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Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a haem–copper protein

Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA¹
Department of Chemistry, Center of Biophysics and Biochemistry, University at Albany, SUNY, Albany, NY 12222, USA²

Author for correspondence: James P. Shapleigh. Tel: +1 607 255 8535. Fax: +1 607 255 3904. e-mail: jps2{at}cornell.edu

Upstream of the nor and nnrR cluster in Rhodobacter sphaeroides 2.4.3 is a previously uncharacterized gene that has been designated nnrS. nnrS is only expressed when 2.4.3 is grown under denitrifying conditions. Expression of nnrS is dependent on the transcriptional regulator NnrR, which also regulates expression of genes required for the reduction of nitrite to nitrous oxide, including nirK and nor. Deletion analysis indicated the sequence 5'-TTGCG(N₄)CACAA-3', which is similar to sequences found in nirK and nor, is required for nnrS expression. Mutation of this sequence to the consensus Fnr-binding sequence by changing two bases in each half site caused nnrS expression to become nitrate independent. Inactivation of nnrS did not affect nitric oxide metabolism, nor did it affect expression of any of the genes involved in nitric oxide metabolism. However, taxis towards nitrate and nitrite was affected by nnrS inactivation. Purification of a histidine-tagged NnrS demonstrated that NnrS is a haem- and copper-containing membrane protein. Genes encoding putative orthologues of NnrS are sometimes but not always found in bacteria encoding nitrite and/or nitric oxide reductase.

Keywords: photosynthetic denitrifier, denitrification, nitrite reductase, nitric oxide reductase, regulation

The GenBank accession number for nnrS is U62403.

^a Present address: Biology and Biomedical Sciences, Washington University, St Louis, MO 63110-9822, USA.

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