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Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR‐1
Author(s) -
Maier Tamara M.,
Myers Judith M.,
Myers Charles R.
Publication year - 2003
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200390034
Subject(s) - fumarate reductase , shewanella oneidensis , biology , reductase , genomic dna , gene , transposon mutagenesis , microbiology and biotechnology , shewanella , biochemistry , mutant , genetics , escherichia coli , transposable element , enzyme , bacteria
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 (Tn phoA ) 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 Tn phoA ‐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 Tn phoA ‐flanking genomic DNA of CMTn‐3. PCR analysis demonstrated that a large deletion (≥9.2 kb and ≤11 kb) of genomic DNA occurred in CMTn‐3 as a result of Tn phoA insertion. This deletion included at least half of the fumarate reductase gene as well as ∼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.