Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone

Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as termin al electron accepters for anaerobic respiration. A previous report describe d a mutant strain (CMTn-1) deficient in CymA a tetraheme cytochrome c. Howe ver, the interpretation of the electron transport role of CymA was complica ted by the fact that CMTn-1 was also markedly deficient in menaquinones. Th is report demonstrates that the depressed menaquinone levels were the resul t of the rifampin resistance phenotype of the parent of CMTn-1 and;not the interruption of the cymA gene, This is the first report of rifampin resista nce leading to decreased menaquinone levels, indicating that rifampin-resis tant strains should be used with caution when analyzing electron transport processes. A site directed gene replacement approach was used to isolate a cymA knockout strain: (MR1-CYMA) directly from MR-1. While MR1-CYMA retaine d menaquinone levels comparable to those of MR-1, it lost the ability to re duce iron(III), manganese(TV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type ef ficacy, and the presence of the cymA transcript and CymA protein was also r estored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone i n these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.