The relA Homolog of Mycobacterium smegmatis Affects Cell Appearance, Viability, and Gene Expression

Abstract

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel Msm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.The ability of Mycobacterium tuberculosis to persist in the human host is a major challenge for both vaccine-and drugbased strategies for controlling the spread of tuberculosis (TB) (17). Persistent M. tuberculosis cells are capable of initiating active growth in the host, a condition known as reactivation TB. Although it is somewhat controversial, it is generally believed that the site of viable M. tuberculosis in the host is inside caseous, necrotic, granulomatous lesions in the lungs. It has been reported that M. tuberculosis cells persisting inside granulomas lack the acid-fast staining characteristic of bacteria recovered from sputa and lesions of patents with active disease, indicating that changes occur in the bacterial cell wall during metabolic adaptation to a state of dormancy in the host (18,22). Various models have been developed to create in vitro growth conditions that mimic the presumed state of M. tuberculosis inside granulomas, including oxygen limitation (32, 33) and nutrient starvation (1,20). For example, when M. tuberculosis cultures are suspended in distilled water, they appear to lose their acid-fast staining ability, but the cells can remain viable for over 2 years in this extreme nutrient-deprived environment (22). This implies that in vitro observations of mycobacterial physiology may provide important insights into understanding how pathogenic mycobacteria survive in hosts. In addition, several mutants of M. tuberculosis that are impaired in de novo biosynthesis of various amino acids and vitamins (14) are attenuated in mouse models of tuberculosis infection, suggesting that the organism resides in a nutrient-poor environment. This supports the significance of in vitro nutrient starvation models for understanding in vivo persistence.Recently, the stringent response of M. tuberculosis has been shown to play an important role in the in vitro and in vivo survival of this bacterium (9, 29). Escherichia coli has two homologous genes that are responsible for initiating the stringent response, namely, relA and spoT. Collectively, RelA and SpoT can sense nutrient deprivation and respond by synthesizing guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which can alter the promoter specificity of RNA polymeras...

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

“…The biological function of Eis in Mtb has been the subject of recent interest (6,7,(22)(23)(24)(25)(26)(27)(28)(29). The biochemical and structural studies described in this report provide clear evidence for an unprecedented multiacetylation capability of Eis that inactivates AG antibiotics.…”

Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis

“…Under such stressful conditions, bacteria survive by producing spores, fruiting bodies, etc. However, in the case of mycobacteria, altered colony morphology and different staining properties have been observed in conditions of nutritional starvation (Nyka, 1974;Dahl et al, 2005). During stationary phase, M. smegmatis forms clumps that increase in size during prolonged starvation, and these may promote survival (Smeulders et al, 1999).…”

“…M. smegmatis, a non-pathogenic fast-growing species, has been widely accepted as an excellent model system for studying various aspects of M. tuberculosis biology (Jacobs et al, 1987;Smeulders et al, 1999;Ojha et al, 2000;Dahl et al, 2005). To understand the physiological role of c-di-GMP in mycobacteria, we cloned, overexpressed and knocked out the GGDEF-EAL domain protein of M. smegmatis.…”

Bacterial Growth and Cell Division: a Mycobacterial Perspective

“…tuberculosis incubated under many of these conditions, as well as M. tuberculosis isolated from infections, has thickened cell walls, an altered morphology (86), and duplicated chromosomes and stops replicating (427,429,436,438). Persistent M. tuberculosis is thought to arrest its growth after completing chromosomal replication (437).…”

“…A relA-null strain of M. smegmatis forms clumpy, elevated colonies that have less pigment than the wild type but no detectable cell wall change or defect in growth (86). However, the mutant bacteria were longer and multiseptated, and some had a pear-like shape (86).…”