et al.; Molecular diagnostic methods using DNA hybridization with specific gene probes are being developed for the monitoring of microbial populations capable of polychlorinated biphenyl (PCB) degradation in contaminated soils . Evaluation of composite samples from contaminated electrical substation soil by gas chromatography (GC) indicated that the PCBs present in the soil (approximately 200 ppm) resulted from contamination with Aroclor 1248 . The PCBs have been weathered or degraded so that the lower molecular weight PCB congeners are no longer present . Microbiological and molecular site characterizations are in progress to determine the abundance of PCB degradative organisms and catabolic genes present . Cloned DNA fragments for the bphC gene (2,3-dihydroxybiphenyl dioxygenase) from the biphenyl/chlorobiphenyl degradative pathways of different organisms were used as gene probes to identify indigenous microorganisms with bphC gene sequences . In colony hybridization experiments, positive signals with the pDA251 gene probe were detected in cultures from both contaminated and uncontaminated soils . The degradative abilities of indigenous microorganisms and an added PCB-degradative bacterial strain were also monitored with {14C}4-chlorobiphenyl mineralization assays and gas chromatography of PCB residues extracted from the soils . Enrichment of the contaminated soil with biphenyl and chlorobiphenyls did not stimulate the indigenous microorganisms to degrade the soil PCB . Nevertheless, enrichment of the contaminated soil with biphenyl and chlorobiphenyl and addition of the PCB-degrading strain Alcaligenes eutrophus GG4202 did result in additional degradation of the soil PCB . The results obtained from these experiments should assist in developing and monitoring a remediation plan for these PCB-contaminated soils.

J Bacteriol, 1994 Apr, 176(8), 2348 - 53
Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134; Matrubutham U et al.; Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid . A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A . R . Harker, R . H . Olsen, and R . J . Seidler, J . Bacteriol . 171:314-320, 1989; B . Kaphammer, J . J . Kukor, and R . H . Olsen, J . Bacteriol . 172:2280-2286, 1990) . When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently . The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F . The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4 . An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII . The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family . The complete ORF represents the regulatory gene tfdR . The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively . This ORF is identified as part of a putative isofunctional gene, tfdDII.

Pathology, 1994 Apr, 26(2), 201 - 7
Evaluation of direct disc diffusion susceptibility testing for bacteriuria using diluted urine against standard CDS method; Mukerjee C et al.; Consecutive urine specimens with > or = 10(9) organisms/L on microscopy were diluted 1:100 and direct disc diffusion susceptibility tests performed . Subsequently, the standard Calibrated Dichotomous Sensitivity (CDS) test was performed on all isolates . Urines with > 2 Isolates or where growth was < 10(8) colony forming units (CFU)/L were excluded . Only Gram negative organisms were considered . 361 urines were evaluated, 324 with one and 37 with 2 isolates, comprising 255 Escherichia coli, 49 klebsiella, 41 proteus, 29 either citrobacter, enterobacter, providencia, serratia or alcaligenes, and 14 Pseudomonas aeruginosa . There were 2272 organism/antimicrobial test comparisons . A concordance of 98.5% was obtained . The results are considered acceptable for routine clinical use.

Appl Environ Microbiol, 1994 Apr, 60(4), 1198 - 205
Production of polyhydroxyalkanoates in sucrose-utilizing recombinant Escherichia coli and Klebsiella strains; Zhang H et al.; The cloned poly-3-hydroxybutyrate (PHB) synthesis pathway from Alcaligenes eutrophus has been introduced into sucrose-utilizing strains of Escherichia coli, Klebsiella aerogenes, and Klebsiella oxytoca . The plasmid-borne genes were well expressed in these environments and were able to mediate the production of significant amounts of PHB when the bacteria were grown with sucrose as the sole carbon source . The molecular weight of the PHB polymer made in K . aerogenes and E . coli was approximately 1 x 10(6) to 2 x 10(6) . Sucrose uptake in K . aerogenes was measured and found to be similar to that found for other Klebsiella strains, but sucrose uptake in the E . coli strain was not detectable . K . aerogenes is able to utilize sugarcane molasses as the sole carbon source to accumulate PHB at the rate of approximately 1 g of PHB per liter of culture fluid per h . A K . oxytoca fadR strain was able to incorporate 3-hydroxyvalerate into a poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-V) polymer to levels as high as 56 mol% when grown in a medium containing propionate . Total PHB-co-V levels could be enhanced by adding propionate at the beginning of stationary phase rather than at the time of inoculation.

Appl Environ Microbiol, 1994 Apr, 60(4), 1106 - 15
Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils; Ka JO et al.; Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations . Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period . The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis) . A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species . Hybridization analysis revealed four groups . Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis . Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I . All group I and II strains had plasmids . Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains . One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area . The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe . The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group . The group III strains were identified as S . paucimobilis . The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses . Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Biol Macromol, 1994 Apr, 16(2), 59 - 63
Nuclear magnetic resonance relaxation studies of poly(hydroxybutyrate) in whole cells and in artificial granules; Shaw GL et al.; The physical state of poly(hydroxybutyrate) (PHB) in whole cells and in the form of artificial biomimetic granules has been probed using 13C nuclear magnetic resonance (NMR) spectroscopy . Studies on varying concentrations of whole cells of Alcaligenes eutrophus show that changes in the line widths of PHB in whole cells do not correlate with changes in transverse relaxation times . Solid-state magic-angle spinning NMR studies demonstrate that the line broadening results from a reduction in the static field homogeneity rather than from intrinsic properties of the PHB within the cells . Transverse and longitudinal relaxation times of PHB in whole cells and in artificial granules are similar, indicating similarities in structure and mobility.

Biochemistry, 1994 Mar 22, 33(11), 3171 - 7
EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015); Howes BD et al.; EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans . The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers . This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field . Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site . The EPR results show large changes in the type 2 site gparallel (which decreases by 0.065) and CuAparallel (which increases by 2.0 mT) while the type 1 site EPR is not affected . In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected . Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues . No 15N ENDOR resonances were observed from 15N nitrite . The accessibility of the copper sites to solvent has been studied using 2H2O . The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.

J Biotechnol, 1994 Mar 15, 33(1), 15 - 9
Expression of the Alcaligenes eutrophus phbA gene in Escherichia coli using a positive selection vector based on phage Lambda lysis genes; Kalousek S et al.; A new positive selection vector, pGS23, based on the Lambda lysis cassette has been designed for efficient expression of homologous and heterologous genes in Escherichia coli . The plasmid permits controlled expression of a gene of interest under transcriptional control of the lac promoter with translation initiation of coding sequences directed by the phage T7 gene 10 ribosome binding site . The application of the vector system was tested for high level expression of the heterologous phbA gene of Alcaligenes eutrophus in E . coli.

Mol Microbiol, 1994 Mar, 11(5), 841 - 7
Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport; Saier MH Jr et al.; Homology has been established for members of two families of functionally related bacterial membrane proteins . The first family (the resistance/nodulation/cell division (RND) family) includes (i) two metal-resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD) . The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA) . We propose that an MFP functions co-operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram-negative bacterial cell envelope.

J Biotechnol, 1994 Feb 14, 32(2), 203 - 11
Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli; Lee SY et al.; Plasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure . Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures . Higher instability was observed when cells were accumulating PHB . Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture . Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively . They showed 100% stability even during PHB synthesis and accumulation over 110 generations . All four plasmids were structurally stable . The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 101.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104) . The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E . coli.

Yakugaku Zasshi, 1994 Feb, 114(2), 63 - 72
{Conjugal transfer of chemolithoautotrophically growing ability from hydrogen-oxidizing bacterium Alcaligenes hydrogenophilus to useful material-producing bacteria}; Miura Y et al.; The plasmid genes encoding the ability to grow chemolithoautotrophically with H2 and CO2 from a H2-oxidizing bacterium Alcaligenes hydrogenophilus were in vivo cloned using the broad host range Inc P1 R-plasmid R68.45 . The genes of H2-oxidation enzymes were expressed by a novel alternative sigma 54-like factor encoded on the chromosome . The sigma 54-like factor was also in vivo cloned using R68.45 . Both R68.45-primes, one carrying plasmid genes and the other carrying chromosome genes, were in vivo recombined and a recombinant plasmid carrying both genes from plasmid and chromosome was obtained . The conjugal transfer of chemolithoautotrophically growing ability was carried out using the resulting recombinant plasmid . Seventeen bacterial strains, including useful material-producing bacteria, grew up to be able to grow with H2 and CO2 as the H2-oxidizing bacteria . Some patent strains registered for the production of antibiotics were ascertained to produce some products which inhibited the growth of the testing-bacteria, under not only heterotrophic conditions but also chemolithoautotrophilic conditions . The results obtained in our studies will be available in the future research for the production of useful material from CO2.

FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 273 - 7
Identification and sequencing of pyrG, the CTP synthetase gene of Azospirillum brasilense Sp7; Zimmer W et al.; An 18.5-kb DNA fragment carrying the trpGDC cluster of Azospirillum brasilense Sp7 was previously cloned, yielding cosmid pAB1005 . Attempts to identify trpA in the vicinity of trpGDC failed but led to the detection of a locus strongly homologous to pyrG, the structural gene for the CTP synthetase . The function of the A . brasilense pyrG gene was verified by complementation of the cytidine-requiring PyrG-deficient mutant JF646 of Escherichia coli . A second open reading frame was identified downstream of pyrG . The deduced amino acid sequence showed homology to dienelactone hydrolases of Pseudomonas and Alcaligenes, enzymes involved in utilization of halogenated aromatic compounds.

Appl Environ Microbiol, 1994 Jan, 60(1), 51 - 5
Aerobic degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5; Nadeau LJ et al.; Biotransformation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5 was demonstrated by analysis of ethyl acetate-extracted products from resting cell cultures . Gas chromatography-mass spectrometry characterization of the neutral extracts revealed two hydroxy-DDT intermediates (m/z = 370) with retention times at 19.55 and 19.80 min that shared identical mass spectra . This result suggested that the hydroxylations occurred at the ortho and meta positions on the aromatic ring . UV-visible spectrum spectrophotometric analysis of a yellow metabolite in the culture supernatant showed a maximum A402 with, under acidic and basic conditions, spectrophotometric characteristics similar to those of the aromatic ring meta-cleavage products . 4-Chlorobenzoic acid was detected by thin-layer chromatography radiochemical scanning in samples from mineralization experiments by comparison of Rf values of {14C}DDT intermediates with that of an authentic standard . These results were further confirmed by gas chromatography-mass spectrometry analysis . This study indicates that DDT appears to be oxidized by a dioxygenase in A . eutrophus A5 and that the products of this oxidation are subsequently subjected to ring fission to eventually yield 4-chlorobenzoic acid as a major stable intermediate.

J Bacteriol, 1994 Jan, 176(2), 469 - 85
Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system; Oppermann FB et al.; Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively . The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined . These genes are clustered on a 6.1-kbp region . Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E . coli was demonstrated . The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system . Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found . In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P . carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes.

J Chem Technol Biotechnol, 1994 Jan, 59(1), 83 - 9
Scale-up of a cyclone bioreactor; Sheppard JD et al.; The operation of a cyclone bioreactor differs from conventional stirred tanks since the agitation is accomplished by means of a pumped recirculation loop . Oxygen transfer can occur across the swirling gas-liquid interface in the cyclone or from bubbles entrained in the recirculation loop . A cyclone bioreactor was scaled-up from a 1 dm3 bench top unit to a 75 dm3 Process Development Unit (PDU) . A reduction in the aspect ratio was compensated for by extending the length of the recirculation loop and providing additional aeration . Performance of the two reactors for the production of microbial poly-beta-hydroxybutyrate (PHB) was compared under various operating conditions . The culture used for PHB production was Alcaligenes eutrophus DSM 545, grown on a mineral salts medium limited by the supply of nitrogen . The levels of dissolved oxygen obtained in the PDU were strongly dependent on the location at which the air was introduced into the reactor . However, with aeration balanced between two injection points and a similar level of power input, 17 J s-1 dm-3, the PDU was able to provide at least as much oxygen transfer capability as the laboratory-scale reactor . Under all conditions tested, the PHB accumulation by A . eutrophus was in excess of 80% of the biomass dry weight, although the yield on glucose was lower in the PDU than in the laboratory-scale reactor.

Appl Environ Microbiol, 1994 Jan, 60(1), 307 - 12
Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90; Bhat MA et al.; Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy . Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium . The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2 . The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1 . The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation . Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase . After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment . The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different . The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

Appl Microbiol Biotechnol, 1994 Jan, 40(5), 669 - 75
A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I; Timm A et al.; Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P . mendocina, Pseudomonas sp . DSM 1650 and Pseudomonas sp . GP4BH1 were cloned from genomic libraries in the cosmid pHC79 . A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P . citronellolis or Pseudomonas sp . DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydoxyalkanoic acids (HAMCL) from octanoate as well as from gluconate to the PHA-negative mutant P . putida GPp104 . An 11.0-kbp EcoRI fragment was cloned from P . mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate . From Pseudomonas sp . GP4BH1 three different genomic fragments encoding PHA synthases were cloned . This indicated that strain GP4BH1 possesses three different functionally active PHA synthases . Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HASCL), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB-4, respectively . The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HAMCL and was expressed in GPp104 but not in PHB-4.

J Mol Biol, 1993 Nov 20, 234(2), 508 - 12
Nucleotide sequence analysis of four genes, hupC, hupD, hupF and hupG, downstream of the hydrogenase structural genes in Bradyrhizobium japonicum; Van Soom C et al.; The nucleotide sequence of a 2.2 kb region downstream of the hydrogenase structural genes in Bradyrhizobium japonicum was determined . Four genes encoding predicted polypeptides of 27.8 (HupC), 21.4 (HupD), 10.6 (HupF) and 15.8 (HupG) kDa were identified, of which the first three probably belong to the same operon as the hup structural genes, hupS and hupL . HupC is homologous to the hydrophobic polypeptides with four potential transmembrane regions that are encoded by open reading frames following the hydrogenase structural genes in Rhodobacter capsulatus, Escherichia coli, Azotobacter vinelandii, Wolinella succinogenes, Rhizobium leguminosarum and Alcaligenes eutrophus . Also HupD, HupF and HupG are homologous to genes involved in processing, maturation, functioning and regulation of hydrogenase activity in various hydrogen-oxidizing bacteria.

J Biol Chem, 1993 Nov 15, 268(32), 24311 - 7
Purification and characterization of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase; Fukumori F et al.; The Alcaligenes eutrophus 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase, encoded by the tfdA gene of plasmid pJP4, is an Fe(II)-dependent enzyme that catalyzes the conversion of 2,4-dichlorophenoxyacetate to 2,4-dichlorophenol and glyoxylate concomitant with the decomposition of alpha-ketoglutarate to form succinate and carbon dioxide (Fukumori, F., and Hausinger, R . P . (1993) J . Bacteriol . 175, 2083-2086) . Using recombinant Escherichia coli cells that overexpress the tfdA gene, the thermolabile enzyme (stable only up to 30 degrees C) was purified to apparent homogeneity (specific activity of 16.9 mumol of substrate converted min-1 mg of protein-1) by a simple two-step procedure . The native protein has an apparent M(r) of 50,000 +/- 2,500, consistent with a homodimeric structure . Ferrous ion is absolutely required for activity and cannot be replaced by several other divalent cations tested . Ascorbic acid stimulates dioxygenase activity and reduces the rate of enzyme inactivation by a metal ion-mediated process . The enzyme exhibits maximum activity at pH 6.5-7, however, it is stable over a pH range of 6.5-11 . Although capable of hydroxylating a wide range of phenoxyacetates and related compounds, the enzyme exhibits the greatest affinity (Km 17.5 +/- 1.0 microM) and highest catalytic efficiency for 2,4-dichlorophenoxyacetate . Similarly, alpha-ketoglutarate is the preferred co-substrate (Km 3.20 +/- 0.54 microM) for the enzyme, but it can utilize a range of other alpha-ketoacids with lower efficiency . Results from chemical modification studies are consistent with the presence of multiple essential histidine residues in the enzyme.

Appl Environ Microbiol, 1993 Nov, 59(11), 3625 - 33
Cloning and expression of the transposable chlorobenzoate-3,4-dioxygenase genes of Alcaligenes sp . strain BR60; Nakatsu CH et al.; Growth on 3-chlorobenzoate was found to induce the enzymes of the protocatechuate meta ring fission pathway in Alcaligenes sp . strain BR60 . The chlorobenzoate catabolic genes, designated cba, were localized to a 3.7-kb NotI-EcoRI fragment within the nonrepeated region of the composite transposon Tn5271 . The cba genes were cloned onto two broad-host-range vectors and expressed in Escherichia coli and Alcaligenes sp . strain BR6024 . In E . coli, expression of the cba genes with the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible tac promoter of the IncQ vector pMMB66HE resulted in the production of protocatechuate and chlorodihydroxybenzoate metabolites of 3-chlorobenzoate . Expression of this construct in one orientation resulted in the formation of two polypeptides 51 and 42 kDa in size . This result was confirmed by subcloning into pGEM3Zf and then incorporating L-35S-methionine into newly synthesized proteins, using the thermally regulated T7 polymerase-promoter system . Introduction of the NotI-EcoRI fragment into Alcaligenes sp . strain BR6024 (Cba-P), using the IncP broad-host-range, mobilizable plasmid pBW13, restored the 3-chlorobenzoate-degradative phenotype and resulted in the accumulation of protocatechuate and chlorodihydroxybenzoate intermediates . The data indicate that a two-component dioxygenase specified by Tn5271 oxidizes 3-chlorobenzoate at the 3,4- or 4,5-positions . This activity extends the range of pathways for chloroaromatic compounds known to be functional in the environment . The new pathway avoids the toxicity attributed to the accumulation of chlorocatechol metabolites in bacteria degrading chlorobenzoates.

Sci Total Environ, 1993 Nov 1, 139-140, 471 - 8
Use of DNA probes and plasmid capture in a search for new interesting environmental genes; Diels L et al.; Adaptation to a stressed environment leads to organisms bearing DNA, encoding defense mechanisms . These mechanisms can be heavy metal resistance, catabolism of organic xenobiotics or stress reactions . Genes responsible for these mechanisms can be used for monitoring changing environments and therefore it can be important to store such bacteria in a bank . DNA-probing will be presented by the use of DNA fragments (of Alcaligenes eutrophus) coding for heavy metal resistance or xenobiotic degradation . Some strains do not grow on petri dishes and accordingly cannot be isolated from soils . In order to isolate plasmids from such strains, coding for heavy metal resistances or xenobiotic degradations, an exogenous plasmid isolation method was developed . In this method, the endogenous population is conjugated with Pseudomonas or Alcaligenes strains bearing a retrotransfer plasmid like RP4 . In that way new plasmids from various sources including non-culturable strains could be obtained . With these methods, a large number of specimens adapted to stressed situations can be isolated or constructed (in the case of the exogenous plasmid isolation method) . They form a source of interesting genetic material that can be used to restore polluted areas in natural areas, if necessary with the aid of genetic engineering (in vitro or in vivo techniques) . Full knowledge of such bacteria and their resistance mechanisms or degradation pathways, can lead to new constructions able to attack recalcitrant mixtures of different organics and to resist heavy metals.

J Bacteriol, 1993 Nov, 175(22), 7329 - 40
The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase; Schaferjohann J et al.; The two highly homologous cbb operons of Alcaligenes eutrophus H16 that are located on the chromosome and on megaplasmid pHG1 contain genes encoding several enzymes of the Calvin carbon reduction cycle . Sequence analysis of a region from the promoter-distal part revealed two open reading frames, designated cbbT and cbbZ, at equivalent positions within the operons . Comparisons with known sequences suggested cbbT to encode transketolase (TK; EC 2.2.1.1) as an additional enzyme of the cycle . No significant overall sequence similarities were observed for cbbZ . Although both regions exhibited very high nucleotide identities, 93% (cbbZ) and 96% (cbbT), only the chromosomally encoded genes were heterologously expressed to high levels in Escherichia coli . The molecular masses of the observed gene products, CbbT (74 kDa) and CbbZ (24 kDa), correlated well with the values calculated on the basis of the sequence information . TK activities were strongly elevated in E . coli clones expressing cbbT, confirming the identity of the gene . Strains of E . coli harboring the chromosomal cbbZ gene showed high levels of activity of 2-phosphoglycolate phosphatase (PGP; EC 3.1.3.18), a key enzyme of glycolate metabolism in autotrophic organisms that is not present in wild-type E . coli . Derepression of the cbb operons during autotrophic growth resulted in considerably increased levels of TK activity and the appearance of PGP activity in A . eutrophus, although the pHG1-encoded cbbZ gene was apparently not expressed . To our knowledge, this study represents the first cloning and sequencing of a PGP gene from any organism.

J Bacteriol, 1993 Nov, 175(21), 6745 - 54
Purification and characterization of maleylacetate reductase from Alcaligenes eutrophus JMP134(pJP4); Seibert V et al.; Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway . The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134 . Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa . The pI was determined to be at pH 5.4 . There was no indication of a flavin prosthetic group . The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol . Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively) . NADH was preferred to NADPH as the cosubstrate . Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH . These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway . In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.

Mol Microbiol, 1993 Nov, 10(3), 529 - 44
Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine; Poole K et al.; Pseudomonas aeruginosa strain K437 is defective in the production of a 90kDa ferripyoverdine receptor and is unable to grow in an iron-deficient medium in the presence of the non-metabolizable iron chelator 2,2'-dipyridyl (0.25 mM) . An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect . A number of clones restoring growth of K437 on dipyridyl-containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor . A 5.5 kb xhoI-HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor . Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively . Using a phage T7-based expression system, products of 42 kDa and c . 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed . The cloned ORFAB operon was inducible under conditions of iron limitation in both P . aeruginosa and Escherichia coli . In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine-mediated iron-transport system are co-regulated with ORFAB . The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation . In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane-associated plasmid-encoded cation efflux system in Alcaligenes eutrophus . Using in vitro mutagenesis and gene replacement, ORFA- and ORFB-deficient mutants of K372, the parent strain of K437, were constructed . These mutants were unable to grow on iron-deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine-mediated iron uptake . Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA-ORFB-deficient mutants were not defective in septum formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem J, 1993 Oct 15, 295 ( Pt 2), 587 - 93
Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp . xylosoxidans (N.C.I.M.B . 11015): evidence for the presence of both type 1 and type 2 copper centres; Abraham ZH et al.; Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp . xylosoxidans (N.C.I.M.B . 11015), after activation of crude extracts by the addition of copper(II) sulphate . The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE . SDS/PAGE and spray m.s . showed that the enzyme had a subunit M(r) of 36.5 kDa . The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500 . E.p.r . spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data {Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J . Biochem . (Tokyo) 96, 447-454}, where only type 1 copper centres were reported . Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously . As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm . The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases . We conclude that, in contrast with previous reports, this 'blue' nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.

