Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice

Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid-fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.     

Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12.

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 HindIII 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 phi (narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent of fnr. Expression of phi (narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A phi (narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ and fnr+. 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.     

Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum.

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed. Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.     

Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12.

In Escherichia coli, aerobiosis inhibits the synthesis of enzymes for anaerobic respiration (e.g., nitrate reductase and fumarate reductase) and for fermentation (e.g., formate-hydrogen lyase). Anaerobically, nitrate induces nitrate reductase synthesis and inhibits the formation of both fumarate reductase and formate-hydrogen lyase. Previous work has shown that narL+ is required for the effects of nitrate on synthesis of both nitrate reductase and fumarate reductase. Another gene, narK (whose function is unknown), has no observable effect on formation of these enzymes. We report here our studies on the role of nar genes in fumarate reductase and formate-hydrogen lyase gene expression. We observed that insertions in narX (also of unknown function) significantly relieved nitrate inhibition of fumarate reductase gene expression. This phenotype was distinct from that of narL insertions, which abolished this nitrate effect under certain growth conditions. In contrast, insertion mutations in narK and narGHJI (the structural genes for the nitrate reductase enzyme complex) significantly relieved nitrate inhibition of formate-hydrogen lyase gene expression. Insertions in narL had a lesser effect, and insertions in narX had no effect. We conclude that nitrate affects formate-hydrogen lyase synthesis by a pathway distinct from that for nitrate reductase and fumarate reductase.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

The napF and narG Nitrate Reductase Operons in Escherichia coli Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite

Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The napF operon apparently encodes a "low-substrate-induced" reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a "high-substrate-induced" operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered.     

Nitrate enhances the survival of Mycobacterium tuberculosis during inhibition of respiration

When oxygen is slowly depleted from growing cultures of Mycobacterium tuberculosis, they enter a state of nonreplicating persistence that resembles the dormant state seen with latent tuberculosis. In this hypoxic state, nitrate reductase activity is strongly induced. Nitrate in the medium had no effect on long-term persistence during gradual oxygen depletion (Wayne model) for up to 46 days, but significantly enhanced survival during sudden anaerobiosis. This enhancement required a functional nitrate reductase. Thioridazine is a member of the class of phenothiazines that act, in part, by inhibiting respiration. Thioridazine was toxic to both actively growing and nonreplicating cultures of M. tuberculosis. At a sublethal concentration of thioridazine, nitrate in the medium improved the growth. At lethal concentrations of thioridazine, nitrate increased survival during aerobic incubation as well as in microaerobic cultures that had just entered nonreplicating persistence (NRP-1). In contrast, the survival of anaerobic persistent (NRP-2) cultures exposed to thioridazine was not increased by the addition of nitrate. Nitrate reduction is proposed to play a role during the sudden interruption of aerobic respiration due to causes such as hypoxia, thioridazine, or nitric oxide.     

A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Assimilatory nitrate and nitrite reductases convert nitrate through nitrite to ammonium. We report here the molecular cloning of the nasA and nasB genes, which encode assimilatory nitrate and nitrite reductase, respectively. These genes are tightly linked and probably form a nasBA operon. In vivo protein expression and DNA sequence analysis revealed that the nasA and nasB genes encode 92- and 104-kDa proteins, respectively. The NASA polypeptide is homologous to other prokaryotic molybdoenzymes, and the NASB polypeptide is homologous to eukaryotic and prokaryotic NADH-nitrite reductases. The narL gene product positively regulates expression of the structural genes for respiratory nitrate reductase, narGHJI. Surprisingly, we found that the nasBA operon is tightly linked to the narL-narGHJI region in K. pneumoniae, even though the nitrate assimilatory and respiratory enzymes serve different physiological functions.