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 only recently been recognized for their diverse roles in regulation of cellular processes. sRNAs are crucial for development in higher organisms. In eubacteria, sRNAs regulate responses to many environmental stresses. Global screens for sRNAs in Escherichia coli have uncovered more than 70 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 basepairing interactions (Fig. 1).

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 (the first step 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.

SgrS performs a basepairing-dependent mechanism of post-transcriptional regulation like other Hfq-binding sRNAs that have been studied. The basepairing 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 that basepairs with the ptsG mRNA, ultimately causing its degradation by the endoribonuclease RNase E. The molecular mechanisms that control sRNA:mRNA interactions and lead to specific regulatory outcomes are not well understood for any bacterial sRNA. We will use both genetic and biochemical approaches to dissect the SgrS:ptsG mRNA interaction and shed light on this important question.

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, possibly glucose-6-phosphate itself. Binding of this molecule would then change the SgrR protein into an active protein that is able to promote activation of its target genes, such as sgrS. 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. We are now able to purify the SgrR protein and will be able to study its properties in vitro.

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?
• Does SgrS have other targets in addition to the ptsG mRNA?
• What are the requirements for SgrS function?
• Where does Hfq bind?
• What regions of SgrS are critical for interactions with mRNA targets?

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.