Biochim Biophys Acta, 1993 Oct 6, 1202(2), 258 - 64
Overproduction, purification and properties of 2,3-dihydroxyphenylpropionate 1,2-dioxygenase from Escherichia coli; Bugg TD; The mhpB gene encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase in Escherichia coli was subcloned from Clarke-Carbon plasmid pLC20-30 by complementation with an mhpB- strain LW366 . Dioxygenase MhpB was purified using a five-step procedure from an overexpressing construct containing the mhpB gene, giving enzyme of > 95% homogeneity . The purified enzyme appeared as a 36-kDa subunit by SDS-PAGE, and had a native molecular mass of 134 kDa as determined by gel filtration . The apoenzyme obtained after chromatography could be re-activated by addition of Fe(II) and ascorbate to give the holoenzyme with a specific activity of 48 U/mg, which could be readily inactivated by oxidation or complexation of the Fe(II) cofactor, or simply by dilution . The substrate specificity of MhpB was examined, and as well as 2,3-dihydroxyphenylpropionate the enzyme was found to catalyse meta-ring cleavage of 3-methylcatechol and catechol, with reduced catalytic efficiency . The N-terminal sequence obtained for the purified enzyme showed significant sequence similarity with catechol 2,3-dioxygenase from Alcaligenes eutrophus, but none with catechol 2,3-dioxygenases from Pseudomonas.

Rev Argent Microbiol, 1993 Oct-Dec, 25(4), 221 - 6
{2-Naphthalene-sulphonic acid degrading microorganisms}; Vidal CM et al.; Derivatives of anionic surfactants, specially naphthalene sulphonates, when discharged into natural waters are accumulated in waters and sediments because of their poor biodegradability . A four-membered bacterial community, able to degrade the sodium salt of 2-naphthalene-sulphonic acid (2NS), was isolated from Rio Reconquista . All the isolated strains consisted of gram-negative, strictly aerobic rods, with a strong proteolytic activity and resistance to high levels of cations like Cr (III), Hg (II), Cd (II) and Pb (II) . Some of them were resistant to gentamicin, amikacin and chloramphenicol . These strains appeared to be related to the genera Pseudomonas and Alcaligenes . When isolated, if growing on 2NS, a brown-dark pigment is formed by three of them, resulting in an inhibition of growth . The presence of a Pseudomonas aeruginosa strain avoided the production of pigment and resulting in a complete consume of 2NS.

Res Microbiol, 1993 Oct, 144(8), 627 - 31
Characterization of a temperate phage hosted by Alcaligenes eutrophus strain A5; Faelen M et al.; Nineteen strains of Alcaligenes eutrophus were tested for the presence of prophages . One strain that lysed upon mitomycin C treatment produced a phage which could not form plaques on any of the strains available . DNA extracted from partially purified phage lysates was digested with various restriction enzymes which showed that the 42 kb long viral double-stranded DNA circularizes by means of cohesive ends . To our knowledge, this is the first description of a phage for the genus Alcaligenes.

J Mol Biol, 1993 Sep 5, 233(1), 109 - 22
The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli . Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I; Weidner U et al.; The gene locus nuo of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli was identified by means of a DNA probe made by the polymerase chain reaction . The primers used for the reaction were derived from consensus sequences of the NAD(H)-binding subunit of mitochondrial NADH: ubiquinone oxidoreductase and the NAD(+)-reducing hydrogenase of Alcaligenes eutrophus . The nuo locus lies between minutes 49.2 and 49.6 on the E . coli chromosome and contains a cluster of 14 genes . They are bordered upstream by a sequence resembling sigma 70-dependent promoters and downstream by a sequence resembling rho-independent terminators . All 14 proteins derived from the nuo-genes are related to subunits of mitochondrial NADH: ubiquinone oxidoreductase, among which all subunits presumed to bind the substrates and to harbour the redox groups are found, as well as all seven mitochondrially encoded subunits . The E . coli enzyme seems to represent the minimal form of a proton-translocating NADH: ubiquinone oxidoreductase . The gene order in the nuo locus is conserved in comparison with other bacterial genomes and the chloroplast genome of higher plants . To some extent, the gene order correlates with the topological arrangement of the encoded subunits . The conception of modular evolution of NADH: ubiquinone oxidoreductase is further supported by the arrangement of the nuo-genes.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 229 - 35
The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase; Jendrossek D et al.; The lactate dehydrogenase gene, ldh, of Alcaligenes eutrophus H16 was identified on a 14-kbp EcoRI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme . Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp PstI subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E . coli lac promoter . The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions . One represented the ldh gene . The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E . coli sequence of unknown function, respectively . The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh, and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins . ORF3 was located upstream of and colinear to ldh, and its putative gene translational product represented a hydrophobic protein . A sequence, which resembled the A . eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.

J Bacteriol, 1993 Sep, 175(18), 5867 - 76
Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16; Siddiqui RA et al.; Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U . Warnecke-Eberz and B . Friedrich, Arch . Microbiol . 159:405-409, 1993) . The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated . The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors . The nap genes were identified in a library of A . eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits . The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively . Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively . This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm . The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site . The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons . An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability of A . eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration . Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.

FEBS Lett, 1993 Aug 9, 328(1-2), 189 - 92
Metal as a novel type of the enzyme substrate . Metallic cadmium photogenerated in the system CdS-formate as a substrate of the NAD-dependent hydrogenase; Shumilin IA et al.; The process of NAD+ photoreduction under the coupled action of CdS semiconductor and NAD-dependent hydrogenase from hydrogen-oxidizing bacterium Alcaligenes eutrophus may be divided into light and dark stages . At the first stage, illumination of the system leads to the photooxidation of the sacrificial electron donor and results in the reduction of the semiconductor surface . At the second dark stage NAD+ is reduced to NADH in the presence of hydrogenase . Atoms of metallic Cd(0) are shown to be the true substrate of the enzymatic reaction . The prerequisite for the electron transfer from Cd(0) to hydrogenase is enzyme adsorption on the semiconductor surface . The redox center of the hydrogenase reacting with Cd(0) atoms resides on the flavin-containing heterodimer of the protein . The activity of the hydrogenase immobilized on CdS in the reaction of NAD+ reduction by metallic Cd is close to the enzyme activity with the physiological substrates in solution . Thus, the first example of a metal being the substrate of the enzymatic process is presented.

Int J Biol Macromol, 1993 Aug, 15(4), 253 - 5
Biosynthesis of polyesters from various amino acids by Alcaligenes eutrophus; Fujita M et al.; The possibilities of the biosynthesis of polyesters from 20 kinds of naturally occurring L-amino acids by Alcaligenes eutrophus were studied . It was found that several amino acids were used efficiently to synthesize copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3 HV) by A . eutrophus under the 'loose nitrogen-limiting' condition, but the other amino acids were scarcely utilized as a carbon source for the synthesis of polyesters . When L-threonine or L-isoleucine were used as the sole carbon source, copolyesters with higher 3HV content were produced.

J Clin Microbiol, 1993 Aug, 31(8), 2114 - 7
Controlled clinical comparison of two lysis-based blood culture systems, isolator and Septi-Chek Release, for detection of bloodstream infections; Kirkley BA et al.; A controlled clinical comparison was made of the Isolator (Wampole Laboratories, Cranbury, N.J.) and the Septi-Chek Release bottle (Roche Diagnostics, Nutley, N.J.) . From 6,345 blood culture sets fulfilling minimum criteria for volume of blood cultured, 840 strains were isolated, of which only 691 (82%) were considered to be representative of bloodstream infection according to Centers for Disease Control definitions . Statistically significant differences were found between the systems for the following organisms, which were all detected more frequently in the Isolator system: Staphylococcus aureus (P = 0.0001), Alcaligenes xylosoxidans (P = 0.008), Klebsiella pneumoniae (P = 0.05), Salmonella spp . (P = 0.03), and Candida albicans (P = 0.02) . The Septi-Chek Release system required a longer period of time than the Isolator system for detection of the following organisms:S . aureus (P = 0.0001), Enterococcus spp . (P = 0.0001), Enterobacter cloacae (P = 0.03), Escherichia coli (P = 0.0001), Klebsiella oxytoca (P = 0.03), K . pneumoniae (P = 0.02), Pseudomonas aeruginosa (P = 0.002), and C . albicans (P = 0.005) . There were 430 episodes of bloodstream infections identified in the study; of these episodes, only those due to S . aureus were detected significantly more frequently (P = 0.0001) by the Isolator system than by the Septi-Chek Release system . However, episodes of bloodstream infections due to S . aureus, Staphylococcus epidermidis, Enterococcus spp., and E . coli were detected significantly faster by the Isolator system.

Nippon Kyobu Geka Gakkai Zasshi, 1993 Aug, 41(8), 1373 - 7
{A case report of aortic valve and VSD Dacron patch infective endocarditis after VSD patch closure 15 years ago}; Sasaki H et al.; A 39-year-old man who had undergone the patch closure of the VSD 15 years ago, was admitted with a diagnosis of infective endocarditis due to Alcaligenes Xylosoxidans . Echocardiography revealed vegetation of the aortic valve and a high echoic lesion on the ventricular septum . Surgical findings showed vegetation of the aortic valve and a subannular type mycotic aneurysm . In the aneurysm, the infected pledget used in a previous surgery was found . After debridement, direct closure of the aneurysm, aortic valve replacement (AVR) using a #25 SJM prosthetic valve, and mitral annuloplasty were performed . Two months later, fever developed . The patient was diagnosed with prosthetic valve endocarditis and a second surgery was performed . The prosthetic valve was clear, but an infected Dacron patch used for VSD closure 15 years earlier was found . Debridement, patch closure of the ventricular septum, and re-AVR were performed . The post-operative course was uneventful . This is thought to be a rare case, because infection extended from the aortic valve to the VSD type II Dacron patch, and remained to the VSD type II Dacron patch.

Mol Gen Genet, 1993 Aug, 240(2), 181 - 7
Chromosome mapping in Alcaligenes eutrophus CH34; Sadouk A et al.; Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34 . In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated . Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37 degrees C and is characteristic of many metallotolerant strains of A . eutrophus . Plasmid pULB113 (RP4::miniMu) was used to map the available mutations . Twenty-five loci were ordered in a circular map . pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37 degrees C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A . eutrophus CH34 chromosome with a small number of crosses.

J Bacteriol, 1993 Aug, 175(16), 5289 - 93
Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules; Gerngross TU et al.; Antibodies raised against the Alcaligenes eutrophus poly-beta-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules . The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation . From these basic observations and data from the recent literature, a model of granule assembly is proposed.

J Bacteriol, 1993 Aug, 175(15), 4719 - 28
Physiological and biochemical characterization of the soluble formate dehydrogenase, a molybdoenzyme from Alcaligenes eutrophus; Friedebold J et al.; Organoautotrophic growth of Alcaligenes eutrophus on formate was dependent on the presence of molybdate in the medium . Supplementation of the medium with tungstate lead to growth cessation . Corresponding effects of these anions were observed for the activity of the soluble, NAD(+)-linked formate dehydrogenase (S-FDH; EC 1.2.1.2) of the organism . Lack of molybdate or presence of tungstate resulted in an almost complete loss of S-FDH activity . S-FDH was purified to near homogeneity in the presence of nitrate as a stabilizing agent . The native enzyme exhibited an M(r) of 197,000 and a heterotetrameric quaternary structure with nonidentical subunits of M(r) 110,000 (alpha), 57,000 (beta), 19,400 (gamma), and 11,600 (delta) . It contained 0.64 g-atom of molybdenum, 25 g-atom of nonheme iron, 20 g-atom of acid-labile sulfur, and 0.9 mol of flavin mononucleotide per mol . The fluorescence spectrum of iodine-oxidized S-FDH was nearly identical to the form A spectrum of milk xanthine oxidase, proving the presence of a pterin cofactor . The molybdenum-complexing cofactor was identified as molybdopterin guanine dinucleotide in an amount of 0.71 mol/mol of S-FDH . Apparent Km values of 3.3 mM for formate and 0.09 mM for NAD+ were determined . The enzyme coupled the oxidation of formate to a number of artificial electron acceptors and was strongly inactivated by formate in the absence of NAD+ . It was inhibited by cyanide, azide, nitrate, and Hg2+ ions . Thus, the enzyme belongs to a new group of complex molybdo-flavo Fe-S FDH that so far has been detected in only one other aerobic bacterium.

J Mol Biol, 1993 Jul 5, 232(1), 305 - 7
Crystallization and preliminary X-ray data of chloromuconate cycloisomerase from Alcaligenes eutrophus JMP134 (pJP4); Hammer A et al.; The pJP4-encoded chloromuconate cycloisomerase, an enzyme of the 2,4-dichlorophenoxy-acetate degradation pathway, was purified from cell-free extracts of Alcaligenes eutrophus JMP134 with a revised procedure . Tetragonal bipyramidal crystals were grown and characterized with respect to their X-ray diffraction properties . They were assigned to the space group I4, with cell dimensions of a = b = 111.9 A, c = 148.5 A . The crystals scattered to approximately 3 A resolution.

Int J Syst Bacteriol, 1993 Jul, 43(3), 618 - 21
Phylogenetic position of Taylorella equigenitalis determined by analysis of amplified 16S ribosomal DNA sequences; Bleumink-Pluym NM et al.; The 16S ribosomal DNA sequence of Taylorella equigenitalis (formerly Haemophilus equigenitalis), the causative organism of contagious equine metritis, was determined . A phylogenetic analysis of this sequence revealed a phylogenetic position of T . equigenitalis in the beta subclass of the class Proteobacteria apart from the position of Haemophilus influenzae, which belongs to the gamma subclass of Proteobacteria . A close phylogenetic relationship among T . equigenitalis, Alcaligenes xylosoxidans, and Bordetella bronchiseptica was detected; Spirillum volutans and Chromobacterium fluviatile (Iodobacter fluviatile) were in the same group but slightly removed . This relationship is surprising in view of the considerable differences in the G + C contents of the genomes of these bacteria.

Biosci Biotechnol Biochem, 1993 Jul, 57(7), 1149 - 52
Production, purification, and characterization of D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6; Moriguchi M et al.; The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-gamma-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine . The enzyme has been homogeneously purified . The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration . A subunit molecular weight of 52,000 was measured by SDS-PAGE, indicating that the native protein is a monomer . The isoelectric point was 5.2 . The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine . The Km for N-acetyl-D-leucine was 9.8 mM . The optimum pH and temperature were 7.0 and 50 degrees C, respectively . The stabilities of pH and temperature were 8.1 and 40 degrees C . D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.

Biosci Biotechnol Biochem, 1993 Jul, 57(7), 1145 - 8
Purification and characterization of novel N-acyl-D-aspartate amidohydrolase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6; Moriguchi M et al.; Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer . The enzyme was purified to homogeneity . The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be a monomer . The isoelectric point was 4.8 . The enzyme had maximal activity at pH 7.5 to 8.0 and 50 degrees C, and was stable at pH 8.0 and up to 45 degrees C . N-Formyl (Km = 12.5 mM), N-acetyl (Km = 2.52 mM), N-propionyl (Km = 0.194 mM), N-butyryl (Km = 0.033 mM), and N-glycyl (Km = 1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates . The enzyme was inhibited by both divalent cations (Hg2+, Ni2+, Cu2+) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid) . The N-terminal amino acid sequence and amino acid composition were analyzed.

Biochim Biophys Acta, 1993 Jun 4, 1163(3), 315 - 20
An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp . during guanidine denaturation; He RQ et al.; Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp . (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions . The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues . The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH . The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C . The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes . The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution . Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition . It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.

J Bacteriol, 1993 Jun, 175(11), 3388 - 93
Analysis of the periplasmic {NiFe} hydrogenase transcription unit from Desulfovibrio fructosovorans; Rousset M et al.; Two genes, hynA and hynB, encode the two subunits of the periplasmic {NiFe} hydrogenase in Desulfovibrio fructosovorans . Sequencing downstream from hynB revealed a third open reading frame (hynC) that has the potential for encoding a polypeptide showing 21% identity with the HyaD, HoxM, and HupD proteins, belonging to putative operons encoding Escherichia coli hydrogenase 1, Alcaligenes eutrophus H16 membrane-bound hydrogenase, and Rhizobium leguminosarum uptake hydrogenase, respectively . Northern (RNA) blotting with a structural gene probe revealed the existence of a major transcript of 2.9 kb, which is the appropriate length to contain the two hydrogenase subunits only . In addition, two minor 4.4- and 5.8-kb transcripts that could contain hynABC and additional genes were found . The 5' end of the most abundant {NiFe} hydrogenase mRNA was found 170 bp upstream from the translational start site of hynA . The sequences at -10 and -35 relative to the transcriptional starting site showed 55% homology with the consensus sequences of the Escherichia coli sigma 70-type promoter . The cloning of that particular region as a promoter to control transcription of the lacZ gene in E . coli DH5 alpha or the hynA, hynB, and hynC genes in D . fructosovorans MR400 led to strong expression in both systems.

FEMS Microbiol Lett, 1993 Jun 1, 110(1), 59 - 64
Metabolic pathway of homophthalic acid in Pseudomonas alcaligenes; Karigar CS et al.; A microorganism capable of degrading homophthalic acid as a sole carbon source was isolated from garden soil . The strain was identified as Pseudomonas alcaligenes . The organism degraded homophthalate by a pathway which involved phenylacetate and p-hydroxyphenylacetate as intermediates . The intermediates have been identified by physico-chemical methods . A tentative pathway for the degradation of homophthalate is proposed based on isolation of intermediates, oxygen uptake studies and presence of enzymes involved in the degradation.

Microb Releases, 1993 Jun, 2(1), 29 - 34
Rapid method for purification of soil DNA for hybridization and PCR analysis; Dijkmans R et al.; Monitoring of microbial DNA in soils by dot blot hybridization and PCR analysis is a useful technique for gaining insight into the survival and impact of genetically modified micro-organisms released in the environment . Most methods of DNA isolation from soils require a large number of purification steps rendering them unsuitable for quantitative analysis of multiple samples . Here we describe a very rapid method for the isolation and purification of multiple samples of soil DNA that can be used directly for dot blot hybridization and PCR analysis . Soil DNA extracts are prepared by lysozyme/SDS treatment at pH 9.0 and purified by ammonium acetate precipitation and Sephadex G50 gel filtration . In a practical application of this method, sandy soil samples were seeded with Alcaligenes eutrophus cells and exposed to high temperature (42 degrees C) or desiccation . As a result, the number of culturable A . eutrophus cells which could be recovered from the soil samples quickly declined . However, the concentration of a marker gene encoding resistance to cadmium, cobalt and zinc (czc) remained unaltered.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 26 - 31
A new pathway for the degradation of a sesquiterpene alcohol, nerolidol by Alcaligenes eutrophus; Madyastha KM et al.; An oxidative pathway hitherto unknown for the degradation of a sesquiterpene alcohol, nerolidol (I) by Alcaligenes eutrophus is presented . Fermentation of nerolidol (I) by this organism in a mineral salts medium resulted in the formation of geranylacetone (II) and an optically active alcohol (S)-(+)-geranylacetol (III), as major metabolites . Nerolidol (I) induced cells readily transformed 1,2-epoxynerolidol (IV) and 1,2-dihydroxynerolidol (V) into geranylacetone (II) . These cells also exhibited their ability to carry out stereospecific reduction of II into (S)-(+)-geranylacetol (III) . Oxygen uptake studies clearly indicated that nerolidol induced cells oxidized compounds II, III, IV, V and ethyleneglycol . Based on these observations a new oxidative pathway for the degradation of I is suggested which envisages the epoxidation of the terminal double bond, opening of the epoxide and cleavage between C-2 and C-3 in a manner similar to the periodate oxidation of diol.

Appl Environ Microbiol, 1993 May, 59(5), 1560 - 4
Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability; Pedersen JC et al.; A sandy loam soil near field capacity moisture content (psi = -0.050 MPa) or air dried (psi = -300 MPa) was inoculated with about 3 x 10(7) CFU of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) per g and incubated in 40-g portions at 17 degrees C in closed or open Erlenmeyer flasks . In the field-moist soil, selective plating, direct viable counts, and DNA hybridization showed only minor changes in the numbers of E . cloacae and A . eutrophus cells with time (14 days), and the results obtained with the three detection methods generally agreed . In the air-dried soil, the majority of both bacteria were found as intact DNA-carrying cells that were neither culturable nor viable by the methods employed in this study . The numbers of culturable E . cloacae and A . eutrophus cells dropped to 10(5) and 10(2) CFU/g, respectively, 2 h after inoculation . Direct viable counts showed that only about 1% of the cells detected by immunofluorescence microscopy were viable, but a fraction of viable nonculturable cells of both bacteria was present . A . eutrophus did not tolerate desiccation as well as E . cloacae . Only a minor fraction of the two test organisms regained their culturability or viability after rewetting of the air-dried soil; the number of total heterotrophic culturable bacteria, however, increased more than 10-fold and reached 73% of the level found in the field-moist soil at day 14.

Appl Environ Microbiol, 1993 May, 59(5), 1504 - 6
Microbial oxidation of dimethylnaphthalene isomers; Miyachi N et al.; Three bacterial strains, identified as Alcaligenes sp . strain D-59 and Pseudomonas sp . strains D-87 and D-186, capable of growing on 2,6-dimethylnaphthalene (2,6-DMN) as the sole source of carbon and energy were isolated from soil samples . 2,6-Naphthalene dicarboxylic acid was formed in the culture broths of these three strains grown on 2,6-DMN . In addition, 2-hydroxymethyl-6-methylnaphthalene and 6-methylnaphthalene-2-carboxylic acid were detected in the culture broth of strain D-87 . Strain D-87 grew well on 1,2-, 1,3-, 1,4-, 1,5-, 2,3-, and 2,7-DMN as the sole source of carbon and energy and accumulated 2-methylnaphthalene-3-carboxylic acid and 2,3-naphthalene dicarboxylic acid from 2,3-DMN, 4-methylnaphthalene-1-carboxylic acid from 1,4-DMN, and 7-methylnaphthalene-2-carboxylic acid from 2,7-DMN.

