Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron-and Furdependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron-and Fur-dependent regulation appears to represent a novel ironhomeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55 Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe 2؉ -Fur complex.

Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene. A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O 2 , NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the ⌽(hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source. Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression. Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation. Anaerobic, but not aerobic, expression of ⌽(hmp-lacZ)1 was stimulated three-to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter. Iron depletion of rich broth medium by the chelator 22-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr. At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically. Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite. Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes. Nitric oxide (10 to 20 M) stimulated aerobic ⌽(hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect. We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability. Hmp is implicated in reactions with small nitrogen compounds.Escherichia coli is generally considered to consume oxygen by using two membrane-bound terminal oxidases for aerobic respiration, cytochromes boЈ and bd (15,36). Cytochrome boЈ is a member of the heme-copper superfamily of terminal oxidases; it is a proton pump (42) and has a moderately high affinity for oxygen, with a K m in the submicromolar range (11). In contrast, cytochrome bd uses a heme-heme binuclear center to bind oxygen as a surprisingly stable oxygenated form and reduce oxygen to water (20, 39). Cytochrome bd is believed not to be a proton pump but has an extraordinarily high apparent affinity for oxygen, with a K m in vivo as low as 5 nM (12). The distinct properties of these oxidases, and thus their suitability for growth under different aerobic conditions, requires that they be differentially regulated. Cytochromes boЈ and bd are maximally synthesized during growth with high (4) or limited (14) aeration, respectively. Expression of operons comprising the oxidase structural genes (cyoABCDE and cydAB, respectively) are each affected by Fnr and ArcA/ArcB, although dissection of the dire...

SummaryGlobin-like oxygen-binding proteins occur in bacteria, yeasts and other fungi, and protozoa. The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins. Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin-NADP 1 reductase (FNR)-like domain that converts the globin into an NAD(P)H-oxidizing protein with diverse reductase activities. In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses. Here, we review recent advances in E. coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to`NO' stresses. Nitric oxide in biologyNitric oxide (NO, nitrogen monoxide) is a signalling and defence molecule of major importance in biological systems (Cooper, 1999). At concentrations around 10 27 M, NO controls blood pressure in mammals and is a messenger in the central and peripheral nervous systems. Some of these processes may also involve the participation of NO congeners. NO generated by the immune system at higher concentrations and over long time periods inhibits key enzymes in bacterial and other foreign cells. These enzymes include terminal oxidases and other haem enzymes that bind dioxygen. While NO has little effect on iron±sulphur enzymes that catalyse electron transfer, it is more inhibitory towards iron± sulphur centres in enzymes such as aconitase, with non-redox functions, which allow the substrate access to the Fe±S cluster. A study of the interaction of NO (50 mM) with aconitase has shown that NO specifically inhibits the enzyme by attack on the non-cysteine co-ordinated iron site. NO inhibits aconitase more effectively (at low mM concentrations) at lower pH values (Kennedy et al., 1997).

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

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.