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PubMed1996

The Rhodobacter sphaeroides 2.4.1 rho gene: expression and genetic analysis of structure and function.

The gene which encodes transcription termination factor Rho from Rhodobacter sphaeroides 2.4.1, the gram-negative facultative photosynthetic bacterium, has been cloned and sequenced. The deduced protein shows a high level of sequence similarity to other bacterial Rho factors, especially those from proteobacteria. However, several amino acid substitutions in the conserved ATP-binding site have been identified. When expressed in Escherichia coli, the R. sphaeroides rho gene relieves Rho-dependent polarity of the trp operon, indicating interference with the transcription termination machinery of E. coli. A truncated version of R. sphaeroides Rho (Rho') is toxic to a bacterium related to R. sphaeroides, Paracoccus denitrificans, and is lethal to R. sphaeroides. We suggest that toxicity is due to the ability of Rho' to form inactive heteromers with the chromosomally encoded intact Rho. We localized a minimal amino acid sequence within Rho which appears to be critical for its toxic effect and which we believe may be involved in protein-protein interactions. This region was previously reported to be highly conserved and unique among various Rho proteins. The lethality of rho' in R. sphaeroides together with our inability to obtain a null mutation in rho suggests that Rho-dependent transcription termination is essential in R. sphaeroides. This is analogous to what is observed for gram-negative E. coli and contrasts with what is observed for gram-positive Bacillus subtilis. The genetic region surrounding the R. sphaeroides rho gene has been determined and found to be different compared with those of other bacterial species. rho is preceded by orf1, which encodes a putative integral membrane protein possibly involved in cytochrome formation or functioning. The gene downstream of rho is homologous to thdF, whose product is involved in thiophene and furan oxidation.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity.

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb3-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa3-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cyt cbb3 oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt cbb3 oxidase, and unlike in Bradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cyt cbb3 oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cyt cbb3 oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb3 oxidase of R. capsulatus.

Bacterial Proteins↗
PubMed1999

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae.

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

Amino Acid Sequence↗
PubMed2000

A soxA gene, encoding a diheme cytochrome c, and a sox locus, essential for sulfur oxidation in a new sulfur lithotrophic bacterium.

A mobilizable suicide vector, pSUP5011, was used to introduce Tn5-mob in a new facultative sulfur lithotrophic bacterium, KCT001, to generate mutants defective in sulfur oxidation (Sox(-)). The Sox(-) mutants were unable to oxidize thiosulfate while grown mixotrophically in the presence of thiosulfate and succinate. The mutants were also impaired in oxidizing other reduced sulfur compounds and elemental sulfur as evident from the study of substrate oxidation by the whole cells. Sulfite oxidase activity was significantly diminished in the cell extracts of all the mutants. A soxA gene was identified from the transposon-adjacent genomic DNA of a Sox(-) mutant strain. The sequence analysis revealed that the soxA open reading frame (ORF) is preceded by a potential ribosome binding site and promoter region with -10- and -35-like sequences. The deduced nucleotide sequence of the soxA gene was predicted to code for a protein of 286 amino acids. It had a signal peptide of 26 N-terminal amino acids. The amino acid sequence showed similarity with a putative gene product of Aquifex aeolicus, soluble cytochrome c(551) of Chlorobium limicola, and the available partial SoxA sequence of Paracoccus denitrificans. The soxA-encoded product seems to be a diheme cytochrome c for KCT001 and A. aeolicus, but the amino acid sequence of C. limicola cytochrome c(551) revealed a single heme-binding region. Another transposon insertion mutation was mapped within the soxA ORF. Four other independent transposon insertion mutations were mapped in the 4.4-kb soxA contiguous genomic DNA region. The results thus suggest that a sox locus of KCT001, essential for sulfur oxidation, was affected by all these six independent insertion mutations.

Amino Acid Sequence↗
PubMed2001

Gene cluster of Rhodothermus marinus high-potential iron-sulfur Protein: oxygen oxidoreductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper oxidases.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.