Mol Gen Genet, 1993 May, 239(1-2), 235 - 40
Identification of a potential transcriptional regulator of hydrogenase activity in free-living Bradyrhizobium japonicum strains; Van Soom C et al.; In Bradyrhizobium japonicum, Tn5 insertions in a particular chromosomal DNA fragment result in a Hup- phenotype in free-living conditions without affecting hydrogenase (Hup) activity in the symbiotic state . By determination of the nucleotide sequence of this region, we were able to identify the nature of the inactivated genes . The fragment is located 9 kb downstream of the hydrogenase structural genes and contains one incomplete and three complete open reading frames . They are designated hypD', hypE, hoxX and hoxA respectively, since the deduced amino acid sequences display very strong homology with genes involved in the regulation of hydrogenase activity in Escherichia coli, Rhodobacter capsulatus, Azotobacter vinelandii (hypD' and hypE) and Alcaligenes eutrophus (hoxX and hoxA) . This is the first report on transcriptional activators of the hup genes in B . japonicum . Implications of these findings with respect to regulation of hydrogenase synthesis by hydrogen, oxygen and nickel in free-living B . japonicum are discussed.

Carbohydr Res, 1993 Apr 7, 242, 153 - 60
Microbial synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate; Knappmann BR et al.; A microbial route was explored for the synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate (2-keto-3-deoxy-6-phosphogluconate, KDPG) . Two strains of bacteria, Alcaligenes eutrophus H16 F34 (DSM 529) and Escherichia coli DF 71 (CGSC 4880), lacking in KDPG-aldolase activity were tested for excretion of KDPG . Using pyruvate and gluconate as carbon sources, Alcaligenes eutrophus H16 F34 accumulated and excreted 3-deoxy-D-erythro-hexulosonic acid 6-phosphate into the culture broth, while the E . coli strain, using pyruvate and glucuronate, failed . KDPG was isolated from the culture supernatant of Alcaligenes eutrophus H16 F34 in 78% yield and 5 g scale with respect to the consumed gluconate.

Mol Microbiol, 1993 Apr, 8(1), 15 - 29
Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus . Sequence analysis and identification of two hyp regulatory mutants; Colbeau A et al.; A 25 kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced . Coding regions corresponding to 20 open reading frames were identified . The R . capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium leguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11) . The sequential arrangement of the R . capsulatus genes is: hupR2-hupU-hypF-hupS-hupL-hupM-hu pD-hupF-hupG-hupH-hupJ-hupK-hypA- hypB-hupR1- hypC-hypD-hypE-ORF19-ORF20, all contiguous and transcribed from the same DNA strand . The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species . The sequence of the 12 R . capsulatus genes underlined above is presented . The mutation site in two of the Hup- mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD gene (deletion of 54 bp), respectively . The hypF gene product shares 45% identity with the product of hydA from E . coli and the product of hypF from R . leguminosarum . Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins . The G deletion in the C-terminal region of hypF in the RS13 mutant prevented the expression of a hupS::lacZ translational fusion from being stimulated by H2 as it is observed in the wild-type strain B10 . It is inferred that the HypF protein is a factor involved in H2 stimulation of hydrogenase expression.

J Bacteriol, 1993 Apr, 175(7), 2083 - 6
Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase; Fukumori F et al.; The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined . Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity . Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate . The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol . By using {1-14C}alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate . Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D.

FEMS Microbiol Rev, 1993 Apr, 10(3-4), 243 - 69
Microbial hydrogenases: primary structure, classification, signatures and phylogeny; Wu LF et al.; Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function . The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria . The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria . The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria . The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16 . The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli . The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria . The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors . While those of class I, II, IV and V hydrogenases are homologous, i.e . sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes . Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively . These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases . Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected . Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains . The regions of hydrogen activation domains are highly conserved and can be divided into two categories . One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases . It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively . The other is the H-cluster of the Fe-hydrogenases . It might comprise three motifs on the C-terminal half of the large subunits . However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved . They are present essentially on the small subunits in NiFe-hydrogenases . Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biotechnol, 1993 Apr, 28(2-3), 291 - 9
Stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid RP4; Haigermoser C et al.; The genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (PHB) cloned from Alcaligenes eutrophus H16 were used for synthesis of PHB with recombinant Escherichia coli strains . It was recognized that the PHB-biosynthesis genes cause segregational instability to the plasmids used as vectors . Recombinant PHB-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids . Cloning the partitioning region of plasmid RP4 onto such plasmids resulted in a high degree of stabilization . These par-stabilized recombinant PHB-plasmids could be maintained quite efficiently in batch cultivation experiments in the absence of any selection pressure.

Steroids, 1993 Feb, 58(2), 79 - 86
Bile acid transformations by Alcaligenes recti; Mazumder I et al.; Metabolism of cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and deoxycholic acid by the grown cells of the bacterium Alcaligenes recti suspended in water was studied . Each isolated metabolite was characterized by the application of various spectroscopic methods . Cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and deoxycholic acid yielded methylated derivatives 3 alpha-methoxy-7 alpha, 12 alpha-dihydroxy-5 beta-cholanoic acid, 3 alpha-methoxy-7 alpha-hydroxy-5 beta-cholanoic acid, 3 alpha-methoxy-7 beta-hydroxy-5 beta-cholanoic acid, and 3 alpha-methoxy-12 alpha-hydroxy-5 beta-cholanoic acid, respectively . In addition, cholic acid furnished 7 alpha, 12 alpha-dihydroxy-3-oxochol-4-en-24-oic acid; chenodeoxycholic acid gave 7 alpha-hydroxy-3-oxo-5 beta-cholanoic acid and 7 alpha-hydroxy-3-oxochol-4-en-24-oic acid while ursodeoxycholic acid yielded 7 beta-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochola-4,6-dien-24-oic acid . The formation of various metabolites showed that two competitive enzymic reactions, i.e., selective methylation of the 3 alpha-hydroxy group and dehydrogenation in the A/B rings, were operative . The methylation process was found to be enzymic involving an S-adenosyl-L-methionine (AdoMet)-dependent methyl transferase, and this reaction appeared to be inhibitory to the process of degradation of the ring system . In the other reaction sequence, degradation of the ring system was initiated by dehydrogenation of the 3 alpha-hydroxy group . A 7 beta-dehydratase activity producing the delta 6 double bond was also noticeable in the metabolism of ursodeoxycholic acid.

J Bacteriol, 1993 Feb, 175(4), 1019 - 25
Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene; Kusano T et al.; The presence of two sets (rbcL1-rbcS1 and rbcL2-rbcS2) of rbc operons has been demonstrated in Thiobacillus ferrooxidans Fe1 (T . Kusano, T . Takeshima, C . Inoue, and K . Sugawara, J . Bacteriol . 173:7313-7323, 1991) . A possible regulatory gene, rbcR, 930 bp long and possibly translated into a 309-amino-acid protein, was found upstream from the rbcL1 gene as a single copy . The gene is located divergently to rbcL1 with a 144-bp intergenic sequence . As in the cases of the Chromatium vinosum RbcR and Alcaligenes eutrophus CfxR, T . ferrooxidans RbcR is thought to be a new member of the LysR family, and these proteins share 46.5 and 42.8% identity, respectively . Gel mobility shift assays showed that T . ferrooxidans RbcR, produced in Escherichia coli, binds specifically to the intergenic sequence between rbcL1 and rbcR . Footprinting and site-directed mutagenesis experiments further demonstrated that RbcR binds to overlapping promoter elements of the rbcR and rbcL1 genes . The above data strongly support the participation of RbcR in regulation of the rbcL1-rbcS1 operon and the rbcR gene in T . ferrooxidans.

J Bacteriol, 1993 Feb, 175(3), 779 - 84
A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system; Collard JM et al.; Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50 . With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected . The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus . The chromate resistance closely linked to the cnr locus was not affected by these mutations . In the Znr mutants, the resistance to zinc and nickel was constitutively expressed . Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain . Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system . A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt . The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc . Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype . Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.

J Bacteriol, 1993 Feb, 175(3), 767 - 78
Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34; Liesegang H et al.; From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2 . This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A . eutrophus hosts, strains AE104 and H16 . Resistances were not expressed in Escherichia coli . The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E . coli under the control of the bacteriophage T7 promoter . The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 {ORF}; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression . ORF0ab and ORF1 exhibit a codon usage that is not typical for A . eutrophus . The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis . Among 72 insertion mutants, the majority were nickel sensitive . The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts . These genes are considered to be involved in the regulation of cnr . Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights . The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon) . The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.

Arch Microbiol, 1993, 159(2), 182 - 8
Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134; Vollmer MD et al.; 2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate . The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction . Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funneled into the 3-oxoadipate pathway by a second reductive step . There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.

Appl Environ Microbiol, 1993 Jan, 59(1), 334 - 9
Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains; Springael D et al.; Alcaligenes eutrophus strains exhibiting both plasmid-borne heavy metal resistance and haloaromatic-degrading functions were obtained by intraspecific conjugation . The strains which we constructed expressed catabolic and resistance markers together . Degradation of various polychlorinated biphenyl isomers and 2,4-D (2,4-dichlorophenoxyacetic acid) was observed in the presence of 1 mM nickel or 2 mM zinc, provided that the metal resistance determinant was present in the catabolizing strain . Such strains may be useful for decontamination of sites that are polluted with both organic compounds and heavy metals.

Arch Microbiol, 1993, 159(6), 545 - 53
Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16; Dernedde J et al.; In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases . This region lies within the hydrogenase gene cluster of megaplasmid pHG1 . Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms . Mutants of A . eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase . Immunological analysis showed that the mutants contained SH- and MBH-specific antigen . Growing the cells in the presence of 63Ni2+ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type . Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A . eutrophus.

Antonie Van Leeuwenhoek, 1993, 64(1), 9 - 15
Purification and characterization of the E1 component of the Clostridium magnum acetoin dehydrogenase enzyme system; Lorenzl H et al.; In Clostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin . Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR . Native Ao:DCPIP OR exhibited a M(r) of 138,000; it consisted of two different subunits of M(r) alpha 38,500 and M(r) beta 34,000, and it occurred most probably in a tetrameric alpha 2 beta 2 structure . The N-terminal amino acid sequences of the alpha- and beta-subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR from Pelobacter carbinolicus and from Alcaligenes eutrophus; furthermore, the N-terminus of the beta-subunit exhibited homologies to the N-termini of beta-subunits from different 2-oxo acid dehydrogenases.

Annu Rev Microbiol, 1993, 47, 351 - 83
Molecular biology of hydrogen utilization in aerobic chemolithotrophs; Friedrich B et al.; The aerobic bacteria capable of obtaining energy from the oxidation of H2 form a heterogenous group that includes both facultative and obligate chemolithotrophs and representatives of both gram-negative and gram-positive genera . H2-oxidizing aerobes inhabit such diverse biotypes as soil, oceans, and hot springs . The oxidation of H2 in these bacteria is catalyzed by {NiFe} metalloenzymes called hydrogenases . The hydrogenases studied so far belong to two families: dimeric, membrane-bound enzymes (MBH) coupled to electron transport chains and tetrameric, cytoplasmic NAD-reducing enzymes (SH) . Ni2+ is an essential component of the active site contained in the large subunit of the MBH enzymes . The genes for the MBH enzymes are located in conserved clusters of accessory genes, some of which encode maturation functions and hydrogenase-related redox proteins . Maturation of both types of hydrogenase is apparently complex, involving specific nickel incorporation and proteolytic processing steps . In Alcaligenes eutrophus and Rhodobacter capsulatus, hydrogenase expression is regulated by transcriptional activators belonging to the response-regulator family.

Arch Microbiol, 1993, 160(3), 169 - 78
Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134; Pieper DH et al.; 2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 134-1 . It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis,cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite . Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide . Those lactones harboring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.

Appl Microbiol Biotechnol, 1993 Jan, 38(4), 493 - 501
Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea; Liebergesell M et al.; From a genomic library of Thiocystis violaceae strain 2311 in lambda L47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned . The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A . eutrophus . The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis . The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a beta-ketothiolase {phbATv, relative molecular mass (M(r)) 40850}, which exhibited 87.3% amino acid identity with the beta-ketothiolase from Chromatium vinosum . The amino acid sequences of the putative proteins deduced from ORF2Tv (M(r) 41450) and phbCTv (M(r) 39550), which were located upstream of and antilinear to phbATv, exhibited 74.7% and 87.6% amino acid identity, respectively, with the corresponding gene products of C . vinosum . Downstream of and antilinear to phbCTv was located ORF5, which encodes for a protein of high relative molecular mass (M(r) 76428) of unknown function . With respect to the divergent organisation of ORF2Tv and phbCTv on one side and of phbATv on the other side and from the homologies of the putative gene products, this region of the T . violaceae genome resembled very much the corresponding region of C . vinosum, which was identified recently.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 107 - 10
Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria; Tabita FR et al.; Structural and regulatory genes encoding enzymes and proteins of the reductive pentose phosphate pathway have been isolated from a number of bacteria recently . In the phototroph Rhodobacter sphaeroides, and in two chemoautotrophic bacteria, Alcaligenes eutrophus and Xanthobacter flavus, these genes have been found in distinct operons . However, in these three organisms and in other bacteria where certain of these genes have been discovered, a uniform nomenclature to designate these genes has been lacking . This report represents an effort to provide uniformity to the designation of these genes from all bacteria.

Comp Biochem Physiol B, 1992 Dec, 103(4), 759 - 73
Organization of non-vertebrate globin genes; Vinogradov SN et al.; The organization of non-vertebrate globin genes exhibits substantially more variability than the three-exon, two-intron structure of the vertebrate globin genes . (1) The structures of genes of the single-domain globin chains of the annelid Lumbricus and the mollusc Anadara, and the globin gene coding for the two-domain chains of the clam Barbatia, are similar to the vertebrate plan . (2) Genes for single-domain chains exist in bacteria and protozoa . Although the globin gene is highly expressed in the bacterium Vitreoscilla, the putative globin gene hmp in E . coli, which codes for a chimeric protein whose N-terminal moiety of 139 residues contains 67 residues identical to the Vitreoscilla globin, may be either unexpressed or expressed at very low levels, despite the presence of normal regulatory sequences . The DNA sequence of the globin gene of the protozoan Paramecium, determined recently by Yamauchi and collaborators, appears to consist of two exons separated by a short intron . (3) Among the lower eukaryotes, the yeasts Saccharomyces and Candida have chimeric proteins consisting of N-terminal globin and C-terminal flavoprotein moieties of about the same size . The structure of the gene for the chimeric protein of Saccharomyces exhibits no introns . According to Riggs, the presence of chimeric proteins in E . coli and other prokaryotes, such as Alcaligenes and Rhizobium, as well as in yeasts, suggests a previously unrecognized evolutionary pathway for hemoglobin, namely that of a multipurpose heme-binding domain attached to a variety of unrelated proteins with diverse functions . (4) The published globin gene sequences of the insect larva Chironomus have an intron-less structure and are present as clusters of multiple copies; the expression of the globin genes is tissue and developmental stage-specific . Furthermore, the expression of many of these genes has not yet been demonstrated despite the presence of apparently normal regulatory sequences in the two flanking regions . Unexpectedly, Bergtrom and collaborators have recently shown that at least three Ctt globin II beta genes contain putative introns . (5) Pohajdak and collaborators have found a seven-exon and six-intron structure for the globin gene of the nematode Pseudoterranova which codes for a two-domain globin chain . Although the second and fourth introns of the N-terminal domain correspond to the two introns found in vertebrate globin genes, the position of the third intron is close to that of the central intron in plant hemoglobins.(ABSTRACT TRUNCATED AT 400 WORDS)

Int J Biol Macromol, 1992 Dec, 14(6), 321 - 5
Biosynthesis of poly(3-hydroxyalkanoate) from amino acids; Nakamura K et al.; It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions . The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.

FEMS Microbiol Rev, 1992 Dec, 9(2-4), 333 - 8
Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M; Saito T et al.; Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear . The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined . The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl) . Triton X-100 and diisopropylfluorophosphate inhibited this reaction . Addition of the supernatant fraction of Z . ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules . On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z . ramigera cells . The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules . Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.

FEMS Microbiol Rev, 1992 Dec, 9(2-4), 217 - 30
Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria; Steinbuchel A et al.; The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided . Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber . Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure . Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA . In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.

J Bacteriol, 1992 Dec, 174(24), 8102 - 10
CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus; Nies DH; The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized . The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes . The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites . A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A . eutrophus, and expressed under the control of CzcR . Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity . Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc . The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A . Kamizomo, M . Nishizawa, Y . Teranishi, K . Murata, and A . Kimura, Mol . Gen . Genet . 219:161-167, 1989).

Eur J Biochem, 1992 Dec 1, 210(2), 475 - 81
Purification and properties of a novel arylmalonate decarboxylase from Alcaligenes bronchisepticus KU 1201; Miyamoto K et al.; A novel decarboxylase which catalyzes an enantioselective decarboxylation of alpha-aryl-alpha-methylmalonates to alpha-arylpropionates has been purified from a soil bacterium Alcaligenes bronchisepticus KU 1201 . The enzyme was purified 300-fold to homogeneity, judged from the analysis of N-terminal amino acid sequence, and found to be a monomeric enzyme of apparent 24 kDa . The enzyme catalyzes a decarboxylation giving alpha-arylalkanoates from substituted malonates such as alpha-arylmalonate and alpha-alkyl-alpha-arylmalonates . The decarboxylase is not a biotin containing enzyme because avidin have no influence on the enzyme activity . In addition, the enzyme does not require known co-factors (ATP, ADP and coenzyme A) for maximum activity . The enzyme activity was inhibited by sulfhydryl agents . The electronic effect of the substituents on kcat for the enzymic decarboxylation of arylmalonates has been studied . The logarithm of relative value of kcat gave a linear correlation to Hammett's sigma with a rho value of +1.9, for substituted phenylmalonates . Comparing the relative activities, it is clear that the enzyme prefers alpha-arylmalonates to alpha-aryl-alpha-methylmalonates . Thus, the enzyme was tentatively named as arylmalonate decarboxylase.

J Bacteriol, 1992 Dec, 174(24), 8133 - 8
Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351; Dong Q et al.; A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes . The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame . Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome . Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences . One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent . Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A . eutrophus) . The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements . Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.

J Bacteriol, 1992 Dec, 174(24), 8023 - 9
Molecular and genetic characterization of an Alcaligenes eutrophus insertion element; Kung SS et al.; An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1 . ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids . Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids . Most insertion were found in the AT-rich target sites . The distribution of ISAE1 is limited to A . eutrophus H1 (ATCC 17698) and H16 (ATCC 17699) . Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions.

Chem Pharm Bull (Tokyo), 1992 Dec, 40(12), 3292 - 6
Nucleotide sequences of membrane-bound hydrogenase gene in Alcaligenes hydrogenophilus; Yagi K et al.; The nucleotide sequences of membrane-bound hydrogenase small (hupS) and large (hupL) subunit genes of hydrogen bacterium Alcaligenes hydrogenophilus were determined . The hupS and hupL genes encoded polypeptides of 363 and 619 amino acids, respectively . The hupS was located upstream of hupL with 35bp of intergenic region . The consensus ribosome-binding sequences were identified upstream of the start codons of hupS and hupL . Amino acid sequence of hupS is very similar to that of Rhodobacter capsulatus, Bradyrhizobium japonicum, and Azotobacter vinelandii at amino acid levels of 82%, 77%, and 81%, respectively . Similarly, amino acid sequence of HupL is similar to that of R . capsulatus, B . japonicum, and A . vinelandii at amino acid levels of 63%, 65%, and 68%, respectively . Northern hybridization analysis showed that hupS and hupL were co-transcribed, and addition of fructose to the culture medium remarkably decreased the amount of mRNA transcribed from hupS and hupL.

Dtsch Tierarztl Wochenschr, 1992 Dec, 99(12), 478 - 82
{The aerobic bacterial flora of songbird nests}; Mehmke U et al.; In the area around Ober- and Unterschleissheim, a medium term decrease in the singing bird population appears to be happening . Therefore, material from nesting-boxes (used for nesting or sleeping) was examined for bacterial contamination . Mainly gram positive bacteria were isolated which were considered to belong to the normal flora . The occurrence of Streptomyces spp . is described in the nesting material of Passeres probably for the first time . Enterobacteriaceae represent only a fraction of the total isolates, but are demonstrated relatively frequently in the nests of the Nuthatch (Sitta europaea) and nests of other non-identified bird spp., so that colonization of the intestinal tract with Enterobacteriaceae cannot be excluded . Surprisingly, bacteria of the aquatic habitats such as Alcaligenes, Bordetella, Aeromonas, Non-Cholera (NC)-Vibrio were isolated, although in small amounts . The occurrence of NC-Vibrio has not yet been described in singing birds . Nests with high numbers of gram negative rods were successful in most instances, therefore, there was no proof that aerobic bacteria are responsible for the decrease in the population.

Biochem J, 1992 Dec 1, 288 ( Pt 2), 475 - 82
Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp . OH-11242; Ishii-Karakasa I et al.; For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp . OH-11242 (endo-GalNAc-ase-S) {Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem . Biophys . Res . Commun . 151, 422-428}, a method for assaying enzyme activity was established . Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c . The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium . The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography . Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column . In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected . The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5 . Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide {Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc} . When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp . (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

Prikl Biokhim Mikrobiol, 1992 Nov-Dec, 28(6), 907 - 11
{A bioluminescence method of determining the activity of NAD-dependent hydrogenase}; Petushkov VN et al.; An analytical multienzyme system composed of NAD-dependent hydrogenase of Alcaligenes eutrophus, and reductase and luciferase from luminous bacteria was studied . The rate of luminescence increase of this system was found to be proportional to hydrogenase activity . The apparent Michaelis constants for NAD and hydrogen were determined (5 and 40 microM, respectively) . The pH optimum is 7.5-9.0 . Over the NAD concentration range from 20 to 100 microM, the rate of luminescence increase changed by less than 10% . At higher concentrations of NAD a monotonous decreasing of the rate of luminescence increase was observed . The proposed multienzyme system can be used for measuring the hydrogenase activity and hydrogen concentration . The high sensitivity to hydrogen (0.1 nmol in sample) and to hydrogenase (0.5 mU) and specificity of the system enable its application in the development of a biosensor for rapid detection of hydrogen in a medium.

