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.

Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a "rebound" of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.It is increasingly recognized that the relative concentrations of nucleotides play important roles in prokaryotic cell regulation. The classic example of this is the stringent response in which a hyperphosphorylated guanosine, ppGpp, is produced in response to stalled ribosomes in amino acid-starved cells (8,9,11). ppGpp is produced even under nutrient-rich growth conditions, however, and its presence probably ensures transcriptional balance for different promoters under a variety of physiological states (13,24,26). It is well accepted now that ppGpp is the primary, although not the exclusive, determinant of growth rate dependence: the observation that the amount of rRNA produced in Escherichia coli is proportional to the growth rate.In addition to alarmones such as ppGpp, the relative concentrations of even standard nucleotides such as ATP and GTP affect bacterial physiology. One theory suggests the concentration of initiating nucleotide (iNTP) for rRNA promoters changes under different growth conditions, which in turn affects the transcriptional rate from these promoters (15). Although it appears under different growth rates the concentration of ATP, the iNTP for rRNA promoter P1, does not change, it has been observed that the ATP concentration does decrease as cells enter stationary phase (21,24). Similarly, Fis, a nucleoid-associated protein that affects transcription, is controlled at the promoter level by concentrations of its iNTP, CTP (32). The ratio of the nucleotide triphosphates (NTPs) to diphosphates also has regulatory consequences. In Bacillus subtilis, the ratio of GTP to GDP plays a critical role in the ...

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...