Long-term survival of nonreplicating

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel Mtb ) and responds to nutrient starvation by producing (p)ppGpp. A rel Mtb knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel Mtb mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel Mtb -independent manner. In vitro growth in liquid media represents a condition that benefits from Rel Mtb -mediated adaptation. Long-term survival of the rel Mtb mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anerobic incubation than the wild-type virulent organism. Thus, the Rel Mtb protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. Mycobacterium tuberculosis ͉ respiratory chainT he World Health Organization estimates that two billion people are infected with Mycobacterium tuberculosis (Mtb), and two million people die of the disease each year (1). Most individuals infected with the organism are latent carriers who have a 2-23% lifetime risk of developing reactivation tuberculosis (TB). The risk dramatically increases if the carrier's immune system is suppressed. Also, drug resistance is a serious concern; the isoniazid (INH)-resistance rate is Ϸ10%, and the rifampicin (RIF) resistance rate is Ϸ1%, with lower numbers in countries with effective TB programs and higher numbers in countries with deficient TB programs. The World Health Organization declared TB infections to be a global public health emergency (1), and the need to identify targets for antimicrobial therapy remains urgent.Mtb is capable of establishing persistent infection in the host by using a complex interplay between the host immune system and bacterial survival mechanisms. In the persistent infection, Mtb adapt to depletion of available oxygen and nutrients and enter a stage of nonreplicating persistence (NRP) in granulomatous or necrotic lesions. NRP Mtb are resistant to INH, ethambutol, and RIF, but they become sensitive to metronidazole in vitro (2). Given the critical role of oxygen in the generation of cellular energy and bacterial long-term survival, there is surprisingly little information on oxidative phosphorylation in Mtb. Clearly, oxidative phosphorylation is a central component in the production of ATP and the subsequent growth and pathogenesis of Mtb. Here, we characterize the aerobic respiratory pathway and show that NADH:menaquinone oxidoreductase (Ndh) is a key target for TB agents. Materials and MethodsMedia and Strains. Mtb H 37 R v was a gift from C. Imperatrice (Clinical Infectious Diseases, Hospital of the University of Pennsylvania) and Mycobacterium smegmatis Mc 2 155 was obtained from V. Mizrahi (National Health Laboratory Service, Johannesburg). Bacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose catalase͞0.5% glycerol͞0.05% Tween 80. Solid agar (15 g͞liter) was ad...

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the ␥-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 ؋ 10 2 Pa of pO 2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 ؋ 10 2 Pa of pO 2 or 0.5 to 1% air saturation). These observations were in accordance with the two-to threefold increase in cydAB gene expression observed upon reduction of the pO 2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

Rel(Mtb) of Mycobacterium tuberculosis is responsible for the intracellular regulation of (p)ppGpp and the consequent ability of the organism to survive long-term starvation, indicating a possible role in the pathogenesis of tuberculosis. Purified Rel(Mtb) is a dual-function enzyme carrying out ATP: GTP/GDP/ITP 3'-pyrophosphoryltransferase and (p)ppGpp 3'-pyrophosphohydrolase reactions. Here we show that in the absence of biological regulators, Rel(Mtb) simultaneously catalyzes both transferase and hydrolysis at the maximal rate for each reaction, indicating the existence of two distinct active sites. The differential regulation of the opposing activities of Rel(Mtb) is dependent on the ratio of uncharged to charged tRNA and the association of Rel(Mtb) with a complex containing tRNA, ribosomes, and mRNA. A 20-fold increase in the k(cat) and a 4-fold decrease in K(ATP) and K(GTP) from basal levels for transferase activity occur when Rel(Mtb) binds to a complex containing uncharged tRNA, ribosomes, and mRNA (Rel(Mtb) activating complex or RAC). The k(cat) for hydrolysis, however, is reduced 2-fold and K(m) for pppGpp increased 2-fold from basal levels in the presence of the Rel(Mtb) activating complex. The addition of charged tRNA to this complex has the opposite effect by inhibiting transferase activity and activating hydrolysis activity. Differential control of Rel(Mtb) gives the Mtb ribosomal complex a new regulatory role in controlling cellular metabolism in response to stringent growth conditions that may be present in the dormant Mtb lesion.

No abstract

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.Mycobacterium tuberculosis is an aerobic, gram-positive bacterium containing a high-GϩC-content genome of approximately 4 ϫ 10 6 bp with a relatively long mean generation time of 24 h and a chromosomal replication time (C period) of 11 h (13). M. tuberculosis can remain in a dormant, nonreplicating state in the human host for years and can be reactivated to cause disease under certain immunological and/or environmental conditions (38). The molecular mechanisms that govern these growth patterns and the regulation of DNA synthesis during these growth phases are now the focus of several lines of investigation. DNA polymerase I (14), topoisomerase I (42), and gyrA and gyrB (21, 33) from M. tuberculosis have been cloned and sequenced. In addition, the origins of replication of M. tuberculosis, M. smegmatis, and M. leprae have been analyzed and shown to contain a typical gram-positive pattern of genes, rnpA-rpmH-dnaA-dnaN-recF-gyrB-gyrA (28). Recently, ribonucleotide reductase (RR), potentially the rate-limiting step in DNA replication, was purified from M. tuberculosis, and the gene encoding the large subunit (R1) was isolated, sequenced, and expressed in Escherichia coli (41).The enzymatic reduction of ribonucleotides by RR regulates the deoxynucleotide precursor pool for DNA synthesis in a wide variety of bacteria and ...