K-089. Reduction of Nitrate Occurs in the Absence of NapB in Shewanella oneidensis MR-1

H. Gao1,2, Z. K. Yang3, S. Barua1, S. Reed4, M. F. Romine4, J. K. Fredrickson4, J. M. Tiedje2, J. Zhou1;
1Univ. of Oklahoma, Norman, OK, 2Michigan State Univ., East Lansing, MI, 3Oak Ridge Natl. Lab., Oak Ridge, TN, 4Pacific Northwest Natl. Lab., Richland, WA.

Shewanella oneidensis MR-1 serves as a model for studying anaerobic respiration and electron transport-linked metal reduction. In the genome of S. oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA of the NAP system) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA of the NRF system) have been identified. However, these two systems appear to be atypical because the genome lacks both napC and nrfH, which are essential for reduction of nitrate to nitrite and nitrite to ammonium in most bacteria containing these two systems, respectively. In this study, we demonstrated that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Unexpectedly, the napB mutant exhibited a higher maximum cell density than the wild-type while the napA mutant was defective completely in growth on nitrate. Although reduction of nitrate to ammonium in the napB mutant is also conducted by NAP and NRF systems, nitrite, the intermediate of the reduction, was not detected as a free intermediate. Further investigation suggests that NapB may be the preferred electron acceptor from a membrane-bound protein which delivers electrons from menaquinol pool to a number of terminal reductases. Microarray and mutational analyses suggest that CymA is likely to be functional replacement of both NapC and NrfH in the nitrate reduction.