Physiological function and regulation of flavocytochrome c3, the soluble fumarate reductase from Shewanella putrefaciens NCIMB 400

Abstract

Shewanella putrefaciens produces a soluble flavocytochrome c under anaerobic growth conditions. This protein shares sequence similarity with the catalytic subunits of membrane-bound fumarate reductases from Escherichia coli and other bacteria and the purified protein has fumarate reductase activity. It is shown here that this enzyme, flavocytochrome c 3, is essential for fumarate respiration in vivo since disruption of the chromosomal fccA gene, which encodes flavocytochrome c 3, leads to a specific loss of the ability to grow with fumarate as terminal electron acceptor. Growth with nitrate, trimethylamine N-oxide (TMAO) and other acceptors was unaffected. The fccA gene is transcribed as a 2 kb monocistronic mRNA. An adjacent reading frame that bears limited sequence similarity to one of the membrane anchor subunits of E. coli fumarate reductase is not co-transcribed with fccA. Expression of the fccA gene is regulated by anaerobiosis and by the availability of alternative electron acceptors, particularly nitrate and TMAO. DNA sequences have been identified that are required for this regulation.

Role of the Tetraheme Cytochrome CymA in Anaerobic Electron Transport in Cells of Shewanella putrefaciens MR-1 with Normal Levels of Menaquinone

“…One report indicated that pACYC184 could not replicate in MR-1 (48), but this was subsequently disproven (37). Another report described the use of pJQ200KS to generate a gene replacement knockout of the fumarate reductase of S. putrefaciens (now S. frigidimarina [47]) NCIMB 400 (13). This use cannot be extrapolated to MR-1 because MR-1 maintains pJQ200KS as a free replicon (C. Myers, unpublished data); this is not surprising because it contains the p15A origin of replication from pACYC184 (45).…”

“…This marked effect on menaquinone levels is of concern because spontaneous Rf r mutants are frequently used as recipients in mating protocols designed to generate mutants. Even though the reduced levels of menaquinones in the Rf r strains (MR-1A and CMTn-1) did not affect the ultimate electron acceptor phenotype of a cymA knockout, effects related to the Rf r phenotype must be considered possible contributing factors to the phenotypes of other Shewanella mutants, e.g., those lacking fumarate reductase (13,35) or MtrB (a putative outer membrane protein required for Fe(III) and Mn(IV) reduction) (4). Even if menaquinone levels are not affected by other rpoB mutations, other as yet uncharacterized effects must be considered.…”

Characterization of Transcriptional Regulation ofShewanella frigidimarinaFe(III)-Induced FlavocytochromecReveals a Novel Iron-Responsive Gene Regulation System

“…A major heme-stained band at approximately 64-kDa was detected under all growth conditions investigated. This heme-stained band corresponds to the Fcc 3 protein, the soluble, periplasmic, tetraheme flavocytochrome that functions as a physiological respiratory fumarate reductase in Shewanella (8,9). Fcc 3 was the dominant heme-stained band during microaerobic growth or anaerobic growth with fumarate or nitrate as the sole electron acceptor.…”

“…The outer membrane cytochromes, including OmcA and OmcB, might be localized where they can make direct contact with extracellular metal oxides at the cell surface. The soluble periplasmic cytochromes could be involved in intermembrane electron transfer between the inner and outer membranes and also in the reduction of soluble Fe(III) chelates that cross the outer membrane (2,5,8).…”

Crystallization and preliminary X-ray crystallographic analysis of a periplasmic tetrahaem flavocytochrome c 3 from Shewanella frigidimarina NCIMB400 which has fumarate reductase activity

“…One of these is a 64 kDa tetrahaem¯avo-cytochrome c3, Fcc 3 , which contains bis-Hisligated c 3 -haems and non-covalently bound FAD (Pealing et al, 1995). Fcc 3 has been demonstrated to be the only physiological fumarate reductase present in S. frigidimarina NCIMB400 (Gordon et al, 1998). The sequence of the gene encoding Fcc 3 reveals that this soluble enzyme is related to the catalytic subunit of membrane-bound fumarate reductases from other bacteria (Pealing et al, 1992).…”