J Biochem (Tokyo), 1992 Nov, 112(5), 689 - 93
Treatment of low density lipophorin with lipoprotein lipase: diacylglycerol content has no effect on dissociation of apolipophorin III from low-density lipophorin; Hiraoka T et al.; The mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp . Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation . The "lipase-treated LDLp" thus obtained still contained associated apolipophorin-III (apoLp-III) . These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle . Some physico-chemical properties of the lipase-treated LDLp were measured.

J Bacteriol, 1992 Nov, 174(22), 7337 - 44
The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus; Kusian B et al.; Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb . One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B . Bowien, U . Windhovel, J.-G . Yoo, R . Bednarski, and B . Kusian, FEMS Microbiol . Rev . 87:445-450, 1990) . Corresponding regions of about 2.7 kb from within the operons were sequenced . Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences . The nucleotide identity of the sequences amounted to 94% . Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes . The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information . E . coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme . These data agree with the finding that in A . eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons . No functions could be assigned to CfxX and CfxY.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 234 - 8
Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201; Miyamoto K et al.; We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AM-Dase) gene from Alcaligenes bronchisepticus KU 1201 . The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M(r)) 24734 . The M(r) deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A . bronchisepticus . No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19 . The enzyme produced in E . coli has the same M(r) and the same enzyme activity as that purified from A . bronchisepticus . Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.

FEMS Microbiol Lett, 1992 Oct 15, 76(3), 227 - 33
Identification of a lipoamide dehydrogenase gene as second locus affected in poly(3-hydroxybutyric acid)-leaky mutants of Alcaligenes eutrophus; Pries A et al.; From a genomic library of Alcaligenes eutrophus strain H16 in the broad-host range cosmid pVK100 a 6300-bp EcoRI-fragment was cloned, which restored the wild-type phenotype in transposon-induced poly(3-hydroxybutyric acid)-leaky mutants, derived from A . eutrophus . Nucleotide sequence analysis of the region adjacent to the transposon insertion revealed an open reading frame which complied with all criteria for a coding region . This region was referred to as phbL, and the deduced amino acid sequence from this part of phbL showed 60% amino acid identity in an overlap of 98 residues to the lipoamide dehydrogenase gene (lpd) of Escherichia coli . In addition, the 6300-bp EcoRI-fragment conferred expression of lipoamide dehydrogenase activity to E . coli.

J Bacteriol, 1992 Oct, 174(19), 6290 - 3
Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16; Kortluke C et al.; The formation of the catalytically active membrane-bound hydrogenase (MBH) of Alcaligenes eutrophus H16 requires the genes for the small and large subunits of the enzyme (hoxK and hoxG, respectively) and an accompanying set of accessory genes (C . Kortl ke, K . Horstmann, E . Schwartz, M . Rohde, R . Binsack, and B . Friedrich, J . Bacteriol . 174:6277-6289, 1992) . Other genes located in the adjacent pleiotropic region are also required . In the absence of these genes, MBH is synthesized but is catalytically inactive . Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity . The results suggest that the conversion of the two subunits into the catalytically active membrane-associated heterodimer depends on specific maturation processes mediated by hox genes.

Eur J Biochem, 1992 Oct 1, 209(1), 15 - 30
Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1; Timm A et al.; From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAO1 was cloned and characterised at the molecular level . Two genes coding for polyhydroxyalkanoate synthases, phaC1Pa and phaC2Pa, a polyhydroxyalkanoate depolymerase gene, phaDPa, and four adjacent open reading frames (ORF1, ORF2, ORF3 and ORF4) were identified from the nucleotide sequence . Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for sigma 54 and sigma 70 promoters, were identified experimentally upstream of phaC1Pa, which was shown by Northern blot analysis to constitute an operon together with phaDPa . A third putative promoter resembling the E . coli consensus sequence for sigma 70-dependent promoters was proposed upstream of phaC2Pa, which is in a bicistronic operon with ORF3 . Investigations of rpoN-negative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P . aeruginosa . Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate . The P . aeruginosa PAO1 polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB-4 and in the poly(3-hydroxybutyrate)-accumulating strain P . oleovorans DSM1045 . It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate) . The sequence of the putative translational product of ORF1 was similar to those of the leukotoxin repressor of Pasteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P . aeruginosa and to eukaryotic histone H1 proteins . The proteins of ORF2 and ORF3 appear to be previously unidentified.

Eur J Biochem, 1992 Oct 1, 209(1), 135 - 50
Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D; Liebergesell M et al.; From a genomic library of Chromatium vinosum strain D in lambda L47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis . This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A . eutrophus . A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C . vinosum . The structural genes for biosynthetic acetyl-CoA acyltransferase (beta-ketothiolase; phbACv, 1188 bp) and NADH-dependent acetoacetyl-CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp) . Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3' terminus . The functions of ORF4, ORF5, and ORF7 are not known . The amino acid sequences of beta-ketothiolase and acetoacetyl-CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A . eutrophus . Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a sigma 70-dependent promoter . This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays . These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low M(r) (phbCCv, 1068 bp) . The gene products of ORF2 and phbCCv, with M(r) of 40,525 and 39,730, respectively, were expressed in E . coli applying the T7 RNA polymerase/promoter system . Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A . eutrophus, highly conserved regions were identified.

J Bacteriol, 1992 Oct, 174(19), 6277 - 89
A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16; Kortluke C et al.; One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase . The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G . Eberz, C . Hogrefe, C . Kortluke, A . Kamienski, and B . Friedrich, J . Bacteriol . 168:636-641, 1986) . Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA . We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG) . The nucleotide sequence contains nine additional closely spaced open reading frames . Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane . Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain . Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

J Bacteriol, 1992 Oct, 174(20), 6590 - 9
Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus; Priefert H et al.; The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase {AMP forming}; EC 6.2.1.1) . This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C . Frund, H . Priefert, A . Steinbuchel, and H . G . Schlegel, J . Bacteriol . 171:6539-6548, 1989) . The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence . A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids) . Tn5 insertions in two transposon-induced mutants of A . eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE . The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted . In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity . The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source . An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54.

J Pharm Biomed Anal, 1992 Oct-Dec, 10(10-12), 931 - 6
Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase; Salamoun J et al.; The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine . A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector . The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes . The rate of the enzyme regeneration was also recorded . The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor . The stability of the enzymes is at least 3 weeks at ambient temperature . The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 173 - 8
Hydrogenase mutants of Alcaligenes eutrophus H16 show alterations in the electron transport system; Komen R et al.; Mutations in the genes coding for the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus strain H16 significantly affected the expression of respiratory chain components . In lithoautotrophically grown wild type cells electron flow mainly proceeded via the cytochrome c oxidases . Mutants defective in the membrane-bound hydrogenase contained a 2- to 3-fold higher cytochrome a content than the wild type and cytochrome c oxidase of the aa3-type was preferentially used by these cells for substrate oxidation . Mutants impaired in the soluble hydrogenase revealed slow growth on hydrogen, presumably due to inefficient reverse electron flow mechanisms which provide the cells with NADH for autotrophic CO2-fixation . In this class of mutants the two quinol oxidases of the o- and d-type in addition to the co-type oxidase were the predominant electron-transport branches.

FEMS Microbiol Lett, 1992 Sep 1, 75(1), 93 - 101
Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum; Du L et al.; In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity . Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis . A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL . Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase . The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product . The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively . A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.

FEMS Microbiol Lett, 1992 Sep 1, 75(1), 73 - 9
Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the gram-positive bacterium Rhodococcus ruber; Pieper U et al.; The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404 . The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus . Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371 . The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A . eutrophus and Pseudomonas oleovorans . Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel . A Mr 61,000 protein was identified as the PHA synthase of R . ruber by N-terminal amino acid sequence determination.

Optom Vis Sci, 1992 Sep, 69(9), 678 - 84
Effect of storage time with different lens care systems on in-office hydrogel trial lens disinfection efficacy: a multi-center study; Callender MG et al.; A combined prospective and retrospective study was conducted to evaluate the efficacy of in-office disinfection methods for hydrogel trial contact lenses . Two hundred and twenty-one trial contact lenses, disinfected by four different disinfection methods, were collected from seven Study Centers and cultured for microbial contamination after various storage periods . Negative and positive control lenses were included as an additional Center in this double-masked study . There was a significant difference in the incidence of microbial contamination among the Centers for all storage times (chi 2 p < 0.001) . Contamination of trial lenses in Centers using thermal disinfection with preserved saline (SoftWear Saline) was negative and thermal disinfection with nonpreserved saline (LensPlus Saline) was 8.7% . Lens contamination in Centers using chemical disinfection was 13.6% with ReNu and 40.7% with OptiFree . The degree of contamination ranged from 90 colony forming units (CFU)/ml to over 10 million CFU/ml . Among the microorganisms isolated after the different disinfection methods were Alcaligenes xylosoxidans, Serratia marcescens, Moraxella phenylpyruvica, Enterobacter agglomerans, Pseudomonas stutzeri, and various gram-positive organisms . This study suggests that practitioners should redisinfect all inventory trial lenses at least once a month to minimize the risk of patient infection.

Eur J Biochem, 1992 Aug 15, 208(1), 31 - 40
Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues . Implications for the CuA site of N2O reductase and cytochrome-c oxidase; Zumft WG et al.; The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri {Viebrock, A . & Zumft, W . G . (1988) J . Bacteriol . 170, 4658-4668} and the subunit II of cytochrome-c oxidase (COII) . Both types of enzymes possess the CuA binding site . The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P . stutzeri . The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A . eutrophus) and 70695 Da (P . aeruginosa) . The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport . A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences . Only three out of nine cysteine residues of the NosZ protein (P . stutzeri) are invariant . Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus . Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes . The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences . Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination . Of 23 histidine residues in NosZ (P . stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences . Conserved tryptophan residues are located close to several potential copper ligands . Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.

Sci Total Environ, 1992 Aug 12, 123-124, 267 - 77
Degradation of aliphatic halogen-substituted pesticides by dehalogenase isolated from Pseudomonas alcaligenes . Identification and properties of the enzyme; Busto MD et al.; Some characteristics of a 2,2-dichloropropionate dehalogenase induced in a bacterial strain capable of degrading high concentrations of the herbicide dalapon were studied . Polyacrilamide gel electrophoresis of the crude cell free extracts identified only one type of dehalogenase . The single enzymatic protein showed activity against a variety of chlorinated aliphatic acids but differed in their activity levels . Thus activity in mumol substrate converted (mg protein)-1 min-1 was 2-monochloropropionate 0.65, 2,2-dichloropropionate 0.56, 2-monochloroacetate 1.70 and 2,2-dichloroacetate 1.00 . In the crude extracts, the enzyme activity against 2,2-dichloropropionate was optimal at a broad pH range with a mid-point at pH 9.5 and apparent Km values were within the range 0.23-0.73 mM.

FEBS Lett, 1992 Jul 20, 306(2-3), 125 - 8
Photogeneration of NADH under coupled action of CdS semiconductor and hydrogenase from Alcaligenes eutrophus without exogenous mediators; Shumilin IA et al.; Photoreduction of NAD has been accomplished by a system consisting of the NAD-dependent hydrogenase from Alcaligenes eutrophus immobilized on CdS particles with formate as artificial electron donor . Enzymatically active NADH is formed under illumination of this system by visible light . Accumulation of the coenzyme dimer (NAD)2 was not detected . NAD photoreduction is supposed to proceed via the direct electron transfer from the semiconductor to the enzyme electron transport chain . However, NADH formation as a result of hydrogenase interaction with anion-radicals (CO2.-) formed in the course of formate photooxidation cannot at present be excluded.

J Bacteriol, 1992 Jul, 174(14), 4549 - 57
Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii; Menon AL et al.; Azotobacter vinelandii contains a heterodimeric, membrane-bound {NiFe}hydrogenase capable of catalyzing the reversible oxidation of H2 . The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 {ORF3}) . In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs . These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively . Mutagenesis of the chromosome of A . vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes . Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit . Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation . We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively . The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1325 - 35
Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1; Wohlfarth S et al.; The structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118 . Nucleotide sequence analysis revealed a gene of 936 bp . lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids . The mature protein was predicted to have a M(r) of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases . Furthermore it is highly homologous (greater than 60%) to other lipases from various pseudomonads . The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P . alcaligenes, even under relaxed stringency . Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M(r) 33587) that shows some homology to the limA gene product of P . cepacia . In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P . aeruginosa PAO1.

J Bacteriol, 1992 Jul, 174(13), 4391 - 400
Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16; Kruger N et al.; Two hundred thirty-nine base pairs upstream from acoXABC, which encodes the Alcaligenes eutrophus H16 structural genes essential for cleavage of acetoin, the 2,004-bp acoR gene was identified . acoR encodes a protein of 668 amino acids with a molecular mass of 72.9 kDa . The amino acid sequence deduced from acoR exhibited homologies to the primary structures of transcriptional activators such as NifA of Azotobacter vinelandii, NtrC of Klebsiella pneumoniae, and HoxA of A . eutrophus . Striking similarities to the central domain of these proteins and the presence of a typical nucleotide-binding site (GETGSGK) as well as of a C-terminal helix-turn-helix motif as a DNA-binding site were revealed . Between acoR and acoXABC, two different types of sequences with dual rotational symmetry {CAC-(N11 to N18)-GTG and TGT-(N10 to N14)-ACA} were found; these sequences are similar to NtrC and NifA upstream activator sequences, respectively . Determination of the N-terminal amino acid sequence of an acoR'-'lacZ gene fusion identified the translational start of acoR . S1 nuclease protection assay identified the transcriptional start site 109 bp upstream of acoR . The promoter region (TTGCGC-N18-TACATT) resembled the sigma 70 consensus sequence of Escherichia coli . Analysis of an acoR'-'lacZ fusion and primer extension studies revealed that acoR was expressed at a low level under all culture conditions, whereas acoXABC was expressed only in acetoin-grown cells . The insertions of Tn5 in six transposon-induced acetoin-negative mutants of A . eutrophus were mapped within acoR . On the basis of these studies, it is probable that AcoR represents a regulatory protein which is required for sigma 54-dependent transcription of acoXABC.

FEMS Microbiol Lett, 1992 Jun 15, 72(3), 285 - 90
Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus; Hustede E et al.; From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively . Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4 . These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R . rubrum or R . sphaeroides . Both fragments hybridized with the PHB synthase structural gene of A . eutrophus H16 and conferred the ability to express PHB synthase activity . Only the 15-kbp HindIII fragment from R . rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).

Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5015 - 9
Yeast flavohemoglobin is an ancient protein related to globins and a reductase family; Zhu H et al.; The hemoglobin of yeast is a two-domain protein with both heme and flavin prosthetic groups . The nucleotide sequences of the cDNA and genomic DNA encoding the protein from Saccharomyces cerevisiae show that introns are absent and that both domains are homologous with a flavoheme protein from Escherichia coli . The heme domains are also homologous with those of O2-binding heme proteins from several other distantly related bacteria, plants, and animals; all appear to be members of the same globin superfamily . Although the homologous hemoglobin of the bacterium Vitreoscilla sp . is a single-domain protein, several bacteria have related O2-binding heme proteins whose second domains have different structures and enzymatic activities: dihydropteridine reductase (E . coli), cytochrome c reductase (Alcaligenes eutrophus), and kinase in the O2 sensor of Rhizobium meliloti . This indicates that one evolutionary pathway of hemoglobin is that of a multipurpose domain attached to a variety of unrelated proteins to form molecules with different functions . The flavin domain of yeast hemoglobin is homologous with members of a flavoprotein family that includes ferredoxin reductase, nitric oxide synthase, and cytochrome P-450 reductase . The correspondence of yeast and E . coli flavohemoglobins indicates that the two-domain protein has been conserved intact for as long as 1.8 billion years, the estimated time of divergence of prokaryotes and eukaryotes provided that cross-species gene transfer has not occurred.

Microb Releases, 1992 Jun, 1(1), 23 - 8
Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics; Delgado A et al.; Pseudomonas sp . FR1 (pFRC20P) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylXYZL and xylS genes from the TOL plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from Alcaligenes eutrophus on pFRC20P . The survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil . The recombinant information was stable in bacteria introduced in soils and conferred selective advantage to the host bacterium when the soils were amended with low amounts of p-methylbenzoate . However, relatively high amounts of this chemical significantly reduced survival of the bacterium . Survival was more seriously affected by chlorobenzoates than by methylbenzoates . Evolution of 14CO2 from p-methyl-14C-benzoate in soils confirmed that Pseudomonas sp . FR1 (pFRC20P) functions in a complex system in accordance with its design . Survival of Pseudomonas sp . FR1 (pFRC20P) was better at 4 degrees C and 25 degrees C than at 37 degrees C . On plates, the pFRC20P plasmid was mobilized by helper plasmids to P . putida at a frequency of about 10(-6) transconjugants per recipient . However, no transfer of recombinant DNA integrated on the chromosome was found, nor was lateral transfer of the recombinant DNA borne by Pseudomonas sp . FR1 (pFRC20P) detected in soils.

Antimicrob Agents Chemother, 1992 May, 36(5), 1024 - 7
Susceptibility of Pseudomonas species to the novel antibiotics mureidomycins; Isono F et al.; Strains of Pseudomonas aeruginosa, including imipenem- or ofloxacin-resistant clinical isolates, and some other species in the genus Pseudomonas were inhibited by novel antibiotics of the mureidomycin (MRD) group . On the other hand, almost all other gram-positive and gram-negative bacteria were resistant to MRDs, though the antibiotics potently inhibited the in vitro peptidoglycan synthesis of Escherichia coli and P . aeruginosa . All of the strains in the genus Pseudomonas that were inhibited by less than or equal to 200 micrograms of MRDs per ml were classified into the rRNA groups I and III, and none of the tested strains of rRNA group I were resistant to MRDs, suggesting that these two groups are closely related to each other evolutionary . Among group I strains, P . aeruginosa, P . mendocina, P . stutzeri, and P . alcaligenes were more susceptible than the others, suggesting a closer relationship among these species.

Appl Environ Microbiol, 1992 Apr, 58(4), 1308 - 12
Complete oxidation of linear alkylbenzene sulfonate by bacterial communities selected from coastal seawater; Sigoillot JC et al.; Anionic surfactants, especially alkylbenzene sulfonates, are discharged into marine areas in great quantities . Because of their poor biodegradability, linear alkylbenzene sulfonates accumulate in seawater and sediments . Bacterial communities that can degrade surfactants were selected from coastal seawater contaminated by urban sewage . All the isolated strains consisted of gram-negative, strictly aerobic rods or helical bacteria . Some of these, though isolated from coastal seawater, did not need sodium for growth and appeared to be related to the genera Alcaligenes and Pseudomonas . Complete surfactant biodegradation was achieved by three important steps: terminal oxidation of the alkyl chain, desulfonation, and aromatic-ring cleavage . Only a few strains were able to carry out the first two steps . The aromatic ring was then cleaved by other strains that possess very specific enzymatic activities . Finally, a number of strains grew on short acids that were end-of-metabolism products of the others.

Appl Environ Microbiol, 1992 Apr, 58(4), 1271 - 5
Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4; Neilson JW et al.; Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems . Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4) . Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined . Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103 . Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA . The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA . However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA . This sensitive detection method is more specific than use of traditional indicator media (M . A . Loos, Can . J . Microbiol . 21:104-107, 1975) . An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

Appl Environ Microbiol, 1992 Apr, 58(4), 1089 - 94
Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain; Slater S et al.; An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV) . This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E . coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate . 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture . P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production . A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM . 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth . The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.

Int J Biol Macromol, 1992 Apr, 14(2), 81 - 6
Biosynthesis and n.m.r . studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16; Yoshie N et al.; Alcaligenes eutrophus H16 was grown on mixtures of 1H- and 2H-acetate as carbon sources . The accumulation of deuterated poly(3-hydroxybutyrate) (P(3HB)) was observed . The deuterium distributions in the isolated P(3HB)s were determined from 1H and 2H-n.m.r . spectra and confirmed by 13C-n.m.r . spectra . Although one would expect to synthesize P({2,2,4,4,4-2H5}3HB) when the cells were grown on 2H-acetate as the sole carbon source, the methyl, methylene and methine groups of the P(3HB) contained both deuterium and proton . This observation indicates some substitution from 2H to 1H during the P(3HB) synthesis . The 2H content in the methyl groups was larger than that in the methylene groups, which suggests a kinetic isotope effect in the P(3HB) synthesizing process . The deuterium distributions in the two magnetically non-equivalent methylene protons were determined to be different, which indicates stereoselectivity at the C2 site.

Int J Biol Macromol, 1992 Apr, 14(2), 117 - 8
Biosynthesis of poly(3-hydroxyalkanoates) from threonine; Nakamura K et al.; A series of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) was biosynthesized by Alcaligenes eutrophus from an amino acid, threonine . The 3HV content of these polyesters ranged from less than 0.1% to 30%.

Appl Microbiol Biotechnol, 1992 Apr, 37(1), 28 - 31
Stereoselective epoxidation of phenyl allyl ether by alkene-utilizing bacteria; Mahmoudian M et al.; Eighteen newly isolated ethene- and propene-utilizing bacteria were screened for the ability to produce phenyl glycidyl ether, a common precursor for the synthesis of beta blockers, from phenyl allyl ether . These organisms included Aerococcus, Alcaligenes, Micrococcus and Staphylococcus spp . and a variety of Gram-negative, Gram-positive and Gram-variable mesophilic rods/coccobacilli not yet identified . The majority of ethene- and propene-grown cultures (14 strains) accumulated phenyl glycidyl ether (0.4-1.7 mM) as the sole oxidation product . The bioconversions with the three most promising ethene-utilizers (M26, M90C, M93A) were scaled-up to yield essentially optically pure (enantiomeric excess = 93%) S-(+)-phenyl glycidyl ether . This is currently under investigation for commercial production of optically pure beta blockers.

Microbiol Rev, 1992 Mar, 56(1), 195 - 228
Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria; Silver S et al.; Regulation of chromosomally determined nutrient cation and anion uptake systems shows important similarities to regulation of plasmid-determined toxic ion resistance systems that mediate the outward transport of deleterious ions . Chromosomally determined transport systems result in accumulation of K+, Mg2+, Fe3+, Mn2+, PO4(3-), SO4(2-), and additional trace nutrients, while bacterial plasmids harbor highly specific resistance systems for AsO2-, AsO4(3-), CrO4(2-), Cd2+, Co2+, Cu2+, Hg2+, Ni2+, SbO2-, TeO3(2-), Zn2+, and other toxic ions . To study the regulation of these systems, we need to define both the trans-acting regulatory proteins and the cis-acting target operator DNA regions for the proteins . The regulation of gene expression for K+ and PO4(3-) transport systems involves two-component sensor-effector pairs of proteins . The first protein responds to an extracellular ionic (or related) signal and then transmits the signal to an intracellular DNA-binding protein . Regulation of Fe3+ transport utilizes the single iron-binding and DNA-binding protein Fur . The MerR regulatory protein for mercury resistance both represses and activates transcription . The ArsR regulatory protein functions as a repressor for the arsenic and antimony(III) efflux system . Although the predicted cadR regulatory gene has not been identified, cadmium, lead, bismuth, zinc, and cobalt induce this system in a carefully regulated manner from a single mRNA start site . The cadA Cd2+ resistance determinant encodes an E1(1)-1E2-class efflux ATPase (consisting of two polypeptides, rather than the one earlier identified) . Cadmium resistance is also conferred by the czc system (which confers resistances to zinc and cobalt in Alcaligenes species) via a complex efflux pump consisting of four polypeptides . These two cadmium efflux systems are not otherwise related . For chromate resistance, reduced cellular accumulation is again the resistance mechanism, but the regulatory components are not identified . For other toxic heavy metals (with few exceptions), there exist specific plasmid resistances that remain relatively terra incognita for future exploration of bioinorganic molecular genetics and gene regulation.

Appl Environ Microbiol, 1992 Mar, 58(3), 1027 - 30
Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria; Greer LE et al.; Seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria, including Pseudomonas, Alcaligenes, and Bordetella spp., were compared on the basis of growth kinetics . Estimates of maximum growth rate (mu max, k1) and half-saturation growth constant (Ks, k3) were obtained by fitting substrate depletion curves to a four-parameter version of the integrated Monod equation . Estimates of Ks ranged from 2.2 micrograms/ml (10 microM) to 33.8 micrograms/ml (154 microM), and estimates of mu max ranged from 0.20 h-1 (Td = 3.5 h) to 0.32 h-1 (Td = 2.2 h) . Estimates of mu max, but not Ks, were affected by changes in initial inoculum density . Maximum growth rates (mu max) were also estimated from turbidity measurements . They ranged from 0.10 h-1 (Td = 6.9 h) to 1.0 h-1 (Td = 0.7 h) . There was no correlation between estimates of mu max derived from substrate depletion curves and those derived from turbidity measurements (P = 0.20).

Can J Microbiol, 1992 Mar, 38(3), 241 - 7
Mutational analysis of genes determining antagonism of Alcaligenes sp . strain MFA1 against the phytopathogenic fungus Fusarium oxysporum; Martinetti G et al.; Alcaligenes sp . strain MFA1 inhibits microconidial germination and germination-tube elongation of Fusarium oxysporum f.sp . dianthi and reduces the severity of fusarium wilt of carnation, presumably as a result of its production of a siderophore (G.Y . Yuen and M.N . Schroth . 1986 . Phytopathology, 76:171-176) . Derivative strains of MFA1, deficient in antagonism against F . oxysporum and in iron-limited growth, were obtained by Tn5 mutagenesis . The presence of a single Tn5 insertion in the genomic DNA of each derivative strain was detected by Southern analysis . Marker-exchange mutagenesis of strain MFA1 with DNA fragments, containing Tn5 and flanking sequences cloned from representative mutants, confirmed the association of single Tn5 insertions with the loss of antifungal activity and iron-independent growth of MFA1 . These results are consistent with the involvement of siderophore biosynthesis by MFA1 in the inhibition of F . oxysporum.

J Bacteriol, 1992 Feb, 174(3), 899 - 907
Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus; Priefert H et al.; The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanol-grown cells of Alcaligenes eutrophus was determined . By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C . Frund, H . Priefert, A . Steinbuchel, and H . G . Schlegel, J . Bacteriol . 171:6539-6548, 1989) . A 2.8-kbp PstI subfragment of D, which harbored acoD, was sequenced . It revealed an open reading frame of 1,518 bp, encoding a protein with a relative molecular weight of 54,819 . The insertions of Tn5::mob of two transposon-induced mutants of A . eutrophus, which were impaired in the catabolism of acetoin, were mapped 483 or 1,359 bp downstream from the translational start codon of acoD . The structural gene was preceded by a putative Shine-Dalgarno sequence . The transcriptional start site 57 bp upstream of acoD was identified and was preceded by a sequence which exhibited a striking homology to the enterobacterial sigma 54-dependent promoter consensus sequence . This was in accordance with the observation that the expression of acoD and of other acetoin-catabolic genes depended on the presence of an intact rpoN-like gene . Alignments of the amino acid sequence deduced from acoD with the primary structures of aldehyde dehydrogenases from other sources revealed high degrees of homology, amounting to 46.5% identical amino acids.

Int J Biol Macromol, 1992 Feb, 14(1), 33 - 40
Biodeuteration of poly(beta-hydroxybutyrate); Gross RA et al.; The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O . The growth of Rb . sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4) . In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight . Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A . eutrophus . Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced . Partially deuterated PHB was obtained in each case, using a deuterium enriched culture . Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.

Mol Gen Genet, 1992 Feb, 231(3), 375 - 84
Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutrophus phbB and to Rhizobium meliloti nodG; Vieille C et al.; A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503 . The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R . meliloti NodG . The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the phbB gene and involved in poly-beta-hydroxybutyrate (PHB) synthesis . Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli sigma 70 consensus promoter . An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E . coli, but was functional and constitutively expressed in Azospirillum . Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity . An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains . No ORF3 homologues were found in A . irakense, A . amazonense, A . halopraeferens or in several A . lipoferum strains.

Plasmid, 1992 Jan, 27(1), 65 - 71
Plasmid chromate resistance and chromate reduction; Cervantes C et al.; Compounds of hexavalent chromium (chromates and dichromates) are highly toxic . Plasmid genetic determinants for chromate resistance have been described in several bacterial genera, most notably in Pseudomonas . Resistance to chromate is associated with decreased chromate transport by the resistant cells . The genes for a hydrophobic polypeptide, ChrA, were identified in chromate resistance plasmids of Pseudomonas aeruginosa and Alcaligenes eutrophus . ChrA is postulated to be responsible for the outward membrane translocation of chromate anions . Widespread bacterial reduction of hexavalent chromate to the less toxic trivalent chromic ions is also known . Chromate reduction determinants have not, however, been found on bacterial plasmids or transposons . In different bacteria, chromate reduction is either an aerobic or an anaerobic process (but not both) and is carried out either by soluble proteins or by cell membranes . Chromate reduction may also be a mechanism of resistance to chromate, but this has not been unequivocally shown.

Plasmid, 1992 Jan, 27(1), 17 - 28
Resistance to cadmium, cobalt, zinc, and nickel in microbes; Nies DH; The divalent cations of cobalt, zinc, and nickel are essential nutrients for bacteria, required as trace elements at nanomolar concentrations . However, at micro- or millimolar concentrations, Co2+, Zn2+, and Ni2+ (and "bad ions" without nutritional roles such as Cd2+) are toxic . These cations are transported into the cell by constitutively expressed divalent cation uptake systems of broad specificity, i.e., basically Mg2+ transport systems . Therefore, in case of a heavy metal stress, uptake of the toxic ions cannot be reduced by a simple down-regulation of the transport activity . As a response to the resulting metal toxicity, metal resistance determinants evolved which are mostly plasmid-encoded in bacteria . In contrast to that of the cation Hg2+, chemical reduction of Co2+, Zn2+, Ni2+, and Cd2+ by the cell is not possible or sensible . Therefore, other than mutations limiting the ion range of the uptake system, only two basic mechanisms of resistance to these ions are possible (and were developed by evolution): intracellular complexation of the toxic metal ion is mainly used in eucaryotes; the cadmium-binding components are phytochelatins in plant and yeast cells and metallothioneins in animals, plants, and yeasts . In contrast, reduced accumulation based on an active efflux of the cation is the primary mechanism developed in procaryotes and perhaps in Saccharomyces cerevisiae . All bacterial cation efflux systems characterized to date are plasmid-encoded and inducible but differ in energy-coupling and in the number and types of proteins involved in metal transport and in regulation . In the gram-positive multiple-metal-resistant bacterium Staphylococcus aureus, Cd2+ (and probably Zn2+) efflux is catalyzed by the membrane-bound CadA protein, a P-type ATPase . However, a second protein (CadC) is required for full resistance and a third one (CadR) is hypothesized for regulation of the resistance determinant . The czc determinant from the gram-negative multiple-metal-resistant bacterium Alcaligenes eutrophus encodes proteins required for Co2+, Zn2+, and Cd2+ efflux (CzcA, CzcB, and CzcC) and regulation of the czc determinant (CzcD) . In the current working model CzcA works as a cation-proton antiporter, CzcB as a cation-binding subunit, and CzcC as a modifier protein required to change the substrate specificity of the system from Zn2+ only to Co2+, Zn2+, and Cd2+.

Res Microbiol, 1992 Jan, 143(1), 27 - 36
Identification of a lacZ gene in Vibrio cholerae; Parsot C; Plasmids that express an enzymatically active beta-galactosidase in an Escherichia coli delta lac strain were isolated from libraries of recombinant plasmids containing Vibrio cholerae chromosomal DNA . Deletion analysis localized the gene responsible for beta-galactosidase activity on a 3.1-kb DNA fragment, and the gene product was identified as a protein of approximately 110 kDa . Primary sequence comparisons indicated that this V . cholerae gene is homologous to the E . coli lacZ gene . In contrast to the lac loci of other bacteria, no gene that could specify a lactose transport system was detected in the vicinity of the V . cholerae lacZ gene, which may account for the inability of this species to use lactose . In V . cholerae, portions of open reading frames encoding proteins homologous to the Alcaligenes eutrophus chrA and E . coli galR gene products were detected upstream and downstream from the lacZ gene, respectively.

Mikrobiologiia, 1992 Jan-Feb, 61(1), 76 - 83
{The use of pulsed field electrophoresis using diversely directed electrical fields for establishing the degree of similarity of three Pseudomonas cultures}; Kondrat'eva TF; It has been shown that from the group of three restriction endonucleases (XbaI, Uba 78I, DraI) only XbaI digests the native Pseudomonas genomic DNA into the number of fragments, sufficient for their comparative analysis . Optimal electrophoresis conditions were adjusted for the better DNA restricts separation . Each Pseudomonas culture gives unique patterns of DNA restriction . The calculation of the conservative fragments fraction content in the samples led us to the conclusion that P . fluorescens differs equally from both P . fluorescens 80 and P . alcaligenes . The P . fluorescens 80 DNA contains a relatively larger number of fragments equal in size with those of P . alcaligenes DNA, than DNA of P . fluorescens . We have concluded that the systematics of the Pseudomonas genus is still imperfect and that the analysis of DNA restriction patterns is necessary for the estimation of the similarity level of cultures studied.

Microbios, 1992, 69(280-281), 215 - 21
An unusual yellow pigmented Pseudomonas species isolated from chlorinated municipal town water supply; O'Brien JR; A Gram-negative non-fermentative rod (GN-NF) which produced a yellow non-diffusible pigment is described . The isolate is typical of the heterotropic population of bacteria present in the chlorinated town water, as supplied to the city of Brisbane, Queensland, Australia . The strain could be not satisfactorily identified by Automatic Microbic Systems (Vitek) or API 20 NE protocols . Characterization tests, performed according to conventional methods, including other morphological and physiological characteristics, indicated a close relationship with the genus Pseudomonas . The phenotype was different from any previously described yellow and non-diffusible pigmented species in the genus Pseudomonas, including P . aureofaciens, P . palleronii, P . paucimobilis, and P . alcaligenes . The town water isolate showed a marked sensitivity to the vibriostatic agent 0/129 (2,4-diamino-6,7,diisopropyl-pteridine phosphate) at a concentration of 150 micrograms.

Appl Environ Microbiol, 1992 Jan, 58(1), 399 - 402
Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50; Layton AC et al.; The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate . Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2 . This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp . strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate . The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70.

Arch Microbiol, 1992, 157(3), 301 - 4
The structural genes encoding CO dehydrogenase subunits (cox L, M and S) in Pseudomonas carboxydovorans OM5 reside on plasmid pHCG3 and are, with the exception of Streptomyces thermoautotrophicus, conserved in carboxydotrophic bacteria; Hugendieck I et al.; Employing deoxyoligonucleotide probes and Southern hybridizations, we have examined in carboxydotrophic bacteria the localization on the genome of genes encoding the large, medium and small subunits of CO dehydrogenase (coxL, M and S, respectively) . In Pseudomonas carboxydovorans OM5 coxL, M and S were identified on the plasmid pHCG3; they were absent on the chromosome . This was evident from positive hybridizations with plasmid DNA of the wild-type strain OM5 and the absence of hybridizations with chromosomal DNA from the plasmid cured mutant strain OM5-12 . The genes coxL, M and S were found on plasmids in all other plasmid-containing carboxydotrophic bacteria e.g . Alcaligenes carboxydus, Azomonas B1, Pseudomonas carboxydoflava, Pseudomonas carboxydovorans OM2 and OM4 . Cox L, M and S could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, Pseudomonas carboxydohydrogena, and Pseudomonas carboxydovorans OM3 . These results essentially confirm and extend former reports that cox genes are rather conserved among carboxydotrophic bacteria of distinct taxonomic position . However, Streptomyces thermoautotrophicus is an noteworthy exception since none of the three cox genes could be detected . This refers to a new type of CO dehydrogenase and is in accord with results indicating that the S . thermoautotrophicus CO dehydrogenase has an unusual electron acceptor specificity and some other properties setting it apart from the 'classical' CO dehydrogenases.

J Basic Microbiol, 1992, 32(6), 397 - 403
Selective isolation of fluorescent and nonfluorescent Pseudomonas species from Sohag Governorate (Upper Egypt); Shoreit AA et al.; Three hundred and fourty nine isolates belonging to ten different species of Pseudomonas were isolated and identified from soil and water samples on four selective isolation media: Modified Trypticase soy agar (TSA), ACC-agar containing ampicillin-chloramphenicol-cycloheximide, Nitrate plus organic acid agar and Tryptophan substrate agar . The fluorescent species were P . putida (21.2% of the total Pseudomonas species), P . fluorescens (15.8%), P . aeruginosa (9.7%) and P . aureobaciens (2.6%), while the nonfluorescent species were P . stutzeri (12.4%), P . alcaligenes (11.2%), P . pseudoalcaligenes (9.5%), P . mendocina (8.3%), P . lemoignei (5.7%) and P . ruhlandii (3.4%) . Modified TSA and ACC media were efficient for the isolation of both fluorescent and nonfluorescent species, whereas Nitrate and Tryptophan media were more selective for the isolation of nonfluorescent species . The optimum temperature for the isolation of different species ranged between 20 degrees and 37 degrees C.

J Basic Microbiol, 1992, 32(6), 381 - 7
Immunoelectronmicroscopic study of the nucleoid structure of hydrogen bacteria; Mogilnaya OA et al.; Electron microscopical studies of the nucleoid structure of hydrogen bacteria using ultrahin sections and spread DNA from bacterial cell lysates revealed a different DNA packaging in the cell . A compact state of the major part of DNA at all growth stages and stability of nucleosome-like structures were shown . The use of antibodies to HU protein of E . coli labelled by protein A-colloidal gold demonstrated the immunological relationship between HU protein of E . coli and histone-like proteins of Alcaligenes eutrophus and their possible role in the nucleosome-like DNA packaging in procariotic genome.

Arch Microbiol, 1992, 158(2), 107 - 14
Nucleotide sequence of the rpoN (hno) gene region of Alcaligenes eutrophus: evidence for a conserved gene cluster; Warrelmann J et al.; The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor sigma 54 involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria . In A . eutrophus hydrogen metabolism is under control of rpoN . The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene . The derived amino acid sequence of the A . eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms . Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF greater than 54) of the rpoN gene . A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.

Acta Cient Venez, 1992, 43(3), 154 - 8
{Bacillary necrosis in larvae of the bivalve Euvola ziczac (Linnaeus, 1758) caused by a Pseudomonas sp.}; Lodeiros C et al.; The incidence of bacteria as etiological agents of diseases in larvae of bivalve mollusks, is well documented in the literature . In this study, a series of test were performed to identify and estimate pathogenic level of marine bacteria isolated during an epizootic in a larval culture system of the tropical scallop Euvola ziczac . Such bacteria was identified as Pseudomonas sp . belonging to the group alcaligenes . The bacterium produced not only lethal effects on the scallop larvae through infection (invasive action), but also a possible pathogenic activity through endotoxins associated to bacterial cell-wall . In addition, it is possible that the isolated bacteria could act as a pathogen with members of the genus Vibrio, which was also present during the epizootic outbreak in the culture system . Susceptibility of Pseudomonas sp . to commercial antibiotics was remarkable.

Appl Environ Microbiol, 1992 Jan, 58(1), 314 - 25
Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem; Fulthorpe RR et al.; A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp . strain BR60, into lake water and sediment flowthrough microcosms . Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl . The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method . Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems . The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration . Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp . strain BR60 . Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60 . The importance of plasmid transfer and transposition during chemical adaptation is discussed.

Chin J Biotechnol, 1992, 8(3), 211 - 8
Screening of bacterial strains producing maltotetraose-forming amylase and the conditions for enzyme production; Yan Z et al.; The authors isolated 1380 bacterial strains from 290 soil samples collected in China and 490 strains were received from other research teams in this institute . By screening 707 strains showed starch-hydrolyzing activity . By further screening and paper chromatographic test, three strains with maltotetraose as the major product were obtained . The maltotetraose was further confirmed by treatment with beta-amylase splitting to maltose and with glucoamylase to glucose . The most promising strain was 537.1, which produced maltotetraose about 90% (w/w) in the starch hydrolysate . While the other two strains produced maltose and maltotriose besides maltotetraose . Strain 537.1 was tentatively identified as Alcaligenes sp . The optimum conditions for enzyme production were as follows: medium composition: 1.5% maltose; 0.5% peptone with initial pH of 7.0-7.5; cultured at 27-28 degrees C for 48 hours on rotary shaker . The culture supernatant of the strain 537.1 can hydrolyze starch and different kinds of cereal flour with a high yield of maltotetraose in the hydrolysate.

Arch Biochem Biophys, 1991 Dec, 291(2), 263 - 9
Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase; Lee BG et al.; The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B . Lee and F . R . Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits . Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate . This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits . In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies . The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.

Appl Environ Microbiol, 1991 Dec, 57(12), 3502 - 10
Identification of a locus within the hydrogenase gene cluster involved in intracellular nickel metabolism in Bradyrhizobium japonicum; Fu CL et al.; A 0.6-kb fragment of DNA involved in intracellular Ni metabolism was isolated and cloned from a cosmid containing 23.2 kb of hydrogenase-related genes of Bradyrhizobium japonicum . This locus is located 8.3 kb upstream of the hydrogenase structural genes . The hydrogenase activity of a mutant with a gene-directed mutation at this locus (strain JHK7) showed dependency on nickel provided during hydrogenase derepression . The hydrogenase activity was only 20% of that in the wild-type strain, JH, at a concentration of 0.5 microM NiCl2 . The hydrogenase activity in JH reached its maximum at 3 microM NiCl2, whereas the mutant (JHK7) reached wild-type levels of hydrogenase activity when derepressed in 50 microM NiCl2 . Studies with the hup-lacZ transcriptional fusion plasmid pSY7 in JHK7 showed that the mutant JHK7 expressed less promoter activity under low-nickel conditions than did strain JH . The mutant accumulated less nickel during a 45-h hydrogenase derepression period than did the wild type . However, both JHK7 and the JH wild-type strain had the same short-term Ni transport rates, and the KmS for Ni of both strains were about 62 microM . When incubated under non-hydrogenase-derepression conditions, the mutant accumulated Ni at the same rate as strain JH . However, this stored source of nickel was unable to restore hydrogenase expression ability of the mutant to wild-type levels during derepression without nickel . The results indicate that the locus identified in B . japonicum is not involved in nickel-specific transport; indeed, it was not at all homologous to the "nickel transporter" hoxN gene of Alcaligenes eutrophus.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1991 Nov 1, 201(3), 547 - 50
Determination of the cDNA sequence for the human mitochondrial 75-kDa Fe-S protein of NADH-coenzyme Q reductase; Chow W et al.; A human-hepatoma cDNA lambda gt11 expression library was probed with an antibody to holoenzyme complex I (NADH-CoQ reductase) of the respiratory chain . One of the 30 antibody positive clones was purified to homogeneity, amplified by the polymerase chain reaction (PCR), subcloned and sequenced . It proved to be highly similar to the cDNA sequence for the bovine 75-kDa Fe--S protein . Using the sequence obtained from this library, both sense and antisense oligonucleotides were constructed and used to probe a human kidney cDNA library using PCR amplification with oligonucleotides that flank the polylinker region of the lambda phage . Two further cDNA clones were obtained which overlapped and covered the entire cDNA sequence of 2526 bp . The encoded protein of 727 amino acids has 21 amino acids that differ from the bovine-protein sequence . Northern blot analysis of mRNA from fibroblasts of complex-I deficient patients revealed no abnormalities . We show that this Fe--S protein has significant similarity with (a) the gamma chain of the hydrogen hydrogenase of Alcaligenes eutrophus and (b) the A chain of the formate dehydrogenase of Methanobacterium formicum.

Antimicrob Agents Chemother, 1991 Nov, 35(11), 2410 - 4
Activity of meropenem against antibiotic-resistant or infrequently encountered gram-negative bacilli; Jorgensen JH et al.; Meropenem was compared in vitro with imipenem as well as with several other contemporary beta-lactams, ciprofloxacin, and gentamicin against a group of highly antibiotic resistant members of the family Enterobacteriaceae and a collection of oxidase-positive and/or glucose-nonfermentative gram-negative bacilli . In this study, meropenem was more active than imipenem against isolates of Enterobacter, Klebsiella, Morganella, Providencia, Alcaligenes, Aeromonas, and Pasteurella.

