SEARCH · PubMed Health

Results for “Megaplasmids”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

PubMed1988

Mutants of Rhizobium meliloti defective in succinate metabolism.

We characterized mutants of Rhizobium meliloti SU47 that were unable to grow on succinate as the carbon source. The mutants fell into five groups based on complementation of the succinate mutations by individual recombinant plasmids isolated from a R. meliloti clone bank. Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group II, enolase (Eno); group III, phosphoenolpyruvate carboxykinase (Pck); group IV, glyceraldehyde-3-phosphate dehydrogenase (Gap), and 3-phosphoglycerate kinase (Pgk). Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) activity. Wild-type cells grown on succinate as the carbon source had high Pck activity, whereas no Pck activity was detected in cells that were grown on glucose as the carbon source. It was found that in free-living cells, Pck is required for the synthesis of phosphoenolpyruvate during gluconeogenesis. In addition, the enzymes of the lower half of the Embden-Meyerhoff-Parnas pathway were absolutely required for gluconeogenesis. Eno, Gap, Pck, and one of the Dct loci (ntrA) mapped to different regions of the chromosome; the other Dct locus was tightly linked to a previously mapped thi locus, which was located on the megaplasmid pRmeSU47b.

Chromosome Mapping↗

PubMed1991

Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production.

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.

Amino Acid Sequence↗

PubMed1991

Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha.

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.

Blotting, Southern↗

PubMed1991

Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

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

Alcaligenes↗

PubMed1992

The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

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

Alcaligenes↗

PubMed1993

Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.

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.

Alcaligenes↗

PubMed1993

Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti.

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

Amino Acid Sequence↗

PubMed1993

The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase.

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.

Alcaligenes↗

PubMed1995

Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus.

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

Alcaligenes↗

PubMed1995

Operator binding of the CbbR protein, which activates the duplicate cbb CO2 assimilation operons of Alcaligenes eutrophus.

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

Alcaligenes↗

PubMed1995

Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti. The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R. meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to that of SigA of the closely related bacterium Agrobacterium tumefaciens. Through Southern analysis, we located the gene on the R. meliloti main chromosome rather than on one of the megaplasmids. The sigA locus does not appear to be part of a macromolecular synthesis operon (MMS), as in many other bacterial species, but rather lies downstream of a partial open reading frame showing similarity to the threonine dehydrogenase gene (tdh) of E. coli.

Amino Acid Sequence↗

PubMed1995

Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans.

The CO dehydrogenase structural genes (cox) and orf4 are clustered in the transcriptional order coxM--> coxS--> coxL--> orf4 on the 128-kb megaplasmid pHCG3 of the carboxidotroph Oligotropha carboxidovorans OM5. Sequence analysis suggested association of molybdopterin cytosine dinucleotide and flavin adenine dinucleotide with CoxL and of the [2Fe-2S] clusters with CoxS.

Aldehyde Oxidoreductases↗

PubMed1996

Differentially transcribed regions of Haloferax volcanii genome depending on the medium salinity.

To identify genomic regions involved in osmoregulation in the extremely halophilic archaeon Haloferax volcanii, we used a technique which involves hybridization of cDNAs obtained at different salinities against a cosmid library of the organism. Both low and high salt concentrations trigger differential expression; however, adaptation to low salinities seems to elicit a wider response. The presence of a large domain within the largest of the megaplasmids with a strong response to low salt concentrations is noteworthy.

Blotting, Northern↗

PubMed1996

Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor.

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

Agrobacterium tumefaciens↗

PubMed1996

A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti.

The bacterium Rhizobium meliloti forms N2-fixing root nodules on alfalfa plants. The ndvF locus, located on the 1,700-kb pEXO megaplasmid of R. meliloti, is required for nodule invasion and N2 fixation. Here we report that ndvF contains four genes, phoCDET, which encode an ABC-type transport system for the uptake of Pi into the bacteria. The PhoC and PhoD proteins are homologous to the Escherichia coli phosphonate transport proteins PhnC and PhnD. The PhoT and PhoE proteins are homologous to each other and to the E. coli phosphonate transport protein PhnE. We show that the R. meliloti phoD and phoE genes are induced in response to phosphate starvation and that the phoC promoter contains two elements which are similar in sequence to the PHO boxes present in E. coli phosphate-regulated promoters. The R. meliloti ndvF mutants grow poorly at a phosphate concentration of 2 mM, and we hypothesize that their symbiotic phenotype results from their failure to grow during the nodule infection process. Presumably, the PhoCDET transport system is employed by the bacteria in the soil environment, where the concentration of available phosphate is normally 0.1 to 1 microM.

Amino Acid Sequence↗

PubMed1996

Regulation of CO2 assimilation in Ralstonia eutropha: premature transcription termination within the cbb operon.

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

Alcaligenes↗

PubMed1996

Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability.

Amino Acid Sequence↗

PubMed1997

Purification and molecular characterization of the H2 uptake membrane-bound NiFe-hydrogenase from the carboxidotrophic bacterium Oligotropha carboxidovorans.

The membrane-bound hydrogenase of Oligotropha carboxidovorans was solubilized with n-dodecyl-beta-D-maltoside and purified 28-fold with a yield of 29% and a specific activity of 173 to 178 micromol of H2 x min(-1) x mg(-1). It is the first hydrogenase studied in a carboxidotrophic bacterium. The enzyme acts on artificial electron-accepting dyes, such as methylene blue, but is ineffective with pyridine nucleotides or other soluble physiological electron acceptors. Hydrogenase of O. carboxidovorans belongs to class I of hydrogenases and is a heterodimeric 101,692-Da NiFe-protein composed of the polypeptides HoxL and HoxS. Molecular cloning data revealed, that HoxL comprises 604 amino acid residues and has a molecular mass of 67,163 Da. Pre-HoxS comprises 360 amino acid residues and is synthesized as a precursor protein which is cleaved after alanine at position 45, thus producing a mature HoxS of 33,767 Da. The leader sequence corresponds to the signal peptide of small subunits of hydrogenases. The hydropathy plots of HoxL and HoxS were indicative for the absence of transmembranous helices. HoxZ has four transmembranous helices and is considered the potential membrane anchor of hydrogenase in O. carboxidovorans. Hydrogenase genes show the transcriptional order 5' hoxV --> hoxS --> hoxL --> hoxZ 3'. The hox gene cluster as well as the clustered CO dehydrogenase (cox) and Calvin cycle (cbb) genes are arranged within a 30-kb DNA segment of the 128-kb megaplasmid pHCG3 of O. carboxidovorans.

Amino Acid Sequence↗