Amino Acid Sequence↗
PubMed2002

A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

Bacterial Proteins↗
PubMed2002

Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12.

Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of phi(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that phi(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

Anaerobiosis↗
PubMed2005

Experimental identification and quantification of glucose metabolism in seven bacterial species.

The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, gamma-proteobacteria, and alpha-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively.

Aerobiosis↗
PubMed1997

Cell biology and molecular basis of denitrification.

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

Amino Acid Sequence↗
PubMed1983

Presence of a new cytochrome b - like pigment with a peak at 567 nm in various aerobic bacteria.

Several physiological groups of bacteria were examined for the presence of a cytochrome b - like pigment which is demonstrable in dithionite-reduced minus substrate-reduced difference spectra. This pigment is characterized by an unusually high alpha band at 567 nm, a low concentration relative to conventional cytochromes, and an inability to be fully reduced by endogenous substrates or NADH. Previous studies with one denitrifying and nondenitrifying species of the genus Pseudomonas, in Paracoccus denitrificans, in Alcaligenes faecalis, in Azotobacter vinelandii, in Branhamella catarrhalis, and in Neisseria lactamicus. In all these organisms, the peak of the 567-nm pigment is accompanied by a peak of about equal height at approximately 559 nm, which exhibits similar properties to the 567-nm pigment. The 567-nm pigment was not demonstrable by this technique in Gluconobacter oxydans subspecies suboxydans, Bacillus subtilis, Bacillus licheniformis, Aeromonas hydrophilia, Escherichia coli, a Klebsiella species, Moraxella osloensis, Aquaspirillum itersonii, Micrococcus lysodeikticus, Micrococcus luteus, Agrobacterium tumefaciens, or Rhizobium meliloti.

Aerobiosis↗
PubMed2003

Nitrate reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum MS-1: purification and sequence analyses.

We purified the nitrate reductase from the soluble fraction of Magnetospirillum magnetotacticum MS-1. The enzyme was composed of 86- and 17-kDa subunits and contained molybdenum, non-heme iron, and heme c. These properties are very similar to those of the periplasmic nitrate reductase found in Paracoccus pantotrophus. The M. magnetotacticum nap locus was clustered in seven open reading frames, napFDAGHBC. The phylogenetic analyses of NapA, NapB, and NapC suggested a close relationship between M. magnetotacticum nap genes and Escherichia coli nap genes, which is not consistent with the 16S rDNA data. This is the first finding that the alpha subclass of Proteobacteria possesses a napFDAGHBC-type nap gene cluster. The nap gene cluster had putative fumarate and nitrate reduction regulatory protein (Fnr) and NarL protein binding sites. Furthermore, we investigated the effect of molybdate deficiency in medium on the total iron content of the magnetosome fraction and discussed the physiological function of nitrate reductase in relation to the magnetite synthesis in M. magnetotacticum.

Amino Acid Sequence↗
PubMed1995

Use of antibody fragments (Fv) in immunocytochemistry.

We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.

Antibodies, Monoclonal↗
PubMed1996

Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry.

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.

Escherichia coli↗
PubMed2006

Performances and microbial features of an aerobic packed-bed biofilm reactor developed to post-treat an olive mill effluent from an anaerobic GAC reactor.

