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

Voordouw, Gerrit; Pollock, W. B. R.; Bruschi, Mireille; Guerlesquin, Françoise; Rapp-Giles, Barbara J.; Wall, Judy D.

doi:10.1128/jb.172.10.6122-6126.1990

Cited by 55 publications

(53 citation statements)

References 31 publications

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“…The N-terminal 20 amino acids of the mature cytochrome c 3 reported for this strain (32) correlate exactly with residues 24 through 43 of the deduced polypeptide, confirming the identity of the cycA gene. The 21st residue, reported from the protein sequencing to be a lysine (32), was found instead from the gene sequence to be a histidine, confirming the more recent protein sequencing of Aubert et al (3). When compared with tetraheme cytochromes from several other Desulfovibrio strains (Fig.…”

Section: Resultssupporting

confidence: 67%

“…For the identification of cycA, the derived amino acid sequences were compared to protein sequences in GenBank. Twenty of the amino acid residues reported for the purified mature cytochrome c 3 (32) were found to be part of the deduced polypeptide, confirming the identification of cycA.…”

supporting

confidence: 56%

“…Two fractions, representing a monomeric cytochrome of 13 kDa (isoelectric point, 5.8) and, presumably, a polymeric version thereof, contained 80% of the periplasmic cytochrome (A. Dolla, unpublished data). N-terminal sequencing of 21 and 12 residues from the monomeric and polymeric versions, respectively, established that the proteins were the same as that encoded by the cycA gene and previously reported as cytochrome c 3 from this bacterium (32). The remainder of the cytochrome was found in fractions that corresponded to a putative highmolecular-weight cytochrome and a monoheme cytochrome c 553 .…”

Section: Resultsmentioning

confidence: 66%

“…Voordouw and coworkers (32) had reported 21 residues of the N-terminal amino acid sequence of the mature cytochrome c 3 purified from D. desulfuricans. From that protein sequence, degenerate primers (NT fwd and NT rev [ Table 2]) were devised to generate a PCR probe for the identification of the c 3 gene, cycA.…”

mentioning

confidence: 99%

“…Cytochrome c 3 was purified as previously described (32). Polyclonal antibodies were raised against purified cytochrome c 3 in a female New Zealand White rabbit.…”

mentioning

confidence: 99%

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Cytochrome c ₃ Mutants of Desulfovibrio desulfuricans

Casalot

et al. 2000

Appl Environ Microbiol

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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 hydrogensulfate 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.The anaerobic sulfate-reducing bacteria have several lowpotential c-type cytochromes that are located in the periplasm and presumably function in electron transfer events. Several members of the genus Desulfovibrio have been shown to have three cytochromes in this class, monoheme cytochrome c 553 , tetraheme cytochrome c 3 , and a high-molecular-weight cytochrome c (Hmc) with 16 hemes (20,21). Of these cytochromes, the most abundant, and that for which the most structural information has been gathered, is tetraheme cytochrome c 3 (8, 21).The physiological role of tetraheme cytochrome c 3 remains enigmatic. This cytochrome has been reported to interact effectively with an almost unrealistic list of electron donors and acceptors. In vivo, c 3 has been suggested to be the redox partner of the periplasmic hydrogenases (5, 22), a role supported by the finding that this cytochrome is tightly associated with hydrogenases during purification of the latter (17). A model in which tetraheme cytochrome c 3 shuttles electrons from hydrogenases to the various polyheme cytochromes (Hmc, nineheme cytochrome, or octaheme cytochrome c 3 ) has been proposed (2,15,18). In addition to having periplasmically located redox partners, tetraheme cytochrome c has been shown to form functional complexes with a number of cytosolic electron carriers, including ferredoxin (9), rubredoxin (28), and flavodoxin (16). In vitro studies have shown that tetraheme cytochrome c 3 and an [NiFe] hydrogenase will mediate reduction of metals, including Fe(III), Cr(VI), and U(VI), with hydrogen as the electron donor (13).The reactive nature of tetraheme cytochrome c 3 makes biochemical characterization of redox partners and functional analysis problematic; therefore, a genetic approach was initiated. As a first step, it was necessary to determine whether this cytochrome is essential for cell growth. Construction of a strain with an interrupted gene encoding tetraheme cytochrome c 3 (cycA) allowed confirmation that this cytochrome is not required for growth with lactate as the primary source of carbon and reductant and with sulfate as the terminal electron acceptor. However, the mutant grew poorly, if at all, on pyruvatesulfate medium. We infer that the fi...

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Section: Resultssupporting

confidence: 67%

supporting

confidence: 56%

Section: Resultsmentioning

confidence: 66%

mentioning

confidence: 99%

“…Cytochrome c 3 was purified as previously described (32). Polyclonal antibodies were raised against purified cytochrome c 3 in a female New Zealand White rabbit.…”

mentioning

confidence: 99%

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Cytochrome c ₃ Mutants of Desulfovibrio desulfuricans

Casalot

et al. 2000

Appl Environ Microbiol

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Ferredoxin electron transfer site on cytochrome c₃. Structural hypothesis of an intramolecular electron transfer pathway within a tetra‐heme cytochrome

Dolla¹,

Guerlesquin²,

Bruschi³

et al. 1991

J of Molecular Recognition

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To specify electron exchanges involving Desulfovibrio desulfuricans Norway tetra-heme cytochrome c3, the chemical modification of arginine 73 residue, was performed. Biochemical and biophysical studies have shown that the modified cytochrome retains its ability to both interact and act as an electron carrier with its redox partners, ferredoxin and hydrogenase. Moreover, the chemical modification effects on the cytochrome c3 1H NMR spectrum were similar to that induced by the presence of ferredoxin. This suggests that arginine 73 is localized on the cytochrome c3 ferredoxin interacting site. The identification of heme 4, the closest heme to arginine 73, as the ferredoxin interacting heme helps us to hypothesize about the role of the three other hemes in the molecule. A structural hypothesis for an intramolecular electron transfer pathway, involving hemes 4, 3 and 1, is proposed on the basis of the crystal structures of D. vulgaris Miyazaki and D. desulfuricans Norway cytochromes c3. The unique role of some structural features (alpha helix, aromatic residues) intervening between the heme groups, is proposed.

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