Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12

Abstract

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...

Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coli – Implications for urinary tract infection

“…Small amounts of NO may also be generated non-enzymatically from urinary nitrite [28,29] and by bacterial nitrite reductase [31] or nitrate reductase [46], in particular if the urine becomes acidified and the bacteria experience low oxygen levels during the infection. The hmp gene is also inducible by nitrite [12] and in our study urinary nitrite levels were elevated in 9 out of 14 patients. However, no correlation was found between hmp expression and urinary nitrite levels.…”

“…Oxygen consumption and NO levels were measured until the chamber contents became anaerobic. A NO-saturated solution was made by acidified nitrite as previously described [12]. Respiratory inhibition (in minutes) was calculated from the time point when NO was added to the time point when the respiration resumed.…”

“…The main function of Hmp is to catalyse the conversion of NO to nitrate [9,10], but Hmp can also operate in anaerobic conditions reducing NO to nitrous oxide (N 2 O) [11]. The regulation of hmp transcription is complex involving several regulators including Fnr [12] and MetR [13]. Recently, it was discovered that the regulator NsrR represses hmp transcription in the absence of NO or nitrite [14,15].…”

Vitreoscilla Hemoglobin Binds to Subunit I of Cytochrome bo Ubiquinol Oxidases

“…In any case, it now appears that the roles and functions of VHb are more complex than just the oxygen-transporting one initially believed. These hypothetical functions are different from the NO detoxification function proposed for flavohemoproteins from E. coli (22)(23)(24)(25)(26)(27). A recent study, however, indicates that NO detoxification may be a secondary but important function for VHb (28), whereas its primary function may be oxygen transfer, as supported by the data presented here.…”

The yjeB ( nsrR ) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

“…Treatment with NO sources caused partial or no growth inhibition, depending on growth conditions and the reagent used. Cultures for iron starvation experiments were grown aerobically in L broth as described previously (33). NO was prepared as an aqueous solution by reaction of sodium nitrite with sulfuric acid (33); GSNO and N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (Spermine NONOate) were purchased from Calbiochem.…”