Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient for inducible nitric oxide synthase (iNOS) did not support growth of E. coli by nitrate respiration, suggesting that nitrate generated during inflammation was host-derived. Thus the inflammatory host response selectively enhances growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

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...

Two sensor proteins, NarX and NarQ, mediate nitrate regulation of anaerobic respiratory gene expression.Either of these sensors is sufficient to signal the presence of nitrate to the response regulator protein, NarL, a transcriptional activator and repressor. Two observations suggested the existence of a second response regulator that is also involved in nitrate regulation. First, narL null mutants retain residual nitrate induction offdnG operon expression; this residual induction is absent in narX narQ double-null strains. Second, nitrate induction of aeg-46.5 operon expression is substantially enhanced in narL null strains (M. H. Choe and W. S. Reznikoff, J. Bacteriol. 173:6139-6146, 1991). We found that this nitrate induction requires either the NarX or the NarQ protein, consistent with the existence of a second response regulator. We designate this second regulator NarP. We isolated insertion mutants that are defective in aeg-46.5 operon expression. These insertions are in the narP gene, which encodes a response regulator that is 44% identical to the NarL protein.Null alleles of narP abolished aeg-46.5 induction and also eliminated the residual NarL-independent nitrate induction offdnG operon expression. Both the NarX and NarQ proteins communicate with both the NarP and NarL proteins. We found that the primary signal for NarP-dependent aeg-46.5 operon induction is nitrite rather than nitrate. By contrast, nitrite is a relatively weak signal for NarL-dependent induction. In narX null strains, nitrite was an efficient signal for NarL-dependent induction, and this induction required the NarQ protein. We conclude that, in wild-type strains, the NarQ protein communicates the presence of nitrite to both the NarP and NarL proteins and that the NarX protein inhibits this communication with the NarL protein.Eschenchia coli is a facultative aerobe and can synthesize a variety of respiratory chains during anaerobic growth. Anaerobic induction of respiratory enzyme synthesis is mediated by the activator protein Fnr (reviewed in reference 35). The energetically most efficient anaerobic respiratory chain is formate-nitrate oxidoreductase, which consists of formate dehydrogenase-N (encoded by the fdnGHI operon), quinone, and nitrate reductase (encoded by the narGHJI operon) (reviewed in reference 38). During anaerobic growth, nitrate (NO3-) induces the synthesis of formate dehydrogenase-N and nitrate reductase through the action of the activator protein NarL (2, 37; reviewed in reference 39). The NarL protein also represses the synthesis of alternate respiratory enzymes such as fumarate reductase (encoded by the frdABCD operon) during anaerobic growth with nitrate (17,19,40 This shows that the NarL protein represses aeg-46. 5 expression even in the absence of nitrate. Regulation of the aeg-93 operon resembles that of the nrf operon, encoding formatedependent respiratory nitrite reductase (26). Additionally, the nrf genes map at 93 min (9). Thus, it is likely that the aeg-93 operon fusion resides in one of the nrf genes. Ex...

Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli. We cloned narL on a 5.1-kilobase HindlIl fragment. Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK. Maxicell experiments indicated that narL and narX encode proteins with approximate Mrs of 28,000 and 66,000, respectively. narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold. Expression of 40(narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent offnr. Expression of 'F(narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A 4(narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ andfnr'. Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E. coli genetic map and that narK is transcribed clockwise.Escherichia coli will use several terminal electron acceptors for respiration, including oxygen, nitrate (NO3-), and fumarate. Use of these electron acceptors is hierarchical: aerobiosis prevents nitrate and fumarate reductase synthesis, and nitrate prevents the anaerobic formation of fumarate reductase (18,21,46,53; V. Stewart, Microbiol. Rev., in press).The nitrate reductase enzyme complex consists of three membrane-bound subunits and also contains molybdenum cofactor and heme (Stewart, in press). The structural genes for the polypeptide subunits are termed narC (formerly narG [2]), narH, and narI and are organized in an operon at the nar (formerly chlC) locus at 27 min on the E. coli genetic map (2,9,16,32,38,51; Stewart, in press). Genetic studies indicate that the organization of this operon is hemApromoter-narC-narH-narl -trp (9, 51).Nitrate reductase synthesis is induced by anaerobiosis, and during anaerobic growth, it is further induced by nitrate (46). Studies with F(nar-lac) operon fusions (14, 50) and measurements of nar mRNA synthesis (28) indicate that both anaerobiosis and nitrate act to control narCHI operon transcription. The effect of anaerobiosis is thought to be mediated through FNR (the fnr gene product), a positiveacting transcription factor required for the synthesis of several anaerobic respiratory enzymes (26, 43; Stewart, in press) including nitrate reductase (14, 50) and fumarate reductase (18, 21). The physiological signal for FNR-mediated transcription activation is unknown (56).A mutation termed narL215::TnlO prevents nitrate induction of nitrate reductase synthesis and of F(narC-lacZ) expression (50) but has no effect on control by anaerobiosis. This mutation is complemented in trans by a specialized transducing bacteriophage, k pchlC3 (50). Genetic mapping indicates that narL is located upstream of the nar operon, between hemA and narCHI; a gene of unknown function, narK, maps between narL and...

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumaratenitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.

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Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein.Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate͞nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains. Together with their cognate autokinases, response regulators dominate signal transduction in bacteria (1-5) and are also found upstream of protein kinase cascades in eukarya (6-9). Autokinase͞response regulator pairs, which are referred to as ''two-component'' systems, control bacterial cell division (10), development (11), chemotaxis (12), virulence (13)(14)(15)(16), and the responses to many changes in nutrient availability (17,18). Phosphorylation of an Asp in receiver domains of response regulators is used to modulate the function of their corresponding output domains (19), many of which activate or repress transcription (2, 3). The structures of six nonphosphorylated receiver domains have been determined (20-25), and it has been shown that a substantial conformational change occurs after phosphorylation in several cases (26-28). However, the lability of the acyl phosphate linkages in these domains (half-lives of seconds to hours) (2, 3) has hindered structural studies of their phosphorylated active forms.The response regulator nitrogen regulatory protein C (NtrC), an enhancer-binding protein, functions as a molecular machine to activate transcription by the 54 -holoenzyme form of RNA polymerase (29-31). The NtrC protein of Salmonella typhimurium is composed of three functional domains (32-34): an (amino) N-terminal receiver or regulatory domain that is phosphorylated on Asp-54 (D54), a central output domain that hydrolyzes ATP and activates transcription, and a C-terminal DNA-binding domain. The central domain of NtrC apparently adopts a mononucleotide-binding fold characteristic of a large group of purine nucleotide-binding proteins (35,36). Phosphorylation of D54 allows NtrC to form large oligomers that are essential for ATP hydrolysis and hence, transcriptional activation (30,31,37,38).The receiver domain of NtrC (NtrC r ) is the only such domain for which the structures of both the phosphorylated and nonphosphorylated forms have been determined (D. Kern, B. F. Volkman, P. Luginbuhl, M. J. Nohaile, S.K., and D.E.W., unpublished material; ref. 21). Although it was possible to maintain phosphorylated NtrC r (P-NtrC r ) for long enough to allow determination of its structure b...

We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis or insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::TnlO mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.