The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

In my laboratory we are interested in understanding how bacteria sense stressful environmental conditions and modulate gene expression in a way that allows them to adapt to or recover from the stress and continue to grow and compete with other cells in their environment. We are particularly interested in the roles of small regulatory RNAs in regulation of bacterial stress responses. Small RNAs (sRNAs) have diverse roles in regulation of cellular processes. sRNAs are crucial for development in higher organisms. In bacteria, sRNAs regulate responses to many environmental stresses. Global screens for sRNAs in Escherichia coli have uncovered more than 80 novel sRNAs of unknown function; a subclass of these bacterial sRNAs has a functional requirement for binding to the RNA chaperone Hfq. This type of sRNA has been found to regulate translation or stability of mRNA targets by a mechanism that requires sRNA:mRNA base pairing interactions (Fig. 1).

Figure 1. Mechanisms of regulation by Hfq-dependent bacterial sRNAs. Hfq-dependent small RNAs can positively regulate translation of target mRNAs by binding to an upstream part of mRNA 5’ untranslated regions and preventing formation of a translation-inhibitory hairpin structure (left diagram). Negative regulation by Hfq-dependent sRNAs typically involves base pairing interactions that occlude the ribosome binding sites of mRNAs. This prevents ribosome association and thus represses translation (right diagram). In some cases translation inhibition is coupled to RNA degradation and in other cases it is not.

Understanding the role of small RNAs in glucose-phosphate stress

We study an sRNA called SgrS (sugar stress sRNA). SgrS is required to allow E. coli cells to recover from a condition that we refer to as glucose-phosphate (or sugar-phosphate) stress. Glucose-phosphate stress occurs when cells cannot efficiently metabolize glucose-6P due to a pgi mutation (mutant cells lack the first enzyme of glycolysis), or when wild-type cells are exposed to the non-metabolizable glucose analog α-methyl glucoside (αMG). When these phosphosugars accumulate in the cytoplasm, the growth of wild-type cells is transiently inhibited. In contrast, mutant cells lacking SgrS show a severe and prolonged growth inhibition under glucose-phosphate stress conditions (Fig. 2). We want to understand how the activity of the SgrS small RNA allows cells to recover from glucose-phosphate stress.

Figure 2. Growth of cells with and without SgrS under glucose-phosphate stress conditions. E. coli sgrS mutant cells carrying either the vector control or plasmid expressing sgrS were grown to mid-logarithmic phase and exposed to αMG to induce stress. Growth of both cultures was followed before and after stress induction. The data show that SgrS is absolutely required to allow cells to grow under glucose-phosphate stress conditions.

SgrS performs a base pairing-dependent mechanism of post-transcriptional regulation like other Hfq-binding sRNAs that have been studied. The base pairing functions of SgrS are required for post-transcriptional regulation of the ptsG mRNA, which encodes the major glucose transporter (EIICB^Glc) in E. coli. The EIICB^Glc protein is part of a phosphoenolpyruvate phosphotransferase system (PTS) that recognizes and transports glucose and αMG; phosphorylation of these sugars is coupled to their transport across the cytoplasmic membrane via EIICB^Glc. SgrS is specifically synthesized under glucose-phosphate stress conditions and carries out functions that allow recovery from the stress. SgrS functions as a riboregulator (defined as an RNA performing base pairing-dependent regulation) that base pairs with ptsG and manXYZ mRNAs, ultimately causing their degradation by the endoribonuclease RNase E. The molecular mechanisms that control sRNA:mRNA interactions and lead to specific regulatory outcomes are still well understood. We are using both genetic and biochemical approaches to dissect the SgrS:mRNA interactions and shed light on this important question.

SgrS is unique among Hfq-dependent sRNAs in that it has an additional function.  We discovered that SgrS is the first Hfq-dependent coding sRNA regulator.  SgrS encodes the 43 amino-acid peptide SgrT.  SgrT is capable of inhibiting glucose and aMG uptake through a mechanism that is independent of the base pairing function.  We have shown that SgrT acts by inhibiting the activity of EIICB^Glc.  The finding that SgrS serves as a bifunctional regulator is important as it raises questions regarding how a single sRNA molecule can serve two independent functions and how these two functions are coordinated.

Other players in the glucose-phosphate stress response

Our studies have uncovered additional genes that encode products that play a role in the glucose-phosphate stress response. One of these, setA, encodes an efflux pump. The setA gene is located just downstream of sgrS and is also activated by SgrR under glucose-phosphate stress conditions. We recently showed that SetA is important for growth recovery when E. coli cells are severely stressed in minimal media. Interestingly, SetA does not appear to be responsible for the efflux of sugar-phosphates, as we initially hypothesized. Rather, SetA activity seems to alter the balance of TCA cycle and glycolytic metabolites. We have generated other unpublished data that suggest that the balance of these metabolites helps determine whether cells will continue to grow or will be strongly inhibited under stress conditions.