Mol Microbiol, 1991 Nov, 5(11), 2695 - 705
Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus; Windhovel U et al.; A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants . The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes . CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon . The cfx operon and cfxR are transcribed divergently . The N-terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family . Heterologous expression of cfxR in Escherichia coli was achieved using the pT7-7 system . Mobility shift experiments demonstrated that CfxR is a DNA-binding protein with a target site upstream of both the chromosomal and the plasmid-encoded cfx operons.

Can J Microbiol, 1991 Nov, 37(11), 875 - 7
A zinc-binding protein in a metal-resistant strain, Alcaligenes eutrophus CH34; Remacle J et al.; Synthesis of a zinc-binding protein was induced when Alcaligenes eutrophus CH34 was grown in the presence of high concentrations of zinc (2.3 mM) . The zinc-binding protein has a low content of cysteine and a high content of acidic amino acids and, thus, differs from metallothionein.

Mol Gen Genet, 1991 Nov, 230(1-2), 136 - 44
Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis; Kiel JA et al.; The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli . Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region . A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene . The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance . The B . stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - sigma H) . The glgB promoter was utilized in B . subtilis exclusively in the stationary phase, and only transcribed at low levels in B . subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B . subtilis . In an expression vector, the B . stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E . coli as well as in B . subtilis, with optimal branching activity at 53 degrees C.

J Bacteriol, 1991 Nov, 173(22), 7313 - 23
Evidence for two sets of structural genes coding for ribulose bisphosphate carboxylase in Thiobacillus ferrooxidans; Kusano T et al.; Previously, we reported the cloning of the ribulose-1,5-bisphosphate carboxylase genes (rbcL1-rbcS1) of Thiobacillus ferrooxidans Fe1 (T . Kusano, K . Sugawara, C . Inoue, and N . Suzuki, Curr . Microbiol . 22:35-41, 1991) . With these genes as probes, a second set of ribulose-1,5-bisphosphate carboxylase genes (rbcL2-rbcS2) was identified in the same strain and cloned . rbcL1 and rbcL2 encode the large subunits, and rbcS1 and rbcS2 encode the small subunits . Similar restriction patterns between these gene sets suggested a high level of sequence homology . In fact, sequence analysis showed that a 2.2-kb region, including the entire large and small subunit structural genes, was totally conserved in rbcL1-rbcS1 and rbcL2-rbcS2 . The rbcL1 (rbcL2) and rbcS1 (rbcS2) genes were 1,422 and 333 bp in length and encoded 473- and 110-amino-acid proteins, respectively . The genes were separated by a 90-bp spacer sequence and were preceded by possible ribosome-binding sites . The N-terminal amino acid sequences of the subunit proteins, synthesized in Escherichia coli, were determined by Edman degradation and found to agree with the deduced amino acid sequences, except for the N-terminal methionine residue . The transcriptional start site of the rbc genes was determined by primer extension, and the size of the rbc transcript was estimated to be about 2.1 kb, suggestive of the cotranscription of rbcL1-rbcS1 and/or rbcL2-rbcS2 mRNAs . Comparisons of amino acid sequences of both subunits with those of other organisms revealed that the ribulose-1,5-bisphosphate carboxylase of T . ferrooxidans, a chemoautotrophic bacterium, is phylogenetically closer to the photosynthetic bacterium Chromatium vinosum than to another chemoautotrophic bacterium, Alcaligenes eutrophus.

Plant Mol Biol, 1991 Oct, 17(4), 853 - 63
Evolution of the Rubisco operon from prokaryotes to algae: structure and analysis of the rbcS gene of the brown alga Pylaiella littoralis; Assali NE et al.; The rbcS gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the brown alga Pylaiella littoralis is located within the plastid genome and is transcribed as a single polycistronic mRNA with the gene for the large subunit of Rubisco, rbcL . The structure of the Rubisco operon from P . littoralis was determined . Molecular phylogenies for rbcS and rbcL with a wide range of prokaryotes and eukaryotes were constructed which are congruent with recent evidence for polyphyletic plastid origins . Both rbcL and rbcS of the beta-purple bacterium Alcaligenes eutrophus clearly cluster with the rhodophyte and chromophyte proteins . The data suggest that the Rubisco operons of red algal and chromophytic plastids derive from beta-purple eubacterial antecedents, rather than the cyanobacterial lineage of eubacteria from which other of their genes derive . This implies a lateral transfer of Rubisco genes from beta-purple eubacterial ancestors to the cyanobacterial ancestor of rhodophyte and chromophyte plastids.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8312 - 6
Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences; Nakatsu C et al.; The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described . Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp . strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate . The transposon is flanked by a directly repeated sequence of 3201 base pairs (bp), which in turn is flanked by 110-bp inverted repeats . The 3.2-kb repeated sequence, designated IS1071, exists in multiple copies in the genome of Alcaligenes sp . strain BR60 and is involved in recombination of the catabolic genes into the chromosome of this strain . Sequence analysis revealed that the inverted repeat of IS1071 and the derived amino acid sequence of the single open reading frame within IS1071 are related to the inverted repeats and transposase (TnpA) proteins of the class II (Tn3 family) transposable elements . The absence of a resolvase gene within IS1071 suggests that this element is capable of determining the first step in class II transposition only . This was confirmed by observations on the IS1071-dependent formation of stable cointegrates in a recombination-deficient Escherichia coli . These results support an evolutionary scheme in which the class II transposable elements descended from simple insertion sequences.

FEBS Lett, 1991 Sep 2, 289(1), 44 - 6
Purification and characterization of N-acyl-D-glutamate deacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6; Sakai K et al.; The purification and properties of N-acyl-D-glutamate deacylase from the cell extracts of Alcaligenes xylosoxydans subsp . xylosoxydans A-6 were studied . The two active fractions (peaks I and II) were obtained by a Mono Q column chromatography . The predominant enzyme (peak I) has been purified, 1960-fold to homogeneity and characterized . The enzyme was a monomer with a molecular weight of 59,000 . The optimum pH and the isoelectric point were 8.0 and 5.5, respectively . The enzyme catalyzed the hydrolysis of N-acyl derivatives of D-glutamate . The Kms for N-acetyl, N-butyryl and N-propionyl derivatives of D-glutamate were 0.129, 0.066 and 0.01 mM, respectively.

Mikrobiol Zh, 1991 Sep-Oct, 53(5), 33 - 7
{The autoselection of neustonic forms of bacteria}; Stabnikova EV et al.; Self-breeding of neuston forms of Methylobacterium sp., Pseudomonas putida BC-2, Alcaligenes paradoxus BC-1, Bacillus thuringiensis var . israilensis bacteria as well as of a mixed culture of methylotrophs is shown possible . In spite of ability of hydrophobicity of the cell surface the suggested method of self-breeding may be used to perfect properties of larvicidal biopreparations, and bacterial preparations which intensify self-purification of water bodies.

J Bacteriol, 1991 Sep, 173(18), 5843 - 53
Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli; Pries A et al.; From genomic libraries of Alcaligenes eutrophus H16 in lambda L47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(beta-hydroxybutyric acid) (PHB)-leaky mutants derived from strains H16 and JMP222 . The nucleotide sequence analysis of a 4.5-kb region of one of these fragments revealed two adjacent open reading frames (ORF) which are relevant for the expression of the PHB-leaky phenotype . The 1,799-bp ORF1 represented a gene which was referred to as phbI . The amino acid sequence of the putative protein I (Mr, 65,167), which was deduced from phbI, exhibited 38.9% identity with the primary structure of enzyme I of the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP-PTS) . The upstream 579-bp ORF2 was separated by 50 bp from ORF1 . It included the 270-bp phbH gene which encoded protein H (Mr, 9,469) . This protein exhibited 34.9% identity to the HPr protein of the E . coli PEP-PTS . Insertions of Tn5 in different PHB-leaky mutants were mapped at eight different positions in phbI and at one position in phbH . Mutants defective in phbH or phbI exhibited no pleiotropic effects and were not altered with respect to the utilization of fructose . However, PHB was degraded at a higher rate in the stationary growth phase . The functions of these HPr- and enzyme I-like proteins in the metabolism of PHB are still unknown . Evidence for the involvement of these proteins in regulation of the metabolism of intracellular PHB was obtained, and a hypothetical model is proposed.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 133 - 8
Primary structures of two subunits of NADH: ubiquinone reductase from Neurospora crassa concerned with NADH-oxidation . Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus; Preis D et al.; The primary structures of the nuclear-encoded 51 kDa and 78 kDa subunits of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria were determined by sequencing cDNA and the N-terminus of the mature proteins . Both subunits are related to the soluble NAD-reducing hydrogenase of the bacterium Alcaligenes eutrophus . Sequence comparison between these subunits, the corresponding subunits of the bovine complex I and the bacterial NAD-reducing hydrogenase further confirms the binding sites of NAD(H), FMN and three iron-sulfur clusters.

Appl Microbiol Biotechnol, 1991 Aug, 35(5), 662 - 8
Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp . A 7-2 in soil; Balfanz J et al.; Alcaligenes sp . A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4-chlorophenol in sandy soil . Good adsorption rates on granular clay were achieved using cell suspensions with high titres and media at pH 8.0 . The influence of various parameters such as aeration rate, pH, temperature, concentration of 4-chlorophenol and size of inoculum on the degradation rate were investigated . During fed-batch fermentations under optimal culture conditions, concentrations of 4-chlorophenol up to 160 mg.l-1 could be degraded . Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria . Continuous fermentation was performed with varying 4-chlorophenol concentrations in the feed and different input levels . The maximum degradation rate was 1.64 g.l-1.day-1.

J Bacteriol, 1991 Jul, 173(13), 4056 - 71
Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism; Priefert H et al.; Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus . Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein . The molecular weight of FMP subunits was 39,000 . The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A . eutrophus . With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C . Frund, H . Priefert, A . Steinbuchel, and H . G . Schlegel, J . Bacteriol . 171:6539-6548, 1989) . The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified . Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC) . The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence . The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli . The occurrence of a new enzyme type for the degradation of acetoin is discussed.

Biokhimiia, 1991 Jul, 56(7), 1288 - 95
{Intracellular L-aspartate-beta-decarboxylase of Pseudomonas sp . and Alcaligenes sp . and its immobilization}; Abelian BA et al.; Intracellular L-aspartate-beta-decarboxylase of Pseudomonas sp . and Alcaligenes sp . was isolated, purified and characterized . The cells were destroyed by ultrasonic treatment; the enzymes were precipitated by ammonium sulfate fractionation, dialyzed and lyophylized using Biogel P-150 . After gel electrophoresis homogeneous enzyme preparations were obtained . The activity of L-aspartate-beta-decarboxylase is rather high--up to 92.1 U/min/mg of protein and is maximal at pH 5.5 and at temperatures of 45-55 degrees C . The Km and Vmax values for the Pseudomonas sp . enzyme are 0.1 M and 0.33 mM/min, respectively: those for the Alcaligenes sp . enzyme are 0.15 M and 1.0 mM/min, respectively . The results of amino acid analysis suggest that the enzymes slightly differ from one another with regard to aspartic and glutamic acid, alanine, valine and isoleucine content . Immobilization of the enzymes on various carriers was performed.

Antonie Van Leeuwenhoek, 1991 Jul, 60(1), 61 - 6
Production of poly-D(-)-3-hydroxybutyrate and poly-D(-)-3-hydroxyvalerate by strains of Alcaligenes latus; Chen GQ et al.; Alcaligenes latus strains can accumulate poly-D(-)-3-hydroxybutyrate (PHB) up to about 85% of cell dry weight . The abilities to store poly-D(-)-3-hydroxyvalerate (PHV) of three strains of A . latus were investigated . With Na-propionate as PHV precursor, strain A . latusDSM 1122 had better PHV accumulation ability than strains A . latus DSM 1123 and 1124 . Strain A . latus DSM 1123 could store PHV when Na-valerate but not Na-propionate served as the PHV precursor . PHB and PHV accumulation by A . latus DSM 1124 rapidly increased when propionic acid and acetic acid were together added to the fermentor . This increase was not obtained in the culture shaker flask and fermentor growing the same strain when Na-propionate alone served as a PHV precursor.

FEBS Lett, 1991 May 20, 283(1), 109 - 12
Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus; Wolfram L et al.; The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1 . A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for beta-galactosidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A . eutrophus gene hoxN . An E . coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction . A broad-host-range derivative of pCH307 was shown to complement an A . eutrophus HoxN- mutant.

Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4225 - 9
cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase; Patel SD et al.; A lambda gt10 bovine brain and a lambda gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratory-chain NADH dehydrogenase . Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit . The size and amino acid composition of this protein agree with those determined for the purified 51-kDa subunit . Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur cluster . The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor . A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the alpha subunit of the soluble NAD(+)-reducing hydrogenase from Alcaligenes eutrophus . In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.

J Mol Evol, 1991 May, 32(5), 379 - 95
Sequence analysis and phylogenetic reconstruction of the genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the chlorophyll b-containing prokaryote Prochlorothrix hollandica; Morden CW et al.; Prochlorophytes similar to Prochloron sp . and Prochlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are prokaryotic organisms that possess chlorophyll b (chl b) . We have sequenced the Prochlorothrix genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase(rubisco), rbcL and rbcS, for comparison with those of other taxa to assess the phylogenetic relationship of this species . Length differences in the large subunit polypeptide among all sequences compared occur primarily at the amino terminus, where numerous short gaps are present, and at the carboxy terminus, where sequences of Alcaligenes eutrophus and non-chlorophyll b algae are several amino acids longer . Some domains in the small subunit polypeptide are conserved among all sequences analyzed, yet in other domains the sequences of different phylogenetic groups exhibit specific structural characteristics . Phylogenetic analyses of rbcL and rbcS using Wagner parsimony analysis of deduced amino acid sequences indicate that Prochlorothrix is more closely related to cyanobacteria than to the green plastid lineage . The molecular phylogenies suggest that plastids originated by at least three separate primary endosymbiotic events, i.e., once each leading to green algae and land plants, to red algae, and to Cyanophora paradoxa . The Prochlorothrix rubisco genes show a strong GC bias, with 68% of the third codon positions being G or C . Factors that may affect the GC content of different genomes are discussed.

Antonie Van Leeuwenhoek, 1991 May, 59(4), 263 - 8
Pyrimidine base and ribonucleoside utilization by the Pseudomonas alcaligenes group; West TP; Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of the Pseudomonas alcaligenes group . As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth of Pseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supported Pseudomonas alcaligens ATCC 14909 growth . Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by either P . pseudoalcaligenes or P . alcaligenes as sole carbon sources . The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts of P . pseudoalcaligenes and P . alcaligenes . In P . pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.

J Bacteriol, 1991 Apr, 173(8), 2425 - 34
Sequence analysis of the Pseudomonas sp . strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates; van der Meer JR et al.; Pseudomonas sp . strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes . The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined . The sequence contained five large open reading frames, which were all colinear . The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities . The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity . The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa) . The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134 . The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three . Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place . TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol . ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.

Int J Biol Macromol, 1991 Apr, 13(2), 83 - 8
Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp . NCIMB 40126; Haywood GW et al.; A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources . One of these, Rhodococcus sp . NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source . Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates . With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced . N.m.r . analysis confirmed the composition of these polyesters . The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.

Mol Microbiol, 1991 Mar, 5(3), 535 - 42
Physiology and molecular genetics of poly(beta-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus; Steinbuchel A et al.; The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned . Molecular studies revealed that these genes are organized in a single operon . The A . eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA . The biochemical and physiological capabilities of A . eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.

J Bacteriol, 1991 Mar, 173(6), 1845 - 54
Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus; Eberz G et al.; Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE . Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa . A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene . Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli . HoxA- mutants of A . eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase . In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes . Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level . The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion . Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

Z Naturforsch {C}, 1991 Mar-Apr, 46(3-4), 210 - 6
Marine biosurfactants, III . Toxicity testing with marine microorganisms and comparison with synthetic surfactants; Poremba K et al.; Eight synthetic and nine biogenetic surfactants were tested on their toxicity . Because of their possible application as oil dispersants against oil slicks on sea, the test organisms used were marine microorganisms (mixed and pure cultures of bacteria, microalgae, and protozoa) . Bacterial growth was hardly effected or stimulated, whilst that of algae and flagellates was reduced . All substances tested were biodegraded in sea water . The bioluminescence of Photobacter phosphoreum (Microtox test) was the most sensitive test system used . A ranking shows that most biogenetic surfactants were less toxic than synthetic surfactants . No toxicity could be detected with the glucose-lipid GL, produced by the marine bacterium Alcaligenes sp . MM1.

Z Naturforsch {C}, 1991 Mar-Apr, 46(3-4), 197 - 203
Marine biosurfactants, I . Screening for biosurfactants among crude oil degrading marine microorganisms from the North Sea; Schulz D et al.; Three bacterial strains of marine origin were isolated during a screening for biosurfactants among n-alkane degrading microorganisms . One strain-identified as Alcaligenes sp . MM1-produced a novel glucose lipid . In the case of Arthrobacter sp . EK 1 the well-known trehalose tetraester was found as major component . From another pure culture classified as Arthrobacter sp . SI 1, extracellular emulsifying agents with properties indicating high molecular weight substances were detected . Furthermore trehalose corynomycolates were found at up to 2 g/l . The isolated biosurfactants showed good interfacial and emulsifying properties.

Biochemistry, 1991 Feb 26, 30(8), 2166 - 75
Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase; Pilkington SJ et al.; Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme . The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins . The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters . Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction . The mature protein is 444 amino acids long . Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16 . This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN . The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal . The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN . Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3 . Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits . This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.

J Biol Chem, 1991 Feb 15, 266(5), 3222 - 7
Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus; Eitinger T et al.; High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN . hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1 . An insertional mutation at the hoxN locus led to an increased nickel requirement . In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium . Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000 . Only the larger polypeptide was essential for nickel transport . The hoxN locus was cloned on a 2.2-kilobase pair fragment . Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa . The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function . The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein . Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains . Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A . eutrophus.

J Biol Chem, 1991 Feb 5, 266(4), 2191 - 8
Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans . Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA; Huisman GW et al.; Pseudomonas oleovorans accumulates poly(3-hydroxyalkanoates) (PHAs) after growth on medium chain length hydrocarbons . Large amounts of this polyester are synthesized when cells are grown under nitrogen-limiting conditions . When nitrogen is resupplied in the medium, the accumulated PHA is degraded . In this paper, we describe mutants which are defective in the synthesis or in the degradation of PHA . These mutants were used to select DNA fragments which encode PHA polymerases and a PHA depolymerase . A 25-kilobase (kb) DNA fragment was isolated from P . oleovorans that complements a Pseudomonas putida mutant unable to accumulate PHA . Subcloning resulted in the assignment of a 6.4-kb EcoRI fragment as the pha locus, containing genetic information for PHA synthesis . Mutants in the PHA degradation pathway were also complemented by this fragment, indicating that genes encoding PHA biosynthetic and degradative enzymes are clustered . Analysis of the DNA sequence of the 6.4-kb fragment revealed the presence of two open reading frames encoding PHA polymerases based on homology to the poly(3-hydroxybutyrate) polymerase from Alcaligenes eutrophus . A third open reading frame complemented the PHA degradation mutation and is likely to encode a PHA depolymerase . The presence of two PHA polymerases is due to a 2098-base pair DNA duplication . The PHA polymerases are 53% identical and show 35-40% identity to the poly(3-hydroxybutyrate) polymerase . No clear difference in specificity was found for the PHA polymerases . However, with the pha locus cloned on a multicopy vector, a polymer was accumulated that contains a significantly higher amount of substrate-derived monomers . An increase in the rate of polyester synthesis versus oxidation of the monomers in the beta-oxidation explains these findings.

Gene, 1991 Feb 1, 98(1), 15 - 20
Sequence of the plasmid-encoded catechol 1,2-dioxygenase-expressing gene, pheB, of phenol-degrading Pseudomonas sp . strain EST1001; Kivisaar M et al.; Phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12O), the two first enzymes of the phenol-degradation pathways, are encoded by a 3.4-kb DNA fragment cloned from Pseudomonas sp . EST1001 plasmid DNA . We have previously shown that activation of the cloned genes in Pseudomonas putida PaW85 is controlled by insertion of the 17-kb transposon, Tn4652, from the host chromosome into the plasmid carrying these genes {Kivisaar et al . Plasmid 24 (1990) 25-36} . Transcription of the DNA encoding PMO (pheA) and C12O (pheB) is activated by a promoter located on a 0.2-kb SacI-ClaI fragment from Tn4652 . We have determined the nucleotide sequence of pheB . The 906-bp gene encodes a protein product with a deduced Mr of 33,362 . The relationship between the pheB gene and other C12O-encoding genes has been shown: comparison of the pheB sequence with sequences of catA of Alcaligenes calcoaceticus, tfdC of A . eutrophus and clcA of P . putida demonstrated that there are conserved residues in all the four protein products of these genes.

Gene, 1991 Jan 2, 97(1), 55 - 62
Sequence and expression of genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chromatium vinosum; Kobayashi H et al.; A DNA fragment bearing genes for the large (rbcL) and small (rbcS) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was cloned from the photosynthetic purple sulfur bacterium Chromatium vinosum . Enzymatically fully active RuBisCO was synthesized in Escherichia coli cells when the cloned DNA was placed downstream of tac promoter . Nucleotide (nt) sequences of rbcL-rbcS were more homologous to cyanobacterial counterparts than to those from Alcaligenes eutrophus or higher plants . However, the amino acid (aa) sequence in a domain responsible for CO2 activation in the C . vinosum rbcL product resembled the corresponding aa sequence in higher plant RuBisCos, but not in the cyanobacterial enzymes . Chemically determined aa sequences at the N terminals of both subunits of RuBisCO purified from C . vinosum were not identical to those deduced from the nt sequences, although they were completely the same as aa sequences deduced from rbcA-rbcB, another locus encoding RuBisCO in C . vinosum . Therefore, the rbcL-rbcS locus seems to be barely expressed under a standard condition for photoautotrophic growth . The homology of the nt sequences between rbcL and rbcA was 82%, and that between rbcS and rbcB was 63%, whereas the codon usages of these genes were basically identical . The rbcL-rbcS and rbcA-rbcB loci therefore must have evolved from a common ancestral set of genes after duplication, instead of lateral gene transfer.

