Deciphering the Interplay between Two Independent Functions of the Small RNA Regulator SgrS in Salmonella

Abstract

Bacterial dual-function small RNAs regulate gene expression by RNA-RNA base pairing and also code for small proteins. SgrS is a dual-function small RNA in Escherichia coli and Salmonella that is expressed under stress conditions associated with accumulation of sugar-phosphates, and its activity is crucial for growth during stress. The base-pairing function of SgrS regulates a number of mRNA targets, resulting in reduced uptake and enhanced efflux of sugars. SgrS also encodes the SgrT protein, which reduces sugar uptake by a mechanism that is independent of base pairing. While SgrS base-pairing activity has been characterized in detail, little is known about how base pairing and translation of sgrT are coordinated. In the current study, we utilized a series of mutants to determine how translation of sgrT affected the efficiency of base pairing-dependent regulation and vice versa. Mutations that abrogated sgrT translation had minimal effects on base-pairing activity. Conversely, mutations that impaired base-pairing interactions resulted in increased SgrT production. Furthermore, while ectopic overexpression of sgrS mutant alleles lacking only one of the two functions rescued cell growth under stress conditions, the SgrS base-pairing function alone was indispensable for growth rescue when alleles were expressed from the native locus. Collectively, the results suggest that during stress, repression of sugar transporter synthesis via base pairing with sugar transporter mRNAs is the first priority of SgrS. Subsequently, SgrT is made and acts on preexisting transporters. The combined action of these two functions produces an effective stress response. In the past decade, a vast number of small RNAs (sRNAs) have been identified as important posttranscriptional regulators. In bacteria, sRNAs typically do not encode proteins. Rather, they regulate gene expression by binding to target mRNAs via basepairing interactions, resulting in enhanced (positive regulation) or reduced (negative regulation) translation. With the identification of new sRNAs, it is increasingly clear that some sRNAs, dubbed dual-function sRNAs, not only act as canonical sRNAs by regulating targets via a base-pairing mechanism but also encode regulatory proteins (1). While the noncoding, base-pairing sRNA mechanisms have been studied extensively (reviewed in references 2 and 3), much remains to be learned about the dual-function sRNA regulators. For example, the molecular functions of the proteins encoded by bifunctional sRNAs and the roles of these small proteins in cellular physiology are not well understood. Moreover, the interplay between translation and base-pairing activity has not been characterized for any dual-function sRNA. In this study, we use a dual-function sRNA from Escherichia coli and Salmonella as a model for understanding how a single RNA can serve two mechanistically distinct purposes.SgrS is a bifunctional sRNA expressed during a specific metabolic stress condition called glucose-phosphate stress (4-6). This stress causes growth inhi...

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System

“…The base-pairing activity of SgrS ameliorates glucose-phosphate stress by inhibiting translation and promoting degradation of the ptsG and manXYZ mRNA transcripts, thereby preventing synthesis of more sugar transporters during stress. In contrast, SgrT was shown to have no effect on sugar transporter mRNA levels, but it could still reverse growth inhibition caused by stress (9,22). These and other results suggested that SgrT acted by inhibiting glucose transporter activity (9).…”

“…Like many other sRNAs in bacteria, SgrS carries out base-pairing-dependent regulation of numerous mRNAs (4,(17)(18)(19)(20)(21). Less typical is the second function of SgrS, namely, encoding the 43-amino-acid protein SgrT (9,22). Unlike any other known dual-function sRNA-small protein pair, SgrS and SgrT act independently in the same pathway, albeit by different mechanisms (9,22).…”

“…Less typical is the second function of SgrS, namely, encoding the 43-amino-acid protein SgrT (9,22). Unlike any other known dual-function sRNA-small protein pair, SgrS and SgrT act independently in the same pathway, albeit by different mechanisms (9,22). The base-pairing activity of SgrS ameliorates glucose-phosphate stress by inhibiting translation and promoting degradation of the ptsG and manXYZ mRNA transcripts, thereby preventing synthesis of more sugar transporters during stress.…”

“…SgrT and SgrS base-pairing functions also act at different times during glucose-phosphate stress. The base-pairing function acts immediately, whereas SgrT is not detected until ϳ30 min following the onset of stress (22). The goal of the present study was to examine the target specificity and physiological roles of SgrT in the glucose-phosphate stress response.…”

2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli

“…Growth stress caused by sugar-phosphate build-up is minimized by induction of the small regulatory RNA SgrS; aMG at 0.1 % was shown to induce sgrS 126-fold while the same concentration of 2DG induced just 36-fold (Rice & Vanderpool, 2011). SgrS-dependent reduction of the level of manXYZ mRNA also takes longer than reduction of ptsG mRNA (Balasubramanian & Vanderpool, 2013). Inability of the cells to prevent 2DG-phosphate build-up as effectively as build-up of aMG-phosphate could account for 2DG being a stronger inhibitor of biofilm growth than aMG.…”