Functional characterization of a novel bacterial transcription factor We have discovered that the sgrR gene, which is divergently transcribed from sgrS, encodes a transcription factor that is responsible for the induction of SgrS synthesis under glucose-phosphate stress conditions. SgrR is the first example of a novel class of transcription factors to be characterized. SgrR contains two major domains: an N-terminal DNA binding domain, and a C-terminal solute binding domain. We hypothesize that SgrR may be able to sense the glucose-phosphate stress by binding to a small molecule signal that is present under stress conditions. Binding of this molecule presumably makes the SgrR protein active for promoting transcription of its target genes, such as sgrS and setA. Since SgrR is the first protein of this novel class to be studied, there are many questions to be answered regarding its mechanism of action and the regulation of its expression.

Vanderpool Lab Future Research Directions We now understand some components of the glucose-phosphate stress response pathway and have a model for how E. coli cells sense and respond to this environmental condition (Fig. 3).  However, there are many unanswered questions about the regulation and physiology underlying this stress.  In the future, we will perform studies aimed at answering the following questions:

• What is the signal triggering the stress response that leads to SgrS production?
• Is SgrR a direct sensor of intracellular glucose-P or another stress molecule?
• How does binding a small molecule affect the function of SgrR as a transcription factor?
• Is SgrS the major player in the stress response?
• Do other genes, e.g., members of an SgrR regulon, play a role in the stress response?
• Does SgrS have other targets in addition to the ptsG and manXYZ mRNAs?
• We have evidence that SgrS has additional targets, which raises other questions:
• How are base pairing interactions between SgrS and each target similar and different?
• Does SgrS prioritize regulation of certain targets over others?
• How is this prioritization established?
• What is the mechanism of action of SgrT?
• How are the base pairing function and the mRNA function (coding for SgrT) coordinated?

Figure 3.  Model for the glucose-phosphate stress response. We propose that the stress response involves several components.  First, the transcription factor SgrR that senses the stress and activates target genes that will allow the cell to cope with stress.  These target genes include those encoding the sRNA SgrS and the efflux pump SetA.  SgrS has two primary functions in the glucose-phosphate stress response: 1) it performs a base pairing function that down-regulates synthesis of sugar transporters; 2) it produces the small protein SgrT that interferes with sugar transport by an unknown mechanism.  Together, these functions allow bacterial cells to recover from glucose-phosphate stress by stopping accumulation of phosphosugars that cannot be metabolized.

Summary

There are many advantages in studying E. coli; there is a great deal known about the metabolism of this organism, there are many genetic tools that facilitate construction of mutants and cells that overexpress proteins or RNAs of interest, and the genomic sequence of E. coli K12 and a number of closely related enteric bacteria are readily available.  We therefore have a powerful system in which to carry out studies that will bring us closer to understanding basic bacterial physiology as well as mechanisms of RNA-mediated regulation that will be broadly applicable.

Representative Publications

Richards, G. R. and C. K. Vanderpool. 2012. Induction of the Pho regulon suppresses the growth defect of an Escherichia coli sgrS mutant, connecting phosphate metabolism to the glucose-phosphate stress response. J. Bact. 194:2520.

Vanderpool, C. K., Balasubramanian, D., and C. R. Lloyd. 2011. Dual-function RNA regulators in bacteria. Biochimie. 93:1943.

Richards, G. R. and C. K. Vanderpool. 2011. Molecular call and response: the physiology of bacterial small RNAs. Biochim. Biophys. Acta. 1809:525.

Rice, J. B. and C. K. Vanderpool. 2011. The small RNA SgrS controls sugar-phosphate accumulation by regulating multiple PTS genes. Nucl. Acids Res. 39:3806.

Sun, Y. and C. K. Vanderpool. 2011. Regulation and function of Escherichia coli sugar efflux transporter A (SetA) during glucose-phosphate stress. J. Bact. 193:143.

Horler, R. S. P. and C. K. Vanderpool. 2009.  Homologs of the small RNA SgrS are broadly distributed in enteric bacteria but have diverged in size and sequence.  Nucl. Acids Res.  37:5465.

Wadler, C. S. and C. K. Vanderpool.  2009.  Characterization of homologs of the small RNA SgrS reveals diversity in function.  Nucl. Acids Res.  37:5477.

Wadler, C. S. and C. K. Vanderpool.  2007.  A novel dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide.  Proc. Natl. Acad. Sci. U.S.A. 104:20454-20459.

Vanderpool, C. K.  2007.  Physiological consequences of small RNA-mediated regulation of glucose-phosphate stress.  Curr. Opin. Microbiol. 10:146-151.

Vanderpool, C.K. and S. Gottesman.  2007.  The novel transcription factor SgrR coordinates the response to glucose-phosphate stress.  J. Bact. 189:2238-2248.

Vanderpool, C.K. and Gottesman, S. 2005. Noncoding RNAs at the membrane. Nat Struct Mol Biol., 12(4):285–6. No abstract available.

Vanderpool, C.K. and Gottesman, S. 2004. Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system. Mol Microbiol., 54(4):1076–89. [Abstract]

Complete Publications List