Discovery and dissection of metabolic oscillations in the microaerobic nitric oxide response network of Escherichia coli.

The virulence of many pathogens depends upon their ability to cope with immune-generated nitric oxide (NO·). In Escherichia coli, the major NO· detoxification systems are Hmp, an NO· dioxygenase (NOD), and NorV, an NO· reductase (NOR). It is well established that Hmp is the dominant system under aerobic conditions, whereas NorV dominates anaerobic conditions; however, the quantitative contributions of these systems under the physiologically relevant microaerobic regime remain ill defined. Here, we investigated NO· detoxification in environments ranging from 0 to 50 µM O2, and discovered a regime in which E. coli NO· defenses were severely compromised, as well as conditions that exhibited oscillations in the concentration of NO·. Using an integrated computational and experimental approach, E. coli NO· detoxification was found to be extremely impaired at low O2 due to a combination of its inhibitory effects on NorV, Hmp, and translational activities, whereas oscillations were found to result from a kinetic competition for O2 between Hmp and respiratory cytochromes. Because at least 777 different bacterial species contain the genetic requirements of this stress response oscillator, we hypothesize that such oscillatory behavior could be a widespread phenomenon. In support of this hypothesis,Pseudomonas aeruginosa, whose respiratory and NO· response networks differ considerably from those of E. coli, was found to exhibit analogous oscillations in low O2 environments. This work provides insight into how bacterial NO· defenses function under the low O2 conditions that are likely to be encountered within host environments.

Protection from nitrosative stress: a central role for microbial flavohemoglobin.

Nitric oxide (NO) is an inevitable product of life in an oxygen- and nitrogen-rich environment. This reactive diatomic molecule exhibits microbial cytotoxicity, in large part by facilitating nitrosative stress and inhibiting heme-containing proteins within the aerobic respiratory chain. Metabolism of NO is therefore essential for microbial life. In many bacteria, fungi, and protozoa, the evolutionarily ancient flavohemoglobin (flavoHb) converts NO and O(2) to inert nitrate (NO(3)(-)) and undergoes catalytic regeneration via flavin-dependent reduction. Since its identification, widespread efforts have characterized roles for flavoHb in microbial nitrosative stress protection. Subsequent genomic studies focused on flavoHb have elucidated the transcriptional machinery necessary for inducible NO protection, such as NsrR in Escherichia coli, as well as additional proteins that constitute a nitrosative stress protection program. As an alternative strategy, flavoHb has been heterologously employed in higher eukaryotic organisms such as plants and human tumors to probe the function(s) of endogenous NO signaling. Such an approach may also provide a therapeutic route to in vivo NO depletion. Here we focus on the molecular features of flavoHb, the hitherto characterized NO-sensitive transcriptional machinery responsible for its induction, the roles of flavoHb in resisting mammalian host defense systems, and heterologous applications of flavoHb in plant/mammalian systems (including human tumors), as well as unresolved questions surrounding this paradigmatic NO-consuming enzyme.

The hmp gene encoding the NO-inducible flavohaemoglobin in Escherichia coli confers a protective advantage in resisting killing within macrophages, but not in vitro: links with swarming motility.

Escherichia coli flavohaemoglobin (Hmp) is the best-understood nitric oxide (NO) detoxifying protein and exhibits a robust dioxygenase activity, converting NO to nitrate ion with oxygen as co-substrate. Synthesis of Hmp via transcriptional regulation of hmp gene expression is an adaptive response to NO and related nitrosative stresses since Hmp levels are greatly elevated on exposure in vitro to these agents. Here we show that expression of hmp is greatly enhanced by NO but not by other haem ligands (azide, cyanide and carbon monoxide). Flavohaemoglobins of other pathogenic bacteria have been implicated in conferring resistance to NO in vitro and in macrophage-like cells but the role of the E. coli flavohaemoglobin has not been studied in macrophages. We therefore compared survival of wild-type K-12 E. coli cells and an isogenic hmp mutant after internalisation by human macrophages. Wild-type bacteria survived significantly better than the hmp mutant after incubation with macrophages, despite binding and internalisation rates being similar for both strains. Unexpectedly, however, when grown in MOPS minimal medium, in mixed cultures, more hmp mutant cells were recovered than wild-type. Significantly, an hmp mutant failed to exhibit swarming motility on soft agar and this phenotype was rescued by a plasmid-borne copy of the wild-type hmp(+) gene. Thus, although Hmp constitutes an important mechanism of protection from NO-mediated killing by human macrophages in the model E. coli strain K-12, and probably contributes to the survival of enteropathogenic E. coli during the intestinal inflammatory response, synthesis of Hmp in vitro may represent a selective disadvantage. The lack of swarming motility of the hmp mutant and its aflagellate state suggest that Hmp synthesis is a metabolic burden in the absence of NO-related stresses.

