Molecular dissection of the mycobacterial stringent response protein Rel

Abstract

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel Msm is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Fulllength Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.Keywords: Rel protein; stringent response; ppGpp; multimerization; Rel domains Mycobacterium tuberculosis can be categorized as one of the most successful among human pathogens as several decades of research have not yet been able to completely eradicate tuberculosis (TB), the deadly disease caused by this organism. The major barrier toward complete cure from mycobacterial infection is the unique feature, termed ''latency,'' that these bacteria undergo on infection, leading to overexpression of genes that enable the survival of the pathogen within host organisms under oxygen-(Wayne and Hayes 1996) and nutrient-deprived (Nyka 1974) conditions. Such latent bacteria have been known to be confined in calcified lesions, termed ''granulomas,'' which enable the dormant bacteria to resist conventional antibiotics used against active bacilli. It had been proposed that the morphology and hydrophobicity of the in vivo persistors can be mimicked in laboratory cultures by starving bacteria in vitro (Nyka 1974). Under such stress conditions, adaptation to the Reprint requests to: Dipankar Chatterji, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; e-mail: dipankar@mbu.iisc.ernet.in; fax: +91-80-23600535.Abbreviations: pppGpp, guanosine 39-diphosphate 59-triphosphate; ppGpp, guanosine 39, 59-bis(diphosphate); IPTG, isopropyl b-D-1-thiogalactopyranoside; Ni-NTA, nickel-nitr...

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

“…In Gram-negative organisms, two proteins, RelA and SpoT, control the synthesis and degradation of ppGpp, respectively, and maintain the level of ppGpp, which interacts with RNA polymerase, and downregulates rRNA synthesis under conditions of nutritional starvation (Cashel et al, 1996). Apparently, the protein Rel, which has RelA and SpoT in tandem, and a regulatory domain at its C-terminal, manifests the intradomain interaction into an opposing catalytic activity as a function of environmental conditions (Jain et al, 2006b). However, in the case of MSDGC-1, in order to investigate the interdependence of the domains, we separated and functionally identified each of the individual domains.…”

“…The resulting plasmids pET DGC_2196, pET DGC_2774 and pET DGC_2774 Nter were transformed into E. coli BL21 (DE3) for protein expression. Protein purification was done as described (Jain et al, 2006b lysis buffer containing 50 mM Tris/Cl (pH 7.9), 500 mM NaCl and 1 mM PMSF. After centrifugation, the lysate was loaded onto an Ni-NTA column, and washed with 100 column vols wash buffer containing 10 mM imidazole.…”

Single-molecule investigations of the stringent response machinery in living bacterial cells

“…Recently it became possible to clone and overexpress RelA from several bacterial species (7)(8)(9), reigniting interest in the stringent response. Using overexpressed Escherichia coli RelA, the most extensive in vitro analysis to date of the mechanism was undertaken in 2002 (10), arriving at the so-called hopping model.…”

Molecular Dissection of Phage Endolysin

“…Plasmid purification, restriction digestion, ligation, and transformation were carried out as described (27). Site-directed mutagenesis was carried out following the single primer method (28,29). The gp10 gene was PCR-amplified using a set of primers and D29 genomic DNA as template.…”