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 (>or=9.2 kb and <or=11 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 approximately 8 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.
Maier
Tamara M
TM
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Myers
Judith M
JM
Myers
Charles R
CR
D004251:DNA Transposable Elements; D005650:Fumarates / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000502:physiology
D004251:DNA Transposable Elements; D005650:Fumarates; C032005:fumaric acid; D013385:Succinate Dehydrogenase