Multiple regulators of the Flavohaemoglobin (hmp) gene of Salmonella enterica serovar Typhimurium include RamA, a transcriptional regulator conferring the multidrug resistance phenotype.

Microbial flavohaemoglobins are proteins with homology to haemoglobins from higher organisms, but clearly linked to nitric oxide (NO) metabolism by bacteria and yeast. hmp mutant strains of several bacteria are hypersensitive to NO and related compounds and hmp genes are up-regulated by the presence of NO. The regulatory mechanisms involved in hmp induction by NO and the superoxide-generating agent, methyl viologen (paraquat; PQ), are complex, but progressively being resolved. Here we show for the first time that, in Salmonella enterica serovar Typhimurium, hmp transcription is increased on exposure to PQ and demonstrate that RamA, a homologue of MarA is responsible for most of the hmp paraquat regulation. In addition we demonstrate NO-dependent elevation of Salmonella hmp transcription and Hmp accumulation. In both Escherichia coli and Salmonella modest transcriptional repression of hmp is exerted by the iron responsive transcriptional repressor Fur. Finally, in contrast to previous reports, we show that in E. coli and Salmonella, hmp induction by both paraquat and sodium nitroprusside is further elevated in a fur mutant background, indicating that additional regulators are implicated in this control process.

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Uropathogenic Escherichia coli and tolerance to nitric oxide: the role of flavohemoglobin.

PURPOSE: NO has an important role as part of the innate host response against bacterial infections. Flavohemoglobin, which is encoded by the hmp gene, protects Escherichia coli against nitrosative stress. We compared the NO tolerance of UPEC and nonpathogenic strains, and examined the involvement of flavohemoglobin. MATERIALS AND METHODS: The E. coli K12 derivates HB101 and DH5alpha represent nonpathogenic strains, while J96 and IA2 represent UPEC strains. HB101 was used as the host for a pBR322 plasmid carrying the hmp gene. Bacterial tolerance to NO was evaluated by determining cfu. Flavohemoglobin expression was examined using Northern and Western blot analysis. RESULTS: In the stationary growth phase, J96 was significantly more tolerant to DETA/NO (Alexis Biochemical, Lausen, Switzerland) (1 mM) compared to HB101 (47% +/- 11% vs 6.4% +/- 3.1% cfu). In the exponential growth phase DETA/NO exposure resulted in 98% +/- 4.6% cfu for J96 and 74% +/- 7.6% cfu for IA2 compared to 15% +/- 5.9% for HB101 and 21% +/- 12% for DH5alpha. HB101 over expressing hmp showed increased tolerance to DETA/NO (0.5 mM) compared to WT HB101 (106% +/- 5.6% vs 67 +/- 6.2%, p <0.01). Northern and Western blot analysis demonstrated increased flavohemoglobin expression after DETA/NO exposure and the strongest expression in HB101 carrying hmp on a multicopy plasmid. CONCLUSIONS: UPEC strains were significantly more tolerant to DETA/NO than nonpathogenic strains, which suggests a correlation between virulence and NO tolerance. Flavohemoglobin expression increased after DETA/NO exposure in UPEC and in nonpathogenic strains.

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus.

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

Flavohemoglobin Hmp, but not its individual domains, confers protection from respiratory inhibition by nitric oxide in Escherichia coli.

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.