Ann Rech Vet, 1991, 22(1), 11 - 23
{Resistance of 700 gram-negative bacterial strains to antiseptics and antibiotics}; Maris P; The sensitivity of 701 Gram negative strains, representing 16 species or bacterial genera, towards 4 antiseptics (cetrimide, chlorhexidine, hexachlorophene, mercuric chloride) and 6 antibiotics (ampicillin, streptomycin, erythromycin, chloramphenicol, kanamycin, tetracycline) was analysed . For 3 antiseptics (cetrimide, chlorhexidine, mercuric chloride) minimal inhibitory concentration distribution showed strains to be heterogeneous, particularly in the group Pseudomonas . Most strains were resistant to antibiotics . The statistical analysis of correlation showed positive resistance links between antiseptics (cetrimide, chlorhexidine, hexachlorophene) or between these antiseptics and antibiotics for S marcescens and Alcaligenes . Many strains proved resistant, at the same time, to mercuric chloride and to several antibiotics . These associations of resistance suggest either a common mechanism of action or a crossed resistance.

J Bacteriol, 1991 Jan, 173(1), 6 - 15
Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp . strain P51; van der Meer JR et al.; Pseudomonas sp . strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources . Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization . They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222 . Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase . The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively . The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid . The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.

J Bacteriol, 1991 Jan, 173(1), 168 - 75
Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter; Schubert P et al.; Molecular methods have been applied to analyze the expression of the Alcaligenes eutrophus poly(3-hydroxybutyrate) (PHB) synthase gene (phbC) . The translational initiation codon was identified by analysis of the amino acid sequence of a PHB synthase-beta-galactosidase fusion protein . This protein was purified to almost gel electrophoretic homogeneity by chromatography on DEAE-Sephacel and on aminophenyl-beta-D-thiogalactopyranoside-Sepharose from cells of A . eutrophus which harbored a phbC'-'lacZ fusion gene . A sequence (TTGACA-18N-AACAAT), exhibiting striking homology to the Escherichia coli sigma 70 promoter consensus sequence, was identified approximately 310 bp 5' upstream from the translation initiation codon . An S1 nuclease protection assay mapped the transcription start point of phbC 6 bp downstream from this promoter . The location of the promoter was confirmed by analyzing the expression of active PHB synthase in clones of E . coli harboring 5' upstream deletions of phbC ligated to the promoter of the lacZ gene (lacZp) in a Bluescript vector . Plasmids do181 and do218, which were deleted for the first 108 or 300 bp of the phbC structural gene, respectively, conferred the ability to synthesize large amounts of different truncated PHB synthase proteins to the cells . These proteins contributed to approximately 10% of the total cellular protein as estimated from sodium dodecyl sulfate-polyacrylamide gels . The modified PHB synthase encoded by plasmid do181 was still active . Clones in which the lacZp-'phbC fusion harbored the complete phbC structural gene plus the phbC ribosome binding site did not overexpress PHB synthase.

Bioseparation, 1991, 2(3), 155 - 66
The disruption of Alcaligenes eutrophus by high pressure homogenisation: key factors involved in the process; Harrison ST et al.; The disruption of the Gram-negative bacterium Alcaligenes eutrophus by high pressure homogenisation, using the APV Gaulin 15M 8BA and 30CD homogenisers is reported . The operating parameters such as operating pressure, number of passes, temperature and biomass concentration, mimicked trends previously reported for yeasts . Extension of the study to consider the effect of cell characteristics, including the growth rate, size and shape, illustrated the dominant effect of the growth phase . The improved disruption of bacterial cultures in the logarithmic phase with respect to stationary phase cultures was confirmed by an increased dependence of actively growing cultures on the operating pressure . An increase in size in excess of 30% on the accumulation of the storage product, PHB in the stationary phase caused little change in the ease of disruption . The use of transmission electron microscopy to directly monitor the disruption on multiple passes shed light on the two-stage nature of this disruption process.

Bioseparation, 1991, 2(2), 95 - 105
Combined chemical and mechanical processes for the disruption of bacteria; Harrison ST et al.; Mechanical cell disruption by high pressure homogenisation or high speed bead mills is currently the general method of choice for the large scale disruption of micro-organisms; however, deleterious effects include the high energy requirement, the need for efficient cooling to prevent the excessive heating of the product and the micronisation of cell debris . Certain chemical treatments for microbial cell disruption alter the permeability of bacteria and yeasts, allowing partial release of soluble products . Such treatments are insufficient for the recovery of granular intracellular products . As cell wall strength has been cited as a major factor influencing the requirements for efficient mechanical disruption, the use of chemical pretreatment to decrease cell wall strength prior to mechanical breakage by homogenisation has been considered . The following treatments were shown to increase the sensitivity of the Gram-negative bacterium, Alcaligenes eutrophus, to disruption: alkaline pH shock, the addition of an anionic detergent, increase of the monovalent cation concentration, the addition of EDTA and enzymic lysis by lysozyme . These pretreatments allow equivalent disruption to be achieved at lower operating pressures or fewer passes through the homogeniser . Alkaline pH pretreatment at pH 10.5 allowed a 37.5% increase in soluble protein release on subsequent homogenisation . An increase of some 30% in soluble protein release was found following prior addition of 0.137 M monovalent cations (Na+ or K+) at 60 degrees C . Treatment with an anionic detergent showed a considerable decrease in the number of passes required through the homogeniser . Maximum cell rupture can thus be accomplished at reduced energy inputs.

Bioseparation, 1991, 2(2), 67 - 80
Liquid fluidized bed adsorption of protein in the presence of cells; Draeger NM et al.; The adsorption, in a liquid fluidized bed, of Bovine Serum Albumin (BSA), onto an ion-exchange absorbent, Q-Sepharose Fast Flow, in the presence of Alcaligenes eutrophus cells, has been studied . The expansion of the fluidized bed is greater in the presence than in the absence of cells and obeys the laws of Richardson and Zaki . The effect of cell concentration on the equilibrium adsorption characteristics of the adsorbent has been assessed . The rate of adsorption of BSA onto the adsorbent has been studied in a batch stirred tank, and a fluidized bed system both in the presence and absence of cells . Comparisons have been made with the adsorption of human immunoglobulin G (human IgG), onto an affinity adsorbent, Protein A Sepharose CL-4B . The data from the fluidized bed breakthrough tests have been used to assess the validity of a theoretical model adapted from one that predicts the performance of the adsorption phase in the absence of cells in fixed bed systems . Tests have been done on the washing phase in the fluidized bed adsorption system to establish the most efficient method of washing cells and unadsorbed protein out of the bed.

Biodegradation, 1991-92, 2(4), 253 - 63
Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration; Jacobsen CS et al.; Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (= pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil . Mineralization was measured as 14C-CO2 evolved during degradation of uniformly-ring-labelled 14C-2,4-D . When the strains were inoculated to a level of approximately 10(8) CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h . Mineralization of 0.05 ppm and 10 ppm 2,4-D by the two A . eutrophus strains was identical and rapid whereas mineralization by P . cepacia DBO1(pRO101) occurred more slowly . In contrast, mineralization of 500 ppm 2,4-D by the two A . eutrophus strains was very slow whereas mineralization by P . cepacia DBO1 was more rapid . Comparison of 2,4-D mineralization at different levels of inoculation with P . cepacia DBO1(pRO101) (6 x 10(4), 6 x 10(6) and 1 x 10(8) CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level . The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models . The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil . In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.

Microbiol Rev, 1990 Dec, 54(4), 450 - 72
Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates; Anderson AJ et al.; Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence . They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell . The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago . Copolymers of C4 (3-hydroxybutyrate {3HB}) and C5 (3-hydroxyvalerate {3HV}) monomer units have modified physical properties; e.g., the plastic is less brittle than PHB, whereas PHAs containing C8 to C12 monomers behave as elastomers . This family of materials is the centre of considerable commercial interest, and 3HB-co-3HV copolymers have been marketed by ICI plc as Biopol . The known polymers exist as 2(1) helices with the fiber repeat decreasing from 0.596 nm for PHB to about 0.45 nm for C8 to C10 polymers . Novel copolymers with a backbone of 3HB and 4HB have been obtained . The native granules contain noncrystalline polymer, and water may possibly act as a plasticizer . Although the biosynthesis and regulation of PHB are generally well understood, the corresponding information for the synthesis of long-side-chain PHAs from alkanes, alcohols, and organic acids is still incomplete . The precise mechanisms of action of the polymerizing and depolymerizing enzymes also remain to be established . The structural genes for the three key enzymes of PHB synthesis from acetyl coenzyme A in Alcaligenes eutrophus have been cloned, sequenced, and expressed in Escherichia coli . Polymer molecular weights appear to be species specific . The factors influencing the commercial choice of organism, substrate, and isolation process are discussed . The physiological functions of PHB as a reserve material and in symbiotic nitrogen fixation and its presence in bacterial plasma membranes and putative role in transformability and calcium signaling are also considered.

J Bacteriol, 1990 Dec, 172(12), 7057 - 64
Differential stability of mRNA species of Alcaligenes eutrophus soluble and particulate hydrogenases; Oelmuller U et al.; The functional half-lives of Alcaligenes eutrophus hydrogenase mRNAs were determined by physiological studies . Evidence was obtained for a functional half-life of about 1 h for the soluble NAD-linked hydrogenase (HoxS) mRNA and 14 min for the particulate hydrogenase (HoxP) mRNA . The synthesis of active HoxS continued for about 4 h, albeit at a decreasing rate after inhibition of transcription, e.g., by rifampin . In this strain, the mRNA of HoxS appeared to be stable, while the mRNA of HoxP did not . Different species of hoxS mRNA were detected by the Northern (RNA) hybridization technique using as a probe plasmid pCH139 carrying hoxS structural genes . The sizes of the major hoxS mRNA species were 7.6, 6.2, 5.0, and 0.9 kb . The chemical half-lives of these species ranged from 1 h (5.0-kb mRNA) to 7 h (0.9-kb mRNA) . Evidence for a specific cleavage of the 6.2-kb transcript yielding the 0.9-kb species was obtained from RNA-DNA hybridizations with subcloned hoxS DNA . The chemical half-life of total hoxP mRNA was 8 min.

Appl Environ Microbiol, 1990 Nov, 56(11), 3565 - 75
Identification and catabolic activity of well-derived gasoline-degrading bacteria from a contaminated aquifer; Ridgway HF et al.; Approximately 300 gasoline-degrading bacteria were isolated from well water and core material from a shallow coastal aquifer contaminated with unleaded gasoline . Identification of 244 isolates revealed four genera: Pseudomonas, Alcaligenes, Nocardia, and Micrococcus, with pseudomonads making up 86.9% of bacteria identified . A total of 297 isolates was sorted into 111 catabolic groups on the basis of aerobic growth responses on 15 gasoline hydrocarbons . Each test hydrocarbon was degraded by at least one isolate . Toluene, p-xylene, ethylbenzene, and 1,2,4-trimethylbenzene were most frequently utilized as growth substrates, whereas cyclic and branched alkanes were least utilized . Most isolates were able to grow on 2 or 3 different hydrocarbons, and nearly 75% utilized toluene as a sole source of carbon and energy . Isolates were remarkably specific for hydrocarbon usage, often catabolizing only one of several closely related compounds . A subset of 220 isolates was sorted into 51 groups by polyacrylamide gel electrophoresis . Pseudomonas aeruginosa was partitioned into 16 protein-banding groups (i.e., subspecies) whose catabolic activities were largely restricted to substituted aromatics . Different members of subspecies groups defined by protein-banding pattern analysis often exhibited different growth responses on the same hydrocarbon, implying marked strain diversity . The catabolic activities of well-derived, gasoline-degrading bacteria associated with this contaminated aquifer are consonant with in situ adaptation at the site.

Appl Environ Microbiol, 1990 Nov, 56(11), 3360 - 7
Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads; Timm A et al.; Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer . The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains . Among 55 different strains of 41 Pseudomonas species tested, P . aureofaciens (21.6% of cellular dry matter), P . citronellolis (78.0%), P . chlororaphis (8.5%), P . marginalis (11.4%), P . mendocina (50.7%), P . putida (33.5%), and Pseudomonas sp . strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate . In two strains of P . facilis and P . fluorescens, as well as in one strain of P . syringae, this polymer was detected as a minor constituent (much less than 5%) . All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source . Only a few species (e.g., P . stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all . These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways . The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P . aeruginosa and P . putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.

Biochem J, 1990 Oct 15, 271(2), 529 - 34
Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134; Pieper DH et al.; 4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp . B13, named B13 FR1, carrying the plasmid pFRC2OP . This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source . The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE . In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo{3.3.0}octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate . Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors . Possible reaction mechanisms are discussed.

Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 341 - 7
Release of T-antigen, a carcinoma marker from native human cells, by endo-alpha-N-acetylgalactosaminidase of Alcaligenes sp; Fan JQ et al.; The endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp . could release T-antigen (Ga1 beta 1----3GalNAc), a carcinoma-associated marker from asialo glycophorin and human cells . The released T-antigen from human asialo erythrocytes was determined by thin layer chromatography and gas liquid chromatography . The released T-antigen from human gastric carcinoma cell Kato III was identified by high performance liquid chromatographies with a reverse-phase column and a size fractionation column . Released T-antigen could be analyzed quantitatively by a sensitive method including pyridylamino derivatization and following high performance liquid chromatography . This suggests that the enzyme is useful for detection and determination of T-antigen from cells.

Carbohydr Res, 1990 Oct 10, 206(2), 289 - 96
Structural analysis of an acidic polysaccharide secreted by Xanthobacter sp . (ATCC 53272); O'Neill MA et al.; The structure of an acidic polysaccharide secreted by a Xanthobacter sp . has been investigated by glycosyl-residue and glycosyl-linkage composition analyses, and the characterization of oligoglycosyl fragments of the polysaccharide has been carried out by chemical analyses, 1H-n.m.r . spectroscopy, fast-atom bombardment mass spectrometry, and electron-impact mass spectrometry . The polysaccharide, which contains O-acetyl groups (approximately 5%) that have not been located, has the tetraglycosyl repeating unit 1 and belongs to a group of structurally related polysaccharides synthesized by both Alcaligenes and Pseudomonas species.

Int J Syst Bacteriol, 1990 Oct, 40(4), 384 - 98
Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E . Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb . nov., Acidovorax delafieldii comb . nov., and Acidovorax temperans sp . nov; Willems A et al.; Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas . They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria) . The taxonomic relationships of both of these species, two groups of clinical isolates (E . Falsen {EF} group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization . These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax . We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).

Can J Microbiol, 1990 Oct, 36(10), 718 - 24
Expression of dibenzothiophene-degradative genes in two Pseudomonas species; Foght JM et al.; The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers . This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp . HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype) . Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b . Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b . Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains . DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes . Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.

Appl Microbiol Biotechnol, 1990 Oct, 34(1), 108 - 15
Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system; Beunink J et al.; The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP) . The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation . CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp . TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer . Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate . The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system.

J Bacteriol, 1990 Sep, 172(9), 5119 - 29
Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate; Schlomann M et al.; Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone {(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide} . This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A . eutrophus 335, A . eutrophus JMP134, and P . cepacia as well as by the action of dichloromuconate cycloisomerase from A . eutrophus JMP134 . This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation . 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate . Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible . However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed . Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate . However, experiments with cell extracts of P . putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion . In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.

J Bacteriol, 1990 Sep, 172(9), 5112 - 8
Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria; Schlomann M et al.; Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A . eutrophus H16, A . eutrophus JMP222, A . eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy . P . cepacia also utilizes 3-fluorobenzoate . Except for A . eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R . H . Don and J . M . Pemberton, J . Bacteriol . 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate . Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate . Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols . Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity . (i) Extracts of A . eutrophus 335, A . eutrophus H16, A . eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I) . (ii) The enzyme present in P . cepacia shows the opposite preference for the isomeric substrates (type II) . (iii) Cell extracts of A . eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E . Schmidt and H.-J . Knackmuss, Biochem . J . 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III) . This classification implies that A . eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222.

J Clin Microbiol, 1990 Sep, 28(9), 1982 - 7
Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction; Glare EM et al.; A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19 . This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B . pertussis tested . It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus . The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR) . This procedure was then applied to the rapid (5-h) detection of B . pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis . The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF) . All of the IF-positive samples were PCR positive, as were 63 of the samples from which B . pertussis was eventually cultured . Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay . However, 33 culture- and IF-negative specimens were positive by PCR assay . Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives.

J Bacteriol, 1990 Sep, 172(9), 4844 - 51
Characterization of alcohol dehydrogenase genes of derepressible wild-type Alcaligenes eutrophus H16 and constitutive mutants; Jendrossek D et al.; The nucleotide sequence of the gene that encodes the fermentative, derepressible alcohol dehydrogenase (ADH) in Alcaligenes eutrophus H16 and of adjacent regions was recently determined . Two potential -10 regions resembling the Escherichia coli sigma 70 consensus sequence were identified 77 and 93 nucleotides upstream of the structural gene . By determination of the 5' mRNA terminus of the wild-type adh gene, the proximal -10 region was identified as responsible for adh expression under derepressive conditions . Transcription started seven nucleotides downstream of this region, at position 388 . Sequence analysis of seven mutants expressing the adh gene under aerobic conditions revealed mutations in one or the other potential -10 region . In all seven strains, the mutations restored the invariant T of the E . coli promoter consensus sequence . Mutants altered in the proximal -10 region transcribed the adh gene under aerobic conditions with the same 5' mRNA terminus as in the wild type; gene expression was impaired very little under aerobic conditions . Mutants altered in the distal -10 region also transcribed the adh gene aerobically but were still partially derepressible . The 5' mRNA terminus was seven nucleotides downstream of the distal -10 region, at position 372 . When these mutants were cultivated under conditions of restricted oxygen supply, the adh gene was transcribed from both -10 regions, resulting in the synthesis of two mRNA species with different 5' termini.

Appl Environ Microbiol, 1990 Aug, 56(8), 2471 - 9
Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples; Top E et al.; Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A . eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples . This czc fragment is not expressed in the donor strain, E . coli, but it is expressed in the recipient strain, A . eutrophus . Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes . The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149 . In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell {triparental cross} or in the recipient strain itself {retromobilization}) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively . The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113 . The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils . In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1990 Jul, 28(7), 1628 - 34
Evaluation of autoSCAN-W/A automated microbiology system for the identification of non-glucose-fermenting gram-negative bacilli; Tenover FC et al.; We evaluated the ability of the autoSCAN-W/A (MicroScan Division, Baxter Healthcare Corporation, West Sacramento, Calif.), in conjunction with the dried colorimetric Neg ID type 2 panel (DCP) and new rapid fluorometric Neg ID panel (RFP), to identify non-glucose-fermenting gram-negative bacilli by challenging the system with 310 previously identified reference strains . Of these 310 isolates, 286 organisms were in the DCP data base and 269 were in the RFP data base . Use of the DCP panels resulted in 118 (41.3%) correct and 64 (22.4%) incorrect first choice identifications at greater than or equal to 85% probability, 61 (21.3%) low-probability identifications, and 43 (15.0%) reports of unidentified organisms . The RFP system reported 135 (50.1%) correct and 25 (9.3%) incorrect identifications at greater than or equal to 85% probability and 109 (40.5%) low-probability identifications . Unidentified isolates (DCP system only) and isolates producing low-probability first choice identifications (both systems) required supplementary biochemical testing . Over half (37 of 64 {57.8%}) of the DCP misidentifications were due to four commonly isolated, saccharolytic organisms (Alcaligenes xylosoxidans subsp . xylosoxidans, Pseudomonas putida, Pseudomonas fluorescens, and Xanthomonas maltophilia), while 7 of 25 (28%) of misidentifications in the RFP system were due to P . fluorescens . Of note, the RFP system identified non-glucose-fermenting gram-negative bacilli within 2 h of panel inoculation, allowing additional conventional biochemical tests to be set up the same day on low-probability isolates, whereas only 13.5% of the DCPs could be read at 18 h, with the remainder requiring 42 h of incubation before reading . When organism identifications were recalculated with the updated RFP data base and revised software, only 8.1% of all 310 isolates were misidentified at greater than or equal to 85% probability while 77.1% of the isolates were now correctly reported at this same high probability.

Appl Environ Microbiol, 1990 Jul, 56(7), 2093 - 8
Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids; Ramsay BA et al.; Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid {P(HB-co-HV)} copolymer when supplied with glucose (or sucrose in the case of A . latus) and propionic acid under nitrogen-limited conditions . A fed-batch culture of A . eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions . When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced . The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV) . Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A . eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass . In comparison, A . latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate . When propionic acid was added to the first stage of a two-stage chemostat, A . latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV . In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate . The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed . Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.

Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 751 - 7
Action of endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp . on amino acid-O-glycans: comparison with the enzyme from Diplococcus pneumoniae; Fan JQ et al.; Endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp . released the disaccharide, Gal beta 1----3GalNAc, from both dansylated serine-GalNAc-Gal and threonine-GalNAc-Gal, and showed higher activity on the former than the latter . The Km values were 0.17 mM and 1.43 mM with DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively . The optimum pHs were found to be 4.5-7.5 and 4.5-6.0 on DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively . On the contrary, the enzyme from Diplococcus pneumoniae had low activity to release the disaccharide from the amino acid-O-glycans . The possibility that the same O-glycoside but linked to different aglycon amino acids may play a different biological role in glycoproteins is discussed.

J Appl Bacteriol, 1990 Jun, 68(6), 601 - 18
Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens; Stenstrom IM et al.; One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters . Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages . Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86% . SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps . lundensis (cluster 2; 2% of the field strains), Ps . fluorescens biovar III (cluster 4; 4% of the field strains), Ps . putida biovar A (cluster 5; 3% of the field strains), Ps . fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer . sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish) . Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g . Phenotypic characteristics of major clusters are given . The four S . putrefaciens clusters may be separated by key characteristics . Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters . The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.

J Bacteriol, 1990 Jun, 172(6), 2920 - 9
Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16; Tran-Betcke A et al.; The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced . On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS . Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes . Primer extension experiments indicate that the second promoter is responsible for hoxS transcription . hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity . A putative flavin-binding region is discussed . The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two {4F3-4S}centers . The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS . The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases . The roles of these regions in coordinating iron and nickel are discussed . Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other {NiFe} hydrogenases, the overall amino acid homology is marginal . Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found . Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.

Eur J Biochem, 1990 May 20, 189(3), 529 - 37
Identification of distinct NAD-linked hydrogenase protein species in mutants and nickel-deficient wild-type cells of Alcaligenes eutrophus H16; Hornhardt S et al.; By crossed immunoelectrophoresis with antibodies against the NAD-linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16 . Protein 1 (antigen 1) exhibited NAD-reducing activity and was shown to be identical with the native heterotetrameric enzyme . Protein 2 (antigen 2) was catalytically inactive in the antibody-precipitated form and corresponded to the beta subunit (56 kDa) of the holoenzyme . Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH-oxidizing (diaphorase) activity, suggesting that it either consists of the alpha peptide or of the alpha and gamma subunits of the diaphorase dimer . Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency . Low concentrations of the diaphorase-active antigen 3 were also detected under these conditions . Extracts from mutants defective in the catalytic activity of NAD-reducing hydrogenase still contained the four polypeptides . This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits . However, as observed with nickel-deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70-80%) was the beta peptide.

Appl Environ Microbiol, 1990 May, 56(5), 1357 - 62
Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures; Haugland RA et al.; Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates . These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations . Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134 . High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol . JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism . A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134 . This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.

J Bacteriol, 1990 May, 172(5), 2351 - 9
Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4; Perkins EJ et al.; Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB . Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively . Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R . H . Don, A . J . Wieghtman, H.-J . Knackmuss, and K . N . Timmis, J . Bacteriol . 161:85-90, 1985) . We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B . Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons . The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida . P . putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols . A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols . The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P . putida(pAC27) from utilizing 3,5-dichlorocatechol.

J Biol Chem, 1990 Apr 5, 265(10), 5648 - 53
Nucleotide sequence and expression of a plasmid-encoded chromate resistance determinant from Alcaligenes eutrophus; Nies A et al.; The nucleotide sequence of the 2.6-kilobase pair (kb) EcoRI fragment encoding chromate resistance (Chrr) on plasmid pMOL28 in Alcaligenes eutrophus was determined . Three open reading frames were assigned to three polypeptides which were expressed from this determinant in Escherichia coli under the control of a phage T7 transcription promoter . When the roles of the polypeptides and open reading frames were analyzed with deletion derivatives of the 2.6-kilobase fragment, the membrane-bound ChrA (401 amino acids) and ChrB (196 amino acids) polypeptides were essential for inducible chromate resistance and reduced accumulation of chromate, while the third open reading frame was not needed.

Appl Environ Microbiol, 1990 Apr, 56(4), 1179 - 81
Trichloroethylene degradation by two independent aromatic-degrading pathways in Alcaligenes eutrophus JMP134; Harker AR et al.; The bacterium Alcaligenes eutrophus JMP134(pJP4) degrades trichloroethylene (TCE) by a chromosomal phenol-dependent pathway and by the plasmid-encoded 2,4-dichlorophenoxyacetic acid pathway . The two pathways were independent and exhibited different rates of removal and capacities for quantity of TCE removed . The phenol-dependent pathway was more rapid (0.2 versus 0.06 nmol of TCE removed per min per mg of protein) and consumed all detectable TCE . The 2,4-dichlorophenoxyacetic acid-dependent pathway removed 40 to 60% of detectable TCE.

Int J Biol Macromol, 1990 Apr, 12(2), 102 - 5
Biosynthesis and composition of bacterial poly(hydroxyalkanoates); Anderson AJ et al.; It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism . The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA . Various other bacteria produce similar copolymers from propionic acid and/or valeric acid . A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols.

Biochem J, 1990 Mar 15, 266(3), 877 - 83
Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134; Kuhm AE et al.; Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity . The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE . Mn2+ ions as well as thiol groups are required for activity . A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound . Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate . The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.

Ecotoxicol Environ Saf, 1990 Feb, 19(1), 99 - 105
In vivo effect of the organophosphorus insecticide trichlorphon on immune response of carp (Cyprinus carpio) . II . Effect of high doses of trichlorphon on nonspecific immune response; Siwicki AK et al.; The effect of trichlorphon, one of the most widely used organophosphorus insecticides, on the nonspecific immune response in carp (Cyprinus carpio) was studied . The effect of 20,000 ppm trichlorphon on the immune response was followed for 3 and 56 days after intoxication . The effect of 10,000 ppm trichlorphon on the nonspecific immune response of carp experimentally infected by Pseudomonas alcaligenes and Aeromonas punctata was also examined . Leucocyte number, phagocytic ability of neutrophils, percentage NBT-positive PMN cells, phagocytic index, lysozyme level in serum, and ceruloplasmin activity in plasma were examined on Days 2, 4, 6, 8, 10, 14, 18, 22, and 26 after carp were exposed . After intoxication leukopenia was observed as decreases in phagocytic ability of neutrophils and in phagocytic index . Lysozyme level in serum was also decreased compared to that of control . The percentage of NBT-positive PMN cells decreased when the ceruloplasmin activity in plasma increased in intoxicated fish.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 165 - 9
Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus; Doi Y et al.; The cyclic process of biosynthesis and degradation of poly(3-hydroxyalkanoate) (PHA) was studied in Alcaligenes eutrophus under conditions of nitrogen-limitation of growth . A . eutrophus cells, which had accumulated poly(3-hydroxybutyrate) (PHB) of 55 wt% content within cells from butyric acid, were transferred into a nitrogen-free medium containing pentanoic acid as the sole carbon source and cultivated at 30 degrees C and pH 7.5 . The content of PHB in A . eutrophus cells decreased with time, whereas a copolyester of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) units, P(HB-co-HV), was accumulated in the presence of pentanoic acid . Conversely, when A . eutrophus cells with 50 wt% content of P(HB-co-56% HV) were incubated in a nitrogen-free medium containing butyric acid, the content of P(HB-co-56% HV) decreased with time, whereas PHB was accumulated . These results indicate the cyclic nature of PHA metabolism in A . eutrophus under these conditions.

Appl Biochem Biotechnol, 1990 Spring-Summer, 24-25, 425 - 30
Regeneration of NADH and ketone hydrogenation by hydrogen with the combination of hydrogenase and alcohol dehydrogenase . Scientific note; Okura I et al.; The regeneration of nicotinamide-adenine dinucleotide (reduced form, NADH) by the reaction of NAD with hydrogen gas was carried out in the presence of the hydrogenase from Alcaligenes eutrophus . And the formations of alcohol, CO2, and 6-phospho-gluconate were observed by a combination of the above system and corresponding dehydrogenases . NADH was regenerated by hydrogen gas with the hydrogenase and recycled in these reactions.

Arch Microbiol, 1990, 154(1), 85 - 91
On the operon structure of the cfx gene clusters in Alcaligenes eutrophus; Windhovel U et al.; Three transposon Tn5-induced mutants deficient in autotrophic CO2 fixation were isolated from a megaplasmid pHG1-cured strain of Alcaligenes eutrophus H16 . Their phenotypes were initially characterized by their ability to form both key enzymes of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) . Since the transposon insertions were at different sites within the chromosomal cluster of cfx genes encoding Calvin cycle enzymes, the individual mutants showed different inactivation patterns for Rubisco and PRK synthesis . These data together with already known sequence data and the arrangement of cfx genes suggested that the Rubisco, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase and PRK genes are constituents of the same operon . This was further confirmed by trans complementation analyses which indicated that the very similarly organized pHG1-encoded cfx genes additionally present in wild-type strain H16 are functional and also form a common operon . Each operon may also include a glyceraldehyde-3-phosphate dehydrogenase gene . Thus, the duplicated cfx operons of A . eutrophus H16 are large transcriptional units comprising at least about 8 kilobase pairs (kb) and possibly as much as 11 kb.

Arch Microbiol, 1990, 153(2), 134 - 8
DNA topoisomerase I from Alcaligenes eutrophus H16; Andera L et al.; Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated . Under native conditions the enzyme forms a monomer with a relative molar mass of 98,500 . A rod-like shape of the molecule was derived from the calculated frictional coefficient . The isoelectric point of the enzyme was determined to be in the range of 7.6-8.0 . The enzyme activity is strictly Mg2+ dependent with an optimum at 3 mM Mg2+ . The pH optimum ranges within 7.5-9.0 . A . eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.

J Bacteriol, 1990 Jan, 172(1), 287 - 91
Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505; Cervantes C et al.; The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104 . The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P . aeruginosa PAO1 . Resistance to chromate was not expressed in Escherichia coli . Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P . aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6 . Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced . Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance . This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E . coli under control of the T7 promoter . No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A . Nies, D . Nies, and S . Silver, submitted for publication).

Alcohol Alcohol, 1990, 25(6), 627 - 37
In vivo accelerated acetaldehyde metabolism using acetaldehyde dehydrogenase-loaded erythrocytes; Magnani M et al.; Human erythrocytes were loaded with homogeneous acetaldehyde dehydrogenase (AcDH) purified from Alcaligenes Eutrophus (an enzyme species with an apparent Km for acetaldehyde similar to the mitochondrial enzyme), using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing . The AcDH-overloaded erythrocytes contained 1.55 +/- 0.25 I.U . of AcDH activity per ml of packed erythrocytes, a value 12-15 times higher than that of corresponding unloaded or native red cells . The AcDH-loaded erythrocytes were found to metabolize 4 +/- 0.8 mumol of acetaldehyde/hr/ml of red blood cells, whereas the glycolytic activity was almost unmodified . Estimates of intracellular adenine nucleotides showed 50% ATP decay in the AcDH-loaded cells when incubated in the presence of acetaldehyde concentrations higher than 50 microM, whereas the {NAD+}/{NADH} ratio was strongly decreased but to the same extent as in control cells, suggesting that this was due to the acetaldehyde itself and not to the presence of encapsulated AcDH . Similar results were obtained using mouse erythrocytes . AcDH-overloaded mouse red blood cells from donor animals were also injected intraperitoneally into compatible recipients (Balb/C) and 80 to 85% of these were found to enter into circulation within 24 hr and to circulate with a half-life of 6-7.3 days (normal half-life 11 days) . Following an acute dose of ethanol (2g/kg intraperitoneally), blood levels of acetaldehyde were significantly lower in mice receiving the AcDH-loaded erythrocytes than in controls . Blood levels of ethanol were also lower in the treated mice compared to controls . These results show that AcDH-overloaded erythrocytes can perform in vitro and in vivo as bioreactors improving alcohol and acetaldehyde metabolism, and suggest that administration of these cells to alcoholic patients could be of value in restoring to normal, or improving, alcohol and acetaldehyde metabolism.

Biodegradation, 1990, 1(1), 55 - 64
Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp . strain O-1; Thurnheer T et al.; Alcaligenes sp . strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH4+ or NH4+ plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen . The intracellular desulfonative enzyme did not degrade 3- or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds . We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell . Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of O2 per mol . One mol of O2 was required for a catechol 2,3-dioxygenase . When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of O2 and of NAD(P)H per mol of BS were required for the reaction, and SO3(2-) and catechol were recovered in high yield . Catechol was shown to be formed by dioxygenation in an experiment involving 18O2 . 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO3(2-) and 4-methylcatechol, which was subject to meta cleavage . OS also required 2 mol of O2 per mol and NAD(P)H for degradation, and SO3(2-) and NH4+ were recovered quantitatively . Inhibition of ring cleavage with 3-chlorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO3(2-) (1 mol) and an unidentified organic intermediate, but no NH4+, were observed.

Gene, 1989 Dec 21, 85(1), 247 - 52
Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus; Kossmann J et al.; Two DNA fragments encoding the chromosomal and plasmid copies of the gene (cfxP) encoding phosphoribulokinase (PRK) from the chemoautotrophic bacterium Alcaligenes eutrophus, were sequenced and found to be highly homologous . The gene (cfxF) of another Calvin cycle enzyme, fructose-1,6-bisphosphatase (FBPase), was identified as terminating immediately upstream of cfxP, but was not completely contained on both fragments . A hypothetical, also incompletely contained, open reading frame starts closely downstream from cfxP . Genes cfxF, cfxP, and the third hypothetical gene seem to belong to the same operon . The cfxP genes encode highly homologous PRK isoenzyme subunits consisting of 292 aa residues with calculated Mrs of 33 319 (chromosomal PRKc) and 33 164 (plasmid-encoded PRKp) . There is little overall sequence similarity between the bacterial and plant (spinach) PRK, apart from some structural motifs.

J Bioenerg Biomembr, 1989 Dec, 21(6), 693 - 704
Respiratory Na+ pump and Na+-dependent energetics in Vibrio alginolyticus; Tokuda H; The marine bacterium Vibrio alginolyticus was found to possess the respiratory Na+ pump that generates an electrochemical potential of Na+, which plays a central role in bioenergetics of V . alginolyticus, as a direct result of respiration . Mutants defective in the Na+ pump revealed that one of the two kinds of NADH: quinone oxidoreductase requires Na+ for activity and functions as the Na+ pump . The Na+ pump composed of three subunits was purified and reconstituted into liposomes . Generation of membrane potential by the reconstituted proteoliposomes required Na+ . The respiratory Na+ pump coupled to the NADH: quinone oxidoreductase was found in wide varieties of Gram-negative marine bacteria belonging to the genera Alcaligenes, Alteromonas, and Vibrio, and showed a striking similarity in the mode of electron transfer and enzymic properties . Na+ extrusion seemed to be coupled to a dismutation reaction, which leads to the formation of quinol and quinone from semiquinone radical.

J Bioenerg Biomembr, 1989 Dec, 21(6), 635 - 47
The sodium cycle: a novel type of bacterial energetics; Skulachev VP; The progress of bioenergetic studies on the role of Na+ in bacteria is reviewed . Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na+ can, under certain conditions, substitute for H+ as the coupling ion . Various primary Na+ pumps (delta mu Na+ generators) are described, i.e., Na+ -motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase . The delta mu Na+ formed is shown to be consumed by Na+ driven ATP-synthase, Na+ flagellar motor, numerous Na+, solute symporters, and the methanogenesis-linked reverse electron transfer system . In Vibrio alginolyticus, it was found that delta mu Na+, generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na+, solute symports), and mechanical (rotation of the flagellum) . In Propionigenum modestum, circulation of Na+ proved to be the only mechanism of energy coupling . In other species studied, the Na+ cycle seems to coexist with the H+ cycle . For instance, in V . alginolyticus the initial and terminal steps of the respiratory chain are Na+ - and H+ -motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na+ as well as of H+ . In the alkalo- and halotolerant Bacillus FTU, there are H+ - and Na+ -motive terminal oxidases . Sometimes, the Na+ -translocating enzyme strongly differs from its H+ -translocating homolog . So, the Na+ -motive and H+ -motive NADH-quinone reductases are composed of different subunits and prosthetic groups . The H+ -motive and Na+ -motive terminal oxidases differ in that the former is of aa3-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide . At the same time, both Na+ and H+ can be translocated by one and the same P . modestum ATPase which is of the F0F1-type and sensitive to DCCD . The sodium cycle, i.e., a system composed of primary delta mu Na+ generator(s) and delta mu Na+ consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera: Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcina, etc . Thus, the "sodium world" seems to occupy a rather extensive area in the biosphere.

J Bacteriol, 1989 Dec, 171(12), 6539 - 48
Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus; Frund C et al.; In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth . The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A . eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks . The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants . The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank . Evidence is provided that fragments A (21 kilobase pairs {kb}) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb . Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown . In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob . No structural gene could be assigned to the D or E fragments . In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb) . This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein . The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.

Gene, 1989 Nov 30, 83(2), 225 - 32
Operon structure and nucleotide homology of the chlorocatechol oxidation genes of plasmids pJP4 and pAC27; Ghosal D et al.; Alcaligenes eutrophus harboring plasmid pJP4 (strain JMP134) is capable of growing on both 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba), while Pseudomonas putida carrying plasmid pAC27 (strain AC867) can utilize only 3-Cba as the sole carbon source . The tfdCDEF operon of the pJP4 plasmid and the clcABD operon of plasmid pAC27 each encode enzymes for the degradation of chlorocatechols (Clc), key intermediates in the catabolism of 2,4-D and 3-Cba . Similarities in the nucleotide (nt) sequences of genes tfdC and clcA, encoding pyrocatechases, were reported earlier {Ghosal and You, Mol . Gen . Genet . 211 (1988a) 113-120} . Genes tfdD and clcB, encoding Clc-specific cycloisomerases, have been completely sequenced . The tfdD gene (1107 bp) is slightly smaller than gene clcB (1113 bp) . Comparison of the two cycloisomerase-encoding genes reveals that the nt sequences are 63% homologous with 62% homology in the deduced amino acid (aa) sequences of the polypeptides they encode . Genes tfdD and tfdE are contiguous in the tfdCDEF operon, whereas the corresponding genes, clcB and clcD, of the clcABD operon, are known to be separated by a long open reading frame of unknown function . The predicted N-terminal aa sequences of the two hydrolase-encoding genes, tfdE and clcD, also show homology . The structural and nt homologies between the two Clc operons, tfdCDEF and clcABD, suggest their relatedness.

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7351 - 5
Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus; Nies DH et al.; Resistance to cobalt, zinc, and cadmium specified by the czc determinant on plasmid pMOL30 in Alcaligenes eutrophus results from a cation efflux system . Five membrane-bound polypeptides that were expressed in Escherichia coli from this determinant under the control of a phage T7 promoter were assigned to four open reading frames identified in the nucleotide sequence of the 6881-base-pair fragment containing the czc putative operon . The contributions of the polypeptides to the cation efflux system were analyzed with deletion derivatives of the 6.9-kilobase fragment, constructed, and expressed in E . coli under the control of the phage T7 promoter and in A . eutrophus under the control of the lac promoter.

Appl Environ Microbiol, 1989 Oct, 55(10), 2499 - 504
Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate; Oltmanns RH et al.; Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area . According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium . To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified . Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies . In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate . Alcaligenes sp . strains RHO21 and RHO22 used all three isomers of monofluorobenzoate . Alcaligenes sp . strain RHO22 also grew on 4-chlorobenzoate . Aureobacterium sp . strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released . In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected . All these enzymes were inducible by 4-fluorobenzoate . These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp . strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.

J Biol Chem, 1989 Sep 15, 264(26), 15298 - 303
Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16 . Identification and characterization of the PHB polymerase gene (phbC); Peoples OP et al.; The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A . eutrophus H16 . These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB . Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production . phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.

J Biol Chem, 1989 Sep 15, 264(26), 15293 - 7
Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16 . Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase; Peoples OP et al.; The poly-beta-hydroxybutyrate biosynthetic thiolase gene from Zoogloea ramigera was used as a hybridization probe to screen restriction digests of Alcaligenes eutrophus H16 DNA . Specific hybridization signals were obtained and two fragments (a 2.3-kilobase PstI fragment and a 15-kilobase EcoRI fragment) cloned in the Escherichia coli vector pUC8 (plasmids pAeT3/pAeT10 and pAeT29, respectively) . Biochemical analysis of lysates of E . coli cells containing each plasmid identified significant levels of beta-ketothiolase and acetoacetyl-CoA reductase enzyme activities in lysates of E . coli cells containing plasmids pAeT10 or pAeT29 . Nucleotide sequence analysis of the pAeT10 insert identified two open reading frames which encode polypeptides of Mr = 40,500 and Mr = 26,300 corresponding to the structural genes for beta-ketothiolase (phbA) and acetoacetyl-CoA reductase (phbB), respectively . Amino acid sequence homologies between the two bacterial and two mammalian thiolases are discussed with respect to the chain length specificity exhibited by the different thiolase enzymes.

J Bacteriol, 1989 Sep, 171(9), 5071 - 8
Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp; Siddiqui RA et al.; The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A . eutrophus H16 and subjected to cloning procedures . After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated . HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions . Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19 . Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance . The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34 . The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A . eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance . In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly . The hybrid plasmid metal resistance was not expressed in Escherichia coli . DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.

J Bacteriol, 1989 Sep, 171(9), 5065 - 70
Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus; Nies A et al.; Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102 . A restriction nuclease map of the 30-kb region was generated . The resistances expressed from the hybrid plasmids after transfer back into A . eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28 . Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli . Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment . When the 2.6-kb EcoRI fragment was expressed in E . coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms . The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6968 - 72
Long-range intramolecular electron transfer in azurins; Farver O et al.; The Cu(II) sites of azurins, the blue single copper proteins, isolated from Pseudomonas aeruginosa and Alcaligenes spp . (Iwasaki) are reduced by CO2- radicals, produced by pulse radiolysis, in two distinct reaction steps: (i) a fast bimolecular phase, at the rates (5.0 +/- 0.8) x 10(8) M-1.s-1 (P . aeruginosa) and (6.0 +/- 1.0) x 10(8) M-1.s-1 (Alcaligenes); (ii) a slow unimolecular phase with specific rates of 44 +/- 7 s-1 in the former and 8.5 +/- 1.5 s-1 for the latter (all at 298 K, 0.1 M ionic strength) . Concomitant with the fast reduction of Cu(II), the single disulfide bridge linking cysteine-3 to -26 in these proteins is reduced to the RSSR- radical ion as evidenced by its characteristic absorption band centered at 410 nm . This radical ion decays in a unimolecular process with a rate identical to that of the slow Cu(II) reduction phase in the respective protein, thus clearly suggesting that a long-range intramolecular electron transfer occurs between the RSSR- radicals and the Cu(II) site . The temperature dependence of the internal electron transfer process in both proteins was measured over the 4 degrees C to 42 degrees C range . The activation parameters derived are delta H* = 47.5 +/- 4.0 and 16.7 +/- 1.5 kJ.mol-1; and delta S not equal to = -56.5 +/- 7.0 and -171 +/- 18 J.K-1.mol-1, respectively . Using the Marcus theory, we found that the intramolecular electron transfer rates and their activation parameters observed for the two azurins correlate well with the distances between the reactive sites, their redox potential, and the nature of the separating medium . Thus, azurins with distinct structural and reactivity characteristics isolated from different bacteria or modified by site-directed mutagenesis can be used in comparing long-range electron transfer process between their conserved disulfide bridge and the Cu(II) sites.

Zh Mikrobiol Epidemiol Immunobiol, 1989 Aug, (8), 24 - 8
{Electrophoretic analysis of the exoproducts of bacteria in the genus Pseudomonas}; Volchkevich ZhA et al.; A high degree of similarity (exceeding 90%) between the electrophoregrams of the exoproducts of 31 Pseudomonas strains, including the standard species P . aeruginosa, P . maltophilia, P . putida, P . alcaligenes, P . testosteroni, P . diminuta, P . stutseri, P . fluorescens, has been shown by the method of electrophoresis in polyacrylamide gel.

FEMS Microbiol Lett, 1989 Jul 15, 51(1), 159 - 63
Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy; Gerberding H et al.; Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm . The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides . In face-on view, the particle exhibited bilateral symmetry.

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