The starvation-stress response (SSR) of Salmonella typhimurium includes gene products necessary for starvation avoidance, starvation survival and virulence for this bacterium. Numerous genetic loci induced during carbon-source starvation and required for the long-term-starvation survival of this bacterium have been identified. The SSR not only protects the cell against the adverse effects of long-term starvation but also provides cross-resistance to other environmental stresses, e.g. thermal challenge (55 degrees C) or acid-pH challenge (pH 2.8). One carbon-starvation-inducible lac fusion, designated stiA was previously reported to be a sigma(S)-dependent SSR locus that is phosphate-starvation, nitrogen-starvation and H2O2 inducible, positively regulated by (p)ppGpp in a relA-dependent manner, and negatively regulated by cAMP:cAMP receptor protein complex and OxyR. We have discovered through sequence analysis and subsequent biochemical analysis that the stiA::lac fusion, and a similarly regulated lac fusion designated sti-99, lie at separate sites within the first gene (narZ) of an operon encoding a cryptic nitrate reductase (narZYWV) of unknown physiological function. In this study, it was demonstrated that narZ was negatively regulated by the global regulator Fnr during anaerobiosis. Interestingly, narZ(YWV) was required for carbon-starvation-inducible thermotolerance and acid tolerance. In addition, narZ expression was induced approximately 20-fold intracellularly in Madin-Darby canine kidney epithelial cells and 16-fold in intracellular salts medium, which is believed to mimic the intracellular milieu. Also, a narZ1 knock-out mutation increased the LD50 approximately 10-fold for S. typhimurium SL1344 delivered orally in the mouse virulence model. Thus, the previously believed cryptic and constitutive narZYWV operon is in fact highly regulated by a complex network of environmental-stress signals and global regulatory functions, indicating a central role in the physiology of starved and stressed cells.