Hexavalent chromium reduction in Desulfovibrio vulgaris Hildenborough causes transitory inhibition of sulfate reduction and cell growth.

Desulfovibrio vulgaris Hildenborough is a well-studied sulfate reducer that can reduce heavy metals and radionuclides [e.g., Cr(VI) and U(VI)]. Cultures grown in a defined medium had a lag period of approximately 30 h when exposed to 0.05 mM Cr(VI). Substrate analyses revealed that although Cr(VI) was reduced within the first 5 h, growth was not observed for an additional 20 h. The growth lag could be explained by a decline in cell viability; however, during this time small amounts of lactate were still utilized without sulfate reduction or acetate formation. Approximately 40 h after Cr exposure (0.05 mM), sulfate reduction occurred concurrently with the accumulation of acetate. Similar amounts of hydrogen were produced by Cr-exposed cells compared to control cells, and lactate was not converted to glycogen during non-growth conditions. D. vulgaris cells treated with a reducing agent and then exposed to Cr(VI) still experienced a growth lag, but the addition of ascorbate at the time of Cr(VI) addition prevented the lag period. In addition, cells grown on pyruvate displayed more tolerance to Cr(VI) compared to lactate-grown cells. These results indicated that D. vulgaris utilized lactate during Cr(VI) exposure without the reduction of sulfate or production of acetate, and that ascorbate and pyruvate could protect D. vulgaris cells from Cr(VI)/Cr(III) toxicity.

Cytochrome c(3) mutants of Desulfovibrio desulfuricans.

To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.

New shuttle vectors for the introduction of cloned DNA in Desulfovibrio.

The pBG1 replicon from the cryptic plasmid of Desulfovibrio desulfuricans G100A was inserted into pTZ18U derivatives to generate a new family of shuttle vectors. These plasmids are stable both in Escherichia coli and in Desulfovibrio, they present a large number of unique restriction sites, and colonies of recombinant clones can be identified by blue/white screening in E. coli. The pBMC, pBMK, and pBMS series carry the cat, npt, or strAB genes as selectable markers, respectively. The pBMC6, pBMK6, and pBMS6 plasmids can be introduced both in D. desulfuricans and in Desulfovibrio fructosovorans by electrotransformation, and the pBMC7, pBMK7, and pBMS7 plasmids contain additional mobilization functions which makes them suitable for conjugation.

Transposon mutagenesis in Desulfovibrio desulfuricans: development of a random mutagenesis tool from Tn7.

The transposons Tn5, Tn7, Tn9, and Tn10 or their derivatives have been examined for transposition in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20. Tn7 inserted with a frequency of 10(-4) to 10(-3) into a unique attachment site that shows strong homology with those sites identified in other gram-negative bacteria. Inactivation of the tnsD gene in Tn7, encoding the function directing insertion into the unique site, yielded a derivative that transposed essentially randomly with a frequency of ca. 10(-6) per donor. Derivatives of Tn5, but not wild-type Tn5, were also found to undergo random transposition at a similar frequency. No evidence was obtained for transposition of Tn9 or Tn10.

Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction.

A 2.3-kb plasmid present in about 20 copies per genome was identified in extracts of Desulfovibrio desulfuricans G100A and designated pBG1. It appears to be unable to replicate in Escherichia coli. Although composite plasmids of pBG1 inserted into pTZ18U are stable in E. coli, few if any pBG1-specific transcripts are detectable. The plasmid sequence reveals several features typical of the origin of replication of non-ColE1 enterobacterial plasmids as well as several potential open reading frames. This small replicon has been shown to support the replication of recombinant plasmids in D. desulfuricans G100A and Desulfovibrio fructosovorans. A conjugable shuttle vector has been constructed.

Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement.

The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.

Plasmid transfer by conjugation in Desulfovibrio desulfuricans.

Conjugational transfer of several IncQ plasmids from Escherichia coli to the strictly anaerobic, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G100A was demonstrated. Plasmid DNA from exconjugants was visualized on agarose gels and was used to transform E. coli to the appropriate antibiotic resistances. Neither transfer of IncW and IncP plasmids to strain G100A, nor transfer of any plasmid to D. desulfuricans strain ATCC 27774 was observed. Conjugation of suicide plasmids containing either Tn5 or Tn9 into D. desulfuricans did not result in detectable transposition. Optimal conditions for conjugational transfer and antibiotic resistance levels of strain G100A were examined.

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes.

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria.