Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration.. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (greater than or equal to9.2 kb and less than or equal to11 kb) of genomic DNA occurred in CMTn-3 as a result of TnphoA insertion. This deletion included at least half of the fumarate reductase gene as well as similar to8 kb of upstream DNA. Complementation of CMTn-3 with the fumarate reductase gene plus 0.5-kb of upstream DNA restored growth on fumarate. These studies explicitly define the sole physiological fumarate reductase gene from the several possibilities suggested by the genomic sequence of MR-1. Surprisingly, the fumarate reductase gene plus 0.77-kb upstream DNA from S. frigidimarina NCIMB400 did not complement CMTn-3.