BACKGROUND: Olive mill wastewater (OMW) is the aqueous effluent of olive oil producing processes. Given its high COD and content of phenols, it has to be decontaminated before being discharged. Anaerobic digestion is one of the most promising treatment process for such an effluent, as it combines high decontamination efficiency with methane production. The large scale anaerobic digestion of OMWs is normally conducted in dispersed-growth reactors, where however are generally achieved unsatisfactory COD removal and methane production yields. The possibility of intensifying the performance of the process using a packed bed biofilm reactor, as anaerobic treatment alternative, was demonstrated. Even in this case, however, a post-treatment step is required to further reduce the COD. In this work, a biological post-treatment, consisting of an aerobic biological "Manville" silica bead-packed bed aerobic reactor, was developed, tested for its ability to complete COD removal from the anaerobic digestion effluents, and characterized biologically through molecular tools. RESULTS: The aerobic post-treatment was assessed through a 2 month-continuous feeding with the digested effluent at 50.42 and 2.04 gl(-1)day(-1) of COD and phenol loading rates, respectively. It was found to be a stable process, able to remove 24 and 39% of such organic loads, respectively, and to account for 1/4 of the overall decontamination efficiency displayed by the anaerobic-aerobic integrated system when fed with an amended OMW at 31.74 and 1.70 gl(-1)day(-1) of COD and phenol loading rates, respectively. Analysis of 16S rRNA gene sequences of biomass samples from the aerobic reactor biofilm revealed that it was colonized by Rhodobacterales, Bacteroidales, Pseudomonadales, Enterobacteriales, Rhodocyclales and genera incertae sedis TM7. Some taxons occurring in the influent were not detected in the biofilm, whereas others, such as Paracoccus, Pseudomonas, Acinetobacter and Enterobacter, enriched significantly in the biofilter throughout the treatment. CONCLUSION: The silica-bead packed bed biofilm reactor developed and characterized in this study was able to significantly decontaminate anaerobically digested OMWs. Therefore, the application of an integrated anaerobic-aerobic process resulted in an improved system for valorization and decontamination of OMWs.

Journal Article↗
PubMed2001

A novel C1-using denitrifier alcaligenes sp. STC1 and its genes for copper-containing nitrite reductase and azurin.

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.

Alcaligenes↗
PubMed2003

Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M, and cloning of the gene.

Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.

Amino Acid Sequence↗
PubMed1998

Production of ubiquinone-10 using bacteria.

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.

Journal Article↗
PubMed1994

Molecular characterization of microbial alcohol dehydrogenases.

There is an astonishing array of microbial alcohol oxidoreductases. They display a wide variety of substrate specificities and they fulfill several vital but quite different physiological functions. Some of these enzymes are involved in the production of alcoholic beverages and of industrial solvents, others are important in the production of vinegar, and still others participate in the degradation of naturally occurring and xenobiotic aromatic compounds as well as in the growth of bacteria and yeasts on methanol. They can be divided into three major categories. (1) The NAD- or NADP-dependent dehydrogenases. These can in turn be divided into the group I long-chain (approximately 350 amino acid residues) zinc-dependent enzymes such as alcohol dehydrogenases I, II, and III of Saccharomyces cerevisiae or the plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida; the group II short-chain (approximately 250 residues) zinc-independent enzymes such as ribitol dehydrogenase of Klebsiella aerogenes; the group III "iron-activated" enzymes that generally contain approximately 385 amino acid residues, such as alcohol dehydrogenase II of Zymomonas mobilis and alcohol dehydrogenase IV of Saccharomyces cerevisiae, but may contain almost 900 residues in the case of the multifunctional alcohol dehydrogenases of Escherichia coli and Clostridium acetobutylicum. The aldehyde/alcohol oxidoreductase of Amycolatopsis methanolica and the methanol dehydrogenases of A. methanolica and Mycobacterium gasti are 4-nitroso-N,N-dimethylaniline-dependent nicotinoproteins. (2) NAD(P)-independent enzymes that use pyrroloquinoline quinone, haem or cofactor F420 as cofactor, exemplified by methanol dehydrogenase of Paracoccus denitrificans, ethanol dehydrogenase of Acetobacter and Gluconobacter spp. and the alcohol dehydrogenases of certain archaebacteria. (3) Oxidases that catalyze an essentially irreversible oxidation of alcohols, such as methanol oxidase of Hansenula polymorpha and probably the veratryl alcohol oxidases of certain fungi involved in lignin degradation. This review deals mainly with those enzymes for which complete amino acid sequences are available. The discussion focuses on a comparison of their primary, secondary, tertiary, and quaternary structures and their catalytic mechanisms. The physiological roles of the enzymes and isoenzymes are also considered, as are their probable evolutionary relationships.

Alcohol Dehydrogenase↗