cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae

1 Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester, Box 672, 601 Elmwood Avenue, Rochester, NY 14642, USA 2 Department of Microbiology, Section of Infectious Diseases, Boston University School of Medicine, Boston, MA 02118, USA 3 Department of Medicine, Section of Molecular Medicine, Boston University School of Medicine, Boston, MA 02118, USA Correspondence Virginia L. Clark Ginny_Clark{at}urmc.rochester.edu The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene ( norB ) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended –10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the –10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended –10 motif gave high levels of aerobic expression of a norB : : lacZ fusion. Insertional inactivation of gonococcal nsrR , predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coli , expression of a gonococcal nsrR gene repressed gonococcal norB ; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN , as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site. Abbreviations: FNR, fumarate nitrate reductase regulator Present address: Health and Safety Executive, Hazardous Installations Directorate, Biological Agents Unit, Redgrave Court, Bootle, Merseyside, UK.