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Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3

Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA¹

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

Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity. One of the genes has been designated nirV. Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase. The nirV in 2.4.3 is apparently cotranscribed with nirK. Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity. Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers. However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3. ppaZ is expressed only when oxygen becomes limiting. ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified. Expression of ppaZ is also dependent on the two-component PrrB/PrrA system. Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity. Expression of a maltose-binding protein–PpaZ fusion indicated that the protein could not bind copper. Examination of the genome of the related bacterium R. sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction.

Keywords: photosynthetic denitrifier, denitrification, nitrite reductase, blue copper protein

Abbreviations: MBP, maltose-binding protein

The GenBank accession number for the sequence determined in this work is AF339883.

^a Present address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08540-1014, USA.

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