Professor of Microbiology
Genetics, Genomics, Microbial Physiology, Protein-Nucleic Acid Interactions, Regulation of Gene Expression, RNA Biology, Signal Transduction
B.S. Microbiology, Purdue University, 1998
Ph.D. Microbiology, Immunology and Cancer Biology, University of Minnesota, 1998-2003
Postdoctoral Fellow, National Cancer Institute, 2003-2006
Regulation of Bacterial Stress Responses
Bacteria get stressed. They must constantly adjust and adapt their gene expression to changing, and often stressful, environmental conditions so that they may continue to grow and compete in their environment. While there are many ways to regulate gene expression, our lab is interested in a type of regulation that was only recently discovered and involves small RNAs (sRNAs). sRNAs are typically non-coding RNA molecules that can be found organisms belonging to all domains of life. In bacteria they are typically involved in regulating stress responses and can do so using diverse mechanisms that generally involve base pairing with a target mRNA and enhancing or repressing its stability or translation. (Fig. 1).
Figure 1. Mechanisms of regulation by bacterial sRNAs. 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 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 or other phosphosugars. 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 a molecule (αMG) that induces 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 base pairing-dependent post-transcriptional regulation like other sRNAs that have been studied. The base pairing function of SgrS is required for regulation of the ptsG mRNA, which encodes the major glucose transporter (EIICBGlc) in E. coli. The EIICBGlc protein is part of a phosphoenolpyruvate phosphotransferase system (PTS) that recognizes and transports glucose and other sugars; phosphorylation of these sugars is coupled to their transport across the cytoplasmic membrane via EIICBGlc. 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 not 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 sRNAs in that it has an additional function. We discovered that SgrS is a coding sRNA regulator. SgrS encodes the 43 amino-acid peptide SgrT. SgrT is capable of inhibiting glucose uptake through a mechanism that is independent of the base pairing function. We have shown that SgrT acts by inhibiting the activity of EIICBGlc. 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.
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 SgrS the major player in the stress response?
- Do other genes 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?
- Are there other small RNAs that regulate genes required for phosphotransferase system-mediated carbohydrate transport? (We already have evidence for two additional small RNAs that regulate other pts genes.)
- What physiological signals are controlling the production or activity of other PTS regulating sRNAs?
- What sRNAs are important for controlling virulence properties of enteric pathogens like Salmonella?
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.
There are many advantages in studying E. coli and Salmonella; there is a great deal known about the metabolism of these organisms, there are many genetic tools that facilitate construction of mutants and cells that overexpress proteins or RNAs of interest, and the genomic sequences of E. coli, Salmonella 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.
2103-2014 Helen Corley Petit Scholar, College of Liberal Arts and Sciences
Papenfort, K., Sun, Y., Miyakoshi, M., Vanderpool, C.K. and J. Vogel. 2013. Regulation of glucose homeostasis by small RNA mediated suboperonic mRNA stabilization. Cell 153:426.
Rice, J. B., Balasubramanian, D., and C. K. Vanderpool. 2012. Small RNA binding site multiplicity involved in translational regulation of a polycistronic mRNA. Proc. Natl. Acad. Sci. U.S.A. 109:E2691.
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.
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 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]