Bacteria living in natural environments must adjust their gene expression in response to changing and often stressful conditions in order to continue to grow and compete with their neighbors for limited resources. While bacteria employ different strategies to regulate gene expression, our lab is interested in a type of regulation involving small RNAs (sRNAs). sRNAs are typically non-coding RNA molecules that can be found in organisms belonging to all domains of life. In bacteria, they are often involved in regulating stress responses using mechanisms that involve base pairing with a target mRNA and enhancing or repressing its stability or translation. We are passionate about deciphering the overall impact of sRNAs on cell physiology, metabolism, as well as structure and function.
REGULATION OF BACTERIAL STRESS RESPONSES
Same as us, bacteria get stressed. In order to not only survive but thrive in harsh and often hostile environments, they have to constantly adapt their gene expression to these changing niches. This can be achieved by multiple layers and players involved in gene expression control. However, our lab is fascinated by a certain type of regulation involving small RNAs (sRNAs), which is a fairly recent discovery in the field of molecular microbiology. These typically non-coding RNA molecules are more common than initially assumed and can be found in all domains of life. In bacteria, sRNAs are often involved in the organism's stress response and generally exert their function by basepairing with one or multiple target mRNAs thereby enhancing or repression their respective stability or translation.
GLOBAL DISCOVERY AND CHARACTERIZATION OF SMALL RNA NETWORKS
Bacterial genomes encode tens to hundreds of small RNAs (sRNAs), and we know little or nothing about their functions in most organisms. Discovering the set of mRNA targets that a given sRNA regulates – the sRNA’s “regulon” – helps us begin to understand sRNA function. This is a difficult problem. Although a variety of computational and experimental methods exist for sRNA target identification, the process is labor-intensive and often yields a high false-positive rate. We have attempted to improve the efficiency of sRNA target identification by developing and testing SPOT: sRNA Target Prediction Organizing Tool. This is a pipeline that can integrate computational predictions and experimental evidence to generate sRNA target regulon predictions. SPOT has so far helped us identify new target mRNAs for several sRNAs in E. coli. We will use our results to further refine SPOT and define sRNA target regulons in other bacteria. Our ultimate goal is to have a comprehensive understanding of the roles of sRNAs in bacterial physiology and metabolism.
POST-TRANSCRIPTIONAL CONTROL OF CARBOHYDRATE TRANSPORT AND METABOLISM
Our work on the sRNA SgrS has allowed us to develop one of the top existing models for discovery of new molecular mechanisms of regulation by bacterial sRNAs and sRNA-mediated control of bacterial metabolism and physiology. SgrS (sugar stress sRNA) is produced by enteric bacteria like E. coli and Salmonella during metabolic stress conditions when sugar metabolism can’t keep up with sugar transport. SgrS regulates nine genes to restore metabolic homeostasis and allow stressed cells to recover and continue growing. SgrS represses translation of ptsG and manXYZ mRNAs, encoding sugar transporters, to temporarily block uptake of sugars. SgrS enhances translation of the yigL (sugar phosphatase) mRNA to promote dephosphorylation of sugars and allow them to be pumped out of the cell. SgrS regulates other targets to reroute metabolism around the metabolic block. SgrS prioritizes target regulation in a specific order so that cells rapidly and efficiently adapt to the stress and continue growing.
SMALL RNA-MEDIATED CONTROL OF MEMBRANE STURCTURE AND FUNCTION
Microbial membranes are the first line of defense against environmental stress as well as the site of many metabolic processes, so membrane integrity is key to cell survival. Bacteria control the biophysical properties of membranes using lipid modifications like cyclopropanation. The cyclopropane fatty acid synthase (Cfa) enzyme catalyzes this lipid modification and is regulated in response to stress. The sRNA RydC was previously shown to stabilize cfa mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. We found additional activating (ArrS) and repressing (CpxQ) sRNAs that also directly regulate cfa post-transcriptionally. RydC and CpxQ exhibit pH-dependent regulation of cfa, and RydC-dependent cfa activation protects cells from acid shock. This work illustrates how mRNAs can serve as hubs for signal integration via multiple sRNAs responding to different environmental signals. Future work aims to understand the signals that induce production of each sRNA and more conditions where this sRNA-mediated regulation is beneficial for bacterial cells.
PROPHAGE-ENCODED REGULATORS CONTROL BACTERIAL HOST METABOLISM AND PHYSIOLOGY
Cryptic prophages, that have lost the ability to produce phage progeny, have been discovered within bacterial genomes and harbor genes of unknown function which impact the host cell physiology. We have characterized the sRNA DicF and associated small protein DicB within the cryptic prophage Qin in E. coli K12. DicF and DicB were known to be inhibitors of cell division. We found that DicF sRNA regulates mRNAs encoding cell division and metabolic activities via direct sRNA-mRNA base pairing. We also discovered a new function for the small protein DicB. Transient production of DicB protected E. coli cells from infection by lambda phage. DicB specifically provided protection against phages that use a specific cytoplasmic membrane receptor – ManYZ – for injection of their DNA into the E. coli cytoplasm. Our results suggest that one small prophage-encoded protein can carry out multiple functions that affect bacterial cell division and physiology. Our future work will aim to uncover the molecular mechanisms by which DicB carries out its multiple activities.
GENETICS AND PHYSIOLOGY
OF GUT MICROBES
Complex communities of microbes in our gut have tremendous impacts on our health by contributing metabolic functions, protecting against disease-causing pathogens, educating the immune system, controlling obesity, and even affecting learning and memory. We are interested in understanding the regulatory mechanisms that govern physiology and metabolism of important members of the gut microbiota belonging to the phylum Bacteroidetes. In these organisms, we know very little about RNA-mediated mechanisms of regulation. Work in our laboratory is focused on identification and characterization of novel RNA binding proteins and sRNAs.