NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative stress-responsive regulon that includes both a single-domain and a truncated haemoglobin

Summary Consistent with its role as a nitric oxide (NO)-detoxi- fying globin in Campylobacter jejuni , Cgb ( Campylo- bacter globin) expression is strongly and specifically induced following exposure to nitrosative stress, sug- gesting a previously unrecognized capacity for NO- related stress sensing in this food-borne pathogen. In this study, Fur and PerR have been eliminated as major regulators of cgb , and NssR (Cj0466), a member of the Crp-Fnr superfamily, has been identified as the major positive regulatory factor that controls nitrosa- tive stress-responsive expression of this gene. Accordingly, disruption of nssR resulted in the aboli- tion of inducible cgb expression, which was restored by a complementing chromosomal insertion of the wild-type gene with its indigenous promoter at a sec- ond location. The NssR-deficient mutant was more sensitive to NO-related stress than a cgb mutant and this phenotype most likely arises from the failure of these cells to induce other NO-responsive compo- nents in addition to Cgb. Indeed, analysis of global gene expression, by microarray and confirmatory real-time polymerase chain reaction (PCR) in the wild type and nssR mutant, not only confirmed the depen- dence of inducible cgb expression on NssR, but also revealed for the first time a novel NssR-dependent nitrosative stress-responsive regulon. This regulon of at least four genes includes Cj0465c, a truncated globin. Consistent with NssR being a Crp-Fnr super- family member, an Fnr-like binding sequence (TTAAC- N 4 -GTTAA) was found upstream of each gene at locations - 40.5 to - - - 42.5 relative to the centre of the binding sites and the transcription start point. Site- directed mutagenesis confirmed that this cis -acting motif mediates the nitrosative stress-inducible expression of cgb .