Cytochrome c ₃ Mutants of Desulfovibrio desulfuricans

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 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...

A Molybdopterin Oxidoreductase Is Involved in H 2 Oxidation in Desulfovibrio desulfuricans G20

“…1). The type I tetraheme periplasmic cytochrome c 3 is perhaps the most well studied protein in Desulfovibrio (32), and yet much remains to be learned regarding its function. In Desulfovibrio, the pool of periplasmic cytochrome c 3 has been suggested to act as an electron acceptor for periplasmic hydrogenases and formate dehydrogenases (14,18,23).…”

New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20

“…This small plasmid has provided an SRB replicon for use in construction of shuttle vectors for SRB (29). I2 is a mutant derived from G20 through plasmid insertion and interruption of the gene encoding the type I tetraheme c-type cytochrome TpIc 3 (cycA, Dde_3182) (26). Recombination events have been documented in I2 maintained under plasmid-encoded antibiotic selection that restored a complete cycA gene without removal of the plasmid insert from the genome (26).…”

“…I2 is a mutant derived from G20 through plasmid insertion and interruption of the gene encoding the type I tetraheme c-type cytochrome TpIc 3 (cycA, Dde_3182) (26). Recombination events have been documented in I2 maintained under plasmid-encoded antibiotic selection that restored a complete cycA gene without removal of the plasmid insert from the genome (26). Throughout these studies, care was taken to monitor and minimize the suppressor population by maintaining antibiotic selection and avoiding extensive exposure to electron donor-limiting conditions.…”

“…The work here focuses on Desulfovibrio alaskensis G20 (17) (formerly Desulfovibrio desulfuricans G20) for which mutants lacking the abundant TpIc 3 (Dde_1382; M r , 13,000), CycA mutants, are available (25,26). This tetraheme cytochrome is the most abundant c-type cytochrome in the periplasm of Desulfovibrio spp.…”

Metabolism of H2 by Desulfovibrio alaskensis G20 during syntrophic growth on lactate

“…Another group has generated a mutation in the same cycA gene by plasmid insertion. However, that mutant was able to grow in hydrogen/sulfate medium (Rapp- Giles et al, 2000) and syntrophically with strain JF-1 (unpublished data). The reasons are not clear, although that insertion may be less stable than those described here.…”