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Nitric oxide is not only an obligatory intermediate in denitrification, but also a signalling and defence molecule of major importance. However, the basis of resistance to NO and RNS (reactive nitrogen species) is poorly understood in many microbes. The cellular targets of NO and RNS [e.g. metalloproteins, thiols in proteins, glutathione and Hcy (homocysteine)] may themselves serve as signal transducers, sensing NO and RNS, and resulting in altered gene expression and synthesis of protective enzymes. The properties of a number of such protective mechanisms are outlined here, including globins, flavorubredoxin, diverse enzymes with NO- or S-nitrosothiol-reducing properties and other redox proteins with poorly defined roles in protection from nitrosative stresses. However, the most fully understood mechanism for NO detoxification involves the enterobacterial flavohaemoglobin (Hmp). Aerobically, Hmp detoxifies NO by acting as an NO denitrosylase or ‘oxygenase’ and thus affords inducible protection of growth and respiration, and aids survival in macrophages. The flavohaemoglobin-encoding gene of Escherichia coli, hmp, responds to the presence of NO and RNS in an SoxRS-independent manner. Nitrosating agents, such as S-nitrosoglutathione, deplete cellular Hcy and consequently modulate activity of the MetR regulator that binds the hmp promoter. Regulation of Hmp synthesis under anoxic conditions involves nitrosylation of 4Fe-4S clusters in the global transcriptional regulator, FNR. The foodborne microaerophilic pathogen, Campylobacter jejuni, also expresses a haemoglobin, Cgb, but it does not possess the reductase domain of Hmp. A Cgb-deficient mutant of C. jejuni is hypersensitive to RNS, whereas cgb expression and holoprotein synthesis are specifically increased on exposure to RNS, resulting in NO-insensitive respiration. A ‘systems biology’ approach, integrating the methodologies of bacterial molecular genetics and physiology with post-genomic technologies, promises considerable advances in our understanding of bacterial NO tolerance mechanisms in pathogenesis.

Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparingEscherichia coliMG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO₄concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularlyzntA,zraP, andhydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations ofcusF(involved in Ag and Cu efflux),ais(an Al-inducible gene),asr(encoding an acid shock-inducible periplasmic protein),cpxP(a periplasmic chaperone gene), andbasR. Five up-regulated genes,basRandbasS[encoding a sensor-regulator implicated inSalmonellain Fe(III) sensing and antibiotic resistance],fliM(flagellar synthesis), andycdMandyibD(both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since themdtoperon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions.

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