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V Stewart

Publications and source records attributed to V Stewart.

At least 19 recordsLinked to original sources
PubMed2003

Biochemical Society Special Lecture. Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria.

Nitrate and nitrite are efficient respiratory oxidants for anaerobic growth. In Escherichia coli, the homologous nitrate reductase (Nar) two-component regulatory systems NarX-NarL and NarQ-NarP collaborate to control anaerobic respiratory gene expression in response to nitrate and nitrite. Several other species classified in the gamma and beta subdivisions of the proteobacteria contain only a single Nar two-component regulatory pair. This raises questions concerning the physiology of anaerobic respiration as well as the evolution, function and cross-regulation of two-component regulatory systems. Here, I focus on the sensor histidine kinases NarX and NarQ, and present a comparison of the deduced NarX and NarQ primary sequences from a broad sampling of proteobacteria. This comparison defines shared features, including a large central region of unknown function that appears to be unique to this family of sensor kinases. I then consider the phylogenetic distribution of narX and narQ genes in relation to anaerobic respiratory enzyme repertoire and physiological function. One noteworthy observation is that narXL genes are specifically associated with the structural genes for membrane-bound nitrate reductase, narGHJI, whereas organization and linkage of the narQ and narP genes is quite variable. I conclude with some speculative thoughts on the evolutionary and functional divergence of the NarX-NarL and NarQ-NarP regulatory systems. Overall, this analysis aims to provide a basis for future hypothesis and experimentation in this area.

Amino Acid Sequence↗
PubMed2001

Children. The unwitting target of environmental injustices.

Children have little control over where they live, what they eat, the financial circumstances of their families, or the developmental activities and behaviors that make them vulnerable to environmental contaminants. Minority and poor families disproportionately live in communities with landfills, hazardous waste facilities, incinerators, industrial plants, and old housing with poor indoor air quality and lead-based paint. Residents of these communities are also more likely than are more affluent communities to consume fish on a regular basis from local waters, many of which have banned fishing. Consequently, these children and their families are exposed more frequently than are children in other communities to potentially dangerous chemicals that can affect health. Data indicate that poor and minority children have higher rates of asthma, elevated blood lead levels, learning disabilities, and hyperactivity than do non-minority and more affluent children. When a group of people is exposed unfairly and inequitably to toxins in their communities, workplaces, and schools, a phenomenon called environmental discrimination or environmental racism exists. Environmental justice is a US governmental remedy that requires the application of fair strategies and processes in the resolution of inequality related to environmental contamination. The US response resulted in the establishment of offices of Environmental Justice within the EPA and ATSDR and passage of important legislation and policies, such as the Community Planning and Right-to-Know Act of 1986, Executive Order 12898 (Federal Actions to Address Environmental Justice in Minority Populations), and Executive Order 13045, a parallel order to protect low-income and minority children from actual and potential environmental hazards. Communities and advocacy groups play an important role in promoting healthier environments for children. Frequently, low-income and minority communities are perceived as less powerful, less organized, and ill equipped to defend against actual and potential sources of environmental contamination. Health care professionals are in a strategic position to assist with community development, organizing, and empowerment through educational programming, networking, and supporting other activities that bring attention to the plight of environmentally vulnerable communities.

Child↗
PubMed1999

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.

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 nitrate/nitrite 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.

Aspartic Acid↗
PubMed1999

RNA sequence requirements for NasR-mediated, nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader.

In Klebsiella oxytoca, enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism, in which the nasR gene product responds to nitrate or nitrite and overcomes transcription termination at the factor-independent terminator site located in the nasF upstream leader region. Previous studies led to the hypothesis that the NasR protein mediates transcription antitermination through interaction with nasF leader RNA. Here, we report a DNA sequence comparison that reveals conserved 1:2 and 3:4 RNA secondary structures in the nasF leader RNAs from two Klebsiella species. Additionally, we found that specific binding of the NasR protein to nasF leader RNA was stimulated by nitrate and nitrite. We combined mutational analysis, in vivo and in vitro antitermination assays, and an RNA electrophoretic mobility shift assay to define regions in the nasF leader that are essential for antitermination and for NasR-RNA interaction. Formation of the 1:2 stem structure and the specific sequence of the 1:2 hexanucleotide loop were required for both nitrate induction and for NasR-RNA interaction. Mutations in the 1:2 stem-loop region that abolished nitrate induction also interfered with NasR-leader RNA interaction. Finally, nucleotide alterations or additions in the linker region between the 1:2 and 3:4 stem-loops were deleterious to nasF operon induction but not to NasR-leader RNA interaction. We hypothesize that NasR protein recognizes the 1:2 stem-loop structure in the nasF leader RNA to mediate transcription antitermination in response to nitrate or nitrite.

Bacterial Proteins↗
PubMed1999

Functional similarities among two-component sensors and methyl-accepting chemotaxis proteins suggest a role for linker region amphipathic helices in transmembrane signal transduction.

