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. 2000 Feb;66(2):671-7.
doi: 10.1128/aem.66.2.671-677.2000.

Cytochrome c(3) Mutants of Desulfovibrio Desulfuricans

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Free PMC article

Cytochrome c(3) Mutants of Desulfovibrio Desulfuricans

B J Rapp-Giles et al. Appl Environ Microbiol. .
Free PMC article

Abstract

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.

Figures

FIG. 1
FIG. 1
Diagrammatic representation of the D. desulfuricans G20 chromosome region encoding cycA. (A) Arrangement of genes in wild-type genomic DNA. The vertical arrow indicates the position of a putative promoter. The black bar indicates the position of a putative ORF upstream of cycA that is transcribed left to right, as is cycA in this illustration. t1 and t2 indicate the locations of possible transcriptional terminators. (B) Plasmid pBSC2 (Table 1) used for gene disruption. The SacII-PstI internal cycA fragment is designated ‘cycA’. (C) Plasmid-interrupted chromosomal cycA. The positions of PCR and sequencing primers, B1 cyc primer and Km primer for the leftward junction and T3 primer and cyc2 left primer for the rightward junction, are indicated by arrows. The left copy of cycA lacks 63 bp of the 3′ end of the coding sequence (which totals 393 bp), while the right copy lacks 71 bp of the 5′ end. Note the different scale for panel A versus that for panels B and C.
FIG. 2
FIG. 2
Immunodetection of cytochrome c3 in periplasmic extracts of D. desulfuricans. Polyclonal antibodies raised against purified G20 cytochrome c3 were used to probe periplasmic proteins separated on a denaturing 10 to 22% (wt/vol) polyacrylamide gel. Lane 1, 1 μg of purified cytochrome c3; lane 2, 15 μg of periplasmic proteins from LS-grown G20; lane 3, 15 μg of periplasmic proteins from LS-grown mutant I2; lane 4, protein molecular size standards, with the relative molecular masses (in kilodaltons) indicated on the right. This figure was digitally manipulated in Adobe Photoshop 4.0 to omit two lanes containing irrelevant samples.
FIG. 3
FIG. 3
Alignment of D. desulfuricans G20 cytochrome c3 amino acid sequences with those of other members of the genus. Boxed sequences are the four heme binding sites. The arrow indicates the apparent cleavage site for the signal peptide for the D. desulfuricans G20 proapoprotein. Asterisks mark the conserved histidines that are the sixth axial ligands to the hemes. DdG20, D. desulfuricans G20 (sequence deduced from that of the cloned gene); DvHil, D. vulgaris Hildenborough (sequence deduced from that of the cloned gene [Protein Identification Resource Database, Johns Hopkins University, PIR no. A24799]); DvMiy, D. vulgaris Miyazaki (PIR no. S33874); Ds, D. salexigens (PIR no. A00128); Dg, D. gigas (PIR no. A00126). Levels of identity between the cytochrome sequence of D. desulfuricans G20 and those of D. vulgaris Hildenborough and Miyazaki, D. salexigens, and D. gigas were 66, 69, 35, and 54%, respectively.
FIG. 4
FIG. 4
Sequence of the intergenic region upstream of cycA. Asterisks indicate the termination codon of the putative upstream ORF and the start codon of the cycA gene. Prominent inverted repeats are indicated by arrows. A possible promoter for cycA, the −35 and −10 regions (separated by 17 bp), is boxed.
FIG. 5
FIG. 5
Confirmation, by Southern analysis, of integration of the pBSC2 plasmid into the D. desulfuricans G20 chromosome. Lane 1, G20 total chromosomal DNA digested with KpnI; lanes 2 to 6, total chromosomal DNAs of five kanamycin-resistant strains, derived after the introduction of pBSC2, digested with KpnI. Sizes of hybridizing DNA fragments are indicated on the left (in kilobase pairs). Identical gels were probed with the pCRA amplicon insert coding for the N-terminal region of the mature cytochrome c3 (A) or with the kan cassette from pUC4KIXX (B). KpnI-digested pBSC2 migrated as a 7.5-kbp fragment (not shown). The figure is representative of six independent Southern analyses.
FIG. 6
FIG. 6
Transcription of cycA in D. desulfuricans G20 and constructed strain I3. Total RNA from mid-exponential-phase cultures of G20 (lane 1) and I3 (lane 2) was probed with the pCRA amplicon insert. Transcript sizes (shown to the left) were estimated relative to rRNA migration. The figure is representative of four independent analyses. This figure was digitally manipulated in Adobe Photoshop 4.0 to omit one lane containing an irrelevant sample.
FIG. 7
FIG. 7
Growth curves of D. desulfuricans wild-type strain G20 (●) and mutant strain I2 (■) on LS medium (representative of 14 trials) (A) and on pyruvate-sulfate (representative of three trials) (B). The growth temperature was 37°C.
FIG. 8
FIG. 8
Diagrammatic representation of organic acid oxidation and electron flow to sulfate as the terminal electron acceptor for D. desulfuricans G20. The asterisk indicates the region of the metabolic pathway likely to be affected by the absence of cytochrome c3 in mutant I2, causing poor growth on pyruvate with a buildup of reductant that is released as hydrogen. Arrows do not imply single-electron-transfer components.

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