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-chl