Signal-responsive components of transmembrane signal-transducing regulatory systems include methyl-accepting chemotaxis proteins and membrane-bound, two-component histidine kinases. Prokaryotes use these regulatory networks to channel environmental cues into adaptive responses. A typical network is highly discriminating, using a specific phosphoryl relay that connects particular signals to appropriate responses. Current understanding of transmembrane signal transduction includes periplasmic signal binding with the subsequent conformational changes being transduced, via transmembrane helix movements, into the sensory protein's cytoplasmic domain. These induced conformational changes bias the protein's regulatory function. Although the mutational analyses reviewed here identify a role for the linker region in transmembrane signal transduction, no specific mechanism of linker function has yet been described. We propose a speculative, mechanistic model for linker function based on interactions between two putative amphipathic helices. The model attempts to explain both mutant phenotypes and hybrid sensor data, while accounting for recognized features of amphipathic helices.

Amino Acid Sequence↗
PubMed1999

General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al.

Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of DeltarhaBS::[Phi(nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins. Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988).

Amino Acid Sequence↗
PubMed1998

NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro.

In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. In the presence of nitrate or nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His6-tagged form (His6-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma70-dependent tac promoter for the native sigmaN-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His6-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 microM) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified regions required for both negative regulation (the terminator) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. Finally, electrophoresis mobility shift analysis revealed that His6-NasR bound specifically to nasF leader RNA. This binding was independent of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction with nasF leader RNA.

5' Untranslated Regions↗
PubMed1998

Nitrate assimilation by bacteria.

Nitrate is a significant nitrogen source for plants and microorganisms. Recent molecular genetic analyses of representative bacterial species have revealed structural and regulatory genes responsible for the nitrate-assimilation phenotype. Together with results from physiological and biochemical studies, this information has unveiled fundamental aspects of bacterial nitrate assimilation and provides the foundation for further investigations. Well-studied genera are: the cyanobacteria, including the unicellular Synechococcus and the filamentous Anabaena; the gamma-proteobacteria Klebsiella and Azotobacter; and a Gram-positive bacterium, Bacillus. Nitrate uptake in most of these groups seems to involve a periplasmic binding protein-dependent system that presumably is energized by ATP hydrolysis (ATP-binding cassette transporters). However, Bacillus may, like fungi and plants, utilize electrogenic uptake through a representative of the major facilitator superfamily of transport proteins. Nitrate reductase contains both molybdenum cofactor and an iron-sulfur cluster. Electron donors for the enzymes from cyanobacteria and Azotobacter are ferredoxin and flavodoxin, respectively, whereas the Klebsiella and Bacillus enzymes apparently accept electrons from a specific NAD(P)H-reducing subunit. These subunits share sequence similarity with the reductase components of bacterial aromatic ring-hydroxylating dehydrogenases such as toluene dioxygenase. Nitrite reductase contains sirohaem and an iron-sulfur cluster. The enzymes from cyanobacteria and plants use ferredoxin as the electron donor, whereas the larger enzymes from other bacteria and fungi contain FAD and NAD(P)H binding sites. Nevertheless, the two forms of nitrite reductase share recognizable sequence and structural similarity. Synthesis of nitrate assimilation enzymes and uptake systems is controlled by nitrogen limitation in all bacteria examined, but the relevant regulatory proteins exhibit considerable structural and mechanistic diversity in different bacterial groups. A second level of control, pathway-specific induction by nitrate and nitrite in Klebsiella, involves transcription antitermination. Several issues await further experimentation, including the mechanism and energetics of nitrate uptake, the pathway(s) for nitrite uptake, the nature of electron flow during nitrate reduction, and the action of transcriptional regulatory circuits. Fundamental knowledge of nitrate assimilation physiology should also enhance the study of nitrate metabolism in soil, water and other natural environments, a challenging topic of considerable interest and importance.

Bacteria↗
PubMed1998

Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro.

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

Bacterial Proteins↗
PubMed1998

NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al.

Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the delta nasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.

ATP-Binding Cassette Transporters↗
PubMed1997

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement.

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA-binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and NarP. In this study, DNase I footprinting experiments with the fdnG, nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7-2-7 sites). The NarL protein also binds to these 7-2-7 sites but, unlike NarP, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and NarP at common target operons, such as fdnG and nrfA. To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7-2-7 heptamers was increased (7-n-7 constructs). Increasing the spacing to four or more basepairs abolished NarP binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7-n-7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7-2-7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in NarP.

Bacterial Proteins↗
PubMed1997

Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12.

Anaerobic respiratory gene expression in Escherichia coli is differentially controlled by nitrate and nitrite through dual interacting two-component regulatory systems. The NarX sensor is one of two membrane-spanning sensor kinases that control the phosphorylation state of two DNA-binding response regulators. We have studied NarX autophosphorylation in crude membrane preparations from cells that overexpress NarX protein. The low basal autophosphorylation rate was stimulated about sixfold and threefold by nitrate and nitrite respectively. This demonstrates that nitrate and nitrite differentially activate NarX autokinase activity. We also isolated single-residue substitutions in NarX that affect its ability to respond to or discriminate between nitrate and nitrite. Most of these substitutions affect residues within the conserved P-box sequence in the periplasmic domain. We characterized several of the mutants in vivo, by monitoring ligand-regulated gene expression, and in vitro, by monitoring ligand-responsive autophosphorylation. At least one change, K491 (Lys at position 49 changed to Ile), resulted in a protein with greatly impaired ability to discriminate between nitrate and nitrite. Other changes (H45E and R59K) resulted in proteins that responded normally to nitrate but were unable to respond to nitrite. These results implicate the P-box region in discrimination between subtly different small molecules.

