RelA Protein Is Involved in Induction of Genetic Competence in CertainBacillus subtilisStrains by Moderating the Level of Intracellular GTP

Abstract

We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA ؉ ) strain. Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain. We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography. The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase). Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development. Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression. Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation.When Bacillus subtilis cells encounter adverse nutrient conditions, cells start to approach the stationary growth phase by initiating several development processes that contribute to its survival. Of these processes, genetic competence and sporulation have been studied in great detail. Genetic competence is a physiological state that enables cells to incorporate exogenous DNA, which is then incorporated into the endogenous DNA by crossing over. Competence development is tightly controlled by a complex regulatory network involving kinases, phosphatases, and transcriptional regulators (4). The most critical regulator is a transcriptional factor named ComK, which has been shown to be necessary for inducing the late competence genes such as comG (27,28). It has been demonstrated that the cellular level of ComK is precisely controlled by several regulatory proteins at the transcriptional and posttranscriptional levels (2,5,6,25,26). During the exponential growth phase, ComK activity is inhibited by a complex of two proteins, MecA and ClpC (10, 26). The ComS protein, which is encoded by the srfA operon and is synthesized in response to quorum-sensing oligopeptide pheromones (13, 23), releases cells from ComK inhibition by dissociating the ternary complex with MecA and ClpC (25). In addition, Serror and Sonenshein (21) demonstrated that CodY, which is a global regulator for stationary-phase genes, directly binds to the srfA and comK promoter region. Thus, it is highly likely that CodY acts as a negative regulator for competence genes.One of the most important adaptation s...

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp

“…(Inaoka & Ochi, 2002;Okamoto & Ochi, 1998). Sporulation and competence development of B. subtilis are controlled directly by the GTP level through the GTP-binding protein CodY (Inaoka & Ochi, 2002;Ratnayake-Lecamwasam et al, 2001). The GTP level is closely linked to ppGpp synthesis because (1) ppGpp synthesis consumes GTP as a substrate, and (2) ppGpp potently blocks GTP synthesis by inhibiting IMP dehydrogenase activity.…”

“…In this regard, it should be noted that Rel-mediated effects may also involve indirect effects of ppGpp through the modulation of GTP levels, as reported for B. subtilis and Streptomyces sp. (Inaoka & Ochi, 2002;Okamoto & Ochi, 1998). Sporulation and competence development of B. subtilis are controlled directly by the GTP level through the GTP-binding protein CodY (Inaoka & Ochi, 2002;Ratnayake-Lecamwasam et al, 2001).…”

“…In bacteria, ppGpp markedly inhibits the activity of IMP dehydrogenase, leading to a decrease in GTP pool size (Cashel et al, 1996;Kasai et al, 2006;Ochi, 1987b), and the decrease in GTP has various important biological consequences (Inaoka & Ochi, 2002;Inaoka et al, 2003;Okamoto & Ochi, 1998). In mammalian cells, IMP dehydrogenase is a rate-limiting enzyme involved in de novo GTP biosynthesis.…”

“…In Bacillus subtilis, the stringent response is directly elicited by an abrupt decrease in GTP level. Some physiological processes in B. subtilis, such as sporulation, competence and suppression of rRNA synthesis, result from indirect functions of ppGpp, by which ppGpp potently inhibits inosine monophosphate (IMP) dehydrogenase, leading to a marked decrease in size of the GTP pool (Inaoka & Ochi, 2002;Krásný & Gourse, 2004;Ratnayake-Lecamwasam et al, 2001). …”

“…Importantly, despite the wide or ubiquitous distribution of relA or RSH in bacteria and plants, neither relA and RSH genes nor ppGpp per se have yet been identified in animals or eukaryotic micro-organisms such as yeasts and fungi. Although the mechanism of ppGpp synthesis is rather poorly understood Tozawa & Nomura, 2011;Tozawa et al, 2007), there is accumulating evidence regarding the function of ppGpp in reorienting cellular metabolism and its physiological consequences, including research into sporulation (Balzer & McLean, 2002;Lemos et al, 2004;Ochi et al, 1981), competence (Inaoka & Ochi, 2002), fruiting body formation (Harris et al, 1998), antibiotic production (Bibb, 2005;Hesketh et al, 2001;Hoyt & Jones, 1999;Inaoka et al, 2003;Ochi, 1987aOchi, , 2007Sun et al, 2001), development of persistence (Dahl et al, 2003;Korch et al, 2003), quorum sensing (Baysse et al, 2005;Harris et al, 1998;van Delden et al, 2001), biofilm formation (Balzer & McLean, 2002), pathogenesis (Erickson et al, 2004;Gaynor et al, 2005;Godfrey et al, 2002;Haralalka et al, 2003;Pizarro-Cerdá & Tedin, 2004;Song et al, 2004) and symbiosis (Moris et al, 2005;Wells & Long, 2002;Zhang et al, 2004). The RNA polymerase is the primary target for ppGpp, as confirmed recently by X-ray crystallographic analysis of an RNA polymerase-ppGpp complex (Artsimovitch et al, 2004;Chatterji et al, 1998;Sato et al, 2009).…”