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...

Respiration of

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5U10 S M 3I s 3I to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionitereduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions. z 1999 Federation of European Biochemical Societies.

We report the first example of a gene, hmp, encoding a soluble flavohemoglobin in Escherichia coli K-12, which is up-regulated by paraquat in a SoxRS-independent manner. Unlike what is found for other paraquatinducible genes, high concentrations of paraquat (200 M) were required to increase the level of hmp expression, and maximal induction was observed only after 20 min of exposure to paraquat. Neither a mutation in soxS nor one in soxR prevented the paraquat-dependent increase in ⌽(hmp-lacZ) expression, but either mutant allele delayed full expression of ⌽(hmp-lacZ) activity after paraquat addition. Induction of hmp by paraquat was demonstrated in aerobically grown cultures during exponential growth and the stationary phase, thus revealing two Sox-independent regulatory mechanisms. Induction of hmp by paraquat in the stationary phase was dependent on the global regulator of stationary-phase gene expression, RpoS ( s ). However, a mutation in rpoS did not prevent an increase in hmp expression by paraquat in exponentially growing cells. Induction of s in the exponential phase by heat shock also induced ⌽(hmp-lacZ) expression in the presence of paraquat, supporting the role of s in one of the regulatory mechanisms. Mutations in oxyR or rob, known regulators of several stress promoters in E. coli, had no effect on the induction of hmp by paraquat. Other known superoxide-generating agents (plumbagin, menadione, and phenazine methosulfate) were not effective in inducing hmp expression.

No abstract