Bacterial Proteins↗
PubMed1997

Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases.

Within the CNS and under normal conditions, nitric oxide (.NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that .NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances .NO synthesis may be excessive and .NO may become neurotoxic. Excessive glutamate-receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both .NO and superoxide (O2.-) and hence peroxynitrite (ONOO-) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of .NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of .NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO- interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy-consuming pathway involving poly(ADP-ribose) synthetase. Possible mechanisms whereby ONOO- impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO- production. It is concluded that neurotoxicity elicited by excessive .NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.

Animals↗
PubMed1997

Nitrate- and nitrite-sensing protein NarX of Escherichia coli K-12: mutational analysis of the amino-terminal tail and first transmembrane segment.

Nitrate and nitrite control of anaerobic respiratory gene expression is mediated by dual two-component regulatory systems. The sensors NarX and NarQ each communicate nitrate and nitrite availability to the response regulators NarL and NarP. In the presence of nitrate, the NarX protein acts as a positive regulator ("kinase") of both NarL and NarP activity. In the presence of nitrite, the NarX protein acts primarily as a negative regulator ("phosphatase") of NarL activity but remains a positive regulator of NarP activity. In other topologically similar sensory proteins, such as the methyl-accepting chemotaxis proteins, the transmembrane regions are important for signal transduction. We therefore used localized mutagenesis of the amino-terminal coding region to isolate mutations in narX that confer an altered signaling phenotype. Five of the mutations studied alter residues in the amino-terminal cytoplasmic tail, and five alter residues in the first transmembrane segment. Based on patterns of target operon expression in various regulatory mutant strain backgrounds, most of the mutant NarX proteins appear to have alterations in negative control function. One mutant, with a change of residue Leu-11 to Pro in the cytoplasmic tail, exhibits strikingly altered patterns of NarL- and NarP-dependent gene expression. We conclude that the amino terminus of the NarX protein is important for the differential response to nitrate and nitrite.

Bacterial Proteins↗
PubMed1996

Nitrate and nitrite-mediated transcription antitermination control of nasF (nitrate assimilation) operon expression in Klebsiella pheumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Nitrate is converted through nitrite to ammonium by assimilatory nitrate and nitrite reductase, respectively. Enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon of K. pneumoniae; nasF operon expression is subject to both general nitrogen control and pathway-specific nitrate/nitrite induction, mediated by the NtrC and NasR proteins, respectively. Sequence inspection revealed a presumptive sigmaN (sigma54)-dependent promoter as well as two presumptive upstream NtrC protein binding sites. Site-specific mutational and primer extension analyses confirmed the identity of the sigmaN-dependent promoter. Deletions removing the apparent NtrC protein binding sites greatly reduced NtrC-dependent regulation, indicating that these sites are involved in general nitrogen control. However, deletions removing most of the sequence upstream of the promoter had little effect on nitrate/nitrite regulation, suggesting that the nasF leader region is involved in nitrate/nitrite regulation. The 119 nucleotide long transcribed leader region contains an apparent factor-independent transcription terminator. Promoter replacement experiments demonstrated that the leader region is involved in nitrate/nitrite regulation of nasF operon expression. Deletions removing the transcription terminator structure resulted in a nitrate-blind constitutive phenotype, indicating that the transcription terminator structure serves a negative function. Other deletions, removing proximal portions of the leader region, resulted in an uninducible phenotype, indicating that this region serves a positive function. These results indicate that nitrate/nitrite regulation of nasF operon expression is determined by a transcription attenuation mechanism. We hypothesize that in the absence of nitrate or nitrite, the terminator structure abrogates transcription readthrough into the nasF operon. In the presence of nitrate or nitrite, the NasR protein mediates transcription antitermination, thereby allowing transcription to proceed into the nasF operon.

Bacterial Proteins↗
PubMed1996

Analysis of nitrate regulatory protein NarL-binding sites in the fdnG and narG operon control regions of Escherichia coli K-12.

During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate. The fdnG operon control region contains four NarL-binding sites (termed NarL heptamers) between positions -70 and -130. The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL. We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL-dependent induction. Mutations were examined for their effects on NarL-dependent expression in vivo. Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect. For some positions, alterations in both heptamers had a stronger effect than either of the single changes. The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction. The narG operon control region has at least eight NarL heptamers arranged in two groups. Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions -195 and -89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL-DNA interactions in vitro. Substitutions in heptamers -185 and -101 affected narG operon induction only when the concentration of phospho-NarL was low (during growth in the presence of nitrite). Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL-DNA interactions in vitro.

Bacterial Proteins↗
PubMed1996

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

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 (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the phi (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 phi (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 2'2'-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 microM) stimulated aerobic phi (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.

Aerobiosis↗