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Nagarajan, S. N.

Publications and source records attributed to Nagarajan, S. N..

2 recordsLinked to original sources

(p)ppGpp-dependent activation of gene expression during nutrient limitation

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to stasis or quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that leads to lower cellular energy consumption. Examples of such changes include attenuated transcription of genes encoding ribosome components, in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. In addition, genes encoding proteins that facilitate survival nutrient limitation exhibit increased expression. An example is the hpf gene, encoding a broadly conserved protein responsible for protecting the ribosome from degradation under conditions limiting for ribosome synthesis. Here we show that (p)ppGpp plays a key role in the transcriptional activation of hpf as B. subtilis cells exit rapid growth. Specifically, we demonstrate that hpf transcription during nutrient limitation requires an RNA polymerase holoenzyme containing the alternative sigma factor {sigma}H, encoded by sigH, whose expression is normally inhibited by the AbrB repressor. However, when global protein synthesis decreases, in part dependent on (p)ppGpp, AbrB levels fall, leading to increased sigH transcription and, consequently, hpf activation. This mechanism couples a key physiological consequence of nutrient limitation - reduced protein synthesis - with specific gene activation, thereby linking transcriptional and translational regulation. Finally, we demonstrate that (p)ppGpp is necessary for the gene expression underlying the elaboration of developmental fates including sporulation and genetic competence. ImportanceBacteria often experience nutrient limitation and, in response, they attenuate energetically costly metabolic processes like protein synthesis. At the same time, however, they stimulate the expression of a subset of proteins that facilitate survival under this condition. This study identifies a new molecular mechanism in the model Gram-positive bacterium Bacillus subtilis responsible for gene expression in response to nutrient limitation that couples reduced global protein synthesis with increased transcription of specific genes. This mechanism mediates the elaboration of developmental fates including sporulation and genetic competence that are known responses to nutrient limitation in this organism.

microbiology↗

Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to the non-replicative state of quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that is in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. We have developed a novel reporter of (p)ppGpp abundance in the Gram-positive bacterium Bacillus subtilis based on the recent identification of a riboswitch that binds (p)ppGpp and modulates transcription via regulation of a transcriptional terminator. Placement of an unstable reporter, firefly luciferase, downstream of the riboswitch allows for sensitive and dynamic assessment of (p)ppGpp. We first confirm that the reporter accurately reflects (p)ppGpp abundance in a variety of well-established conditions. We then proceed to use it to demonstrate the physiological importance of several mechanisms of regulation of (p)ppGpp metabolism previously observed only in vitro including allosteric interactions between (p)ppGpp synthesis enzymes and the hydrolytic activity of a (p)ppGpp synthase. (p)ppGpp signaling has been implicated in the regulation of gene expression, and we demonstrate a close temporal association between gene expression and (p)ppGpp abundance, indicating a rapid, and therefore likely direct mechanism of (p)ppGpp dependent gene activation. Thus, this reporter provides a new, comprehensive analysis of (p)ppGpp signaling in vivo and offers the potential ability to sensitively monitor the temporal dynamics of (p)ppGpp abundance under diverse environmental conditions. Author SummaryMost bacteria adapt to stressful conditions such as nutrient limitation by synthesizing a signaling molecule, known as ppGpp, that consists of a hyper-phosphorylated GTP. Synthesis of ppGpp affects most aspects of cellular physiology including replication, transcription and translation. We present here a method that for the first time allows measurement of ppGpp abundance in living cells, greatly facilitating investigation into ppGpp metabolism.

microbiology↗