Robert Britton, Ph.D.

rbritton@msu.edu

Research

Universally conserved essential GTPases

GTPases play pivotal roles in numerous processes in eukaryotes, including regulation of the cell cycle. In bacteria most of the GTPases characterized to date are involved in translation and protein secretion. Comparative analysis of sequenced genomes has uncovered a family of six GTPases that are conserved from bacteria to man. In B. subtilis and E. coli these GTPases have been shown to be essential for growth. A main goal of our research is to understand the cellular function of these conserved essential GTPases in B. subtilis. We are taking a functional genomics approach to uncover the roles of these GTPases in B. subtilis. A combination of methods including DNA microarray, two-hybrid analysis of protein-protein interactions, and genetic suppressor analysis are being utilized.

Role of essential GTPases in ribosome biogenesis

Several GTPases have been implicated in the biogenesis of ribosomes in bacteria and eukaryotes; however the molecular mechanisms by which they assist ribosome formation are poorly understood. Recent work from our laboratory has identified three essential GTPases (RbgA, YphC, and YsxC) that are involved in the assembly of the large ribosomal subunit in Bacillus subtilis. RbgA is a GTPase that when depleted from cells causes a severe reduction in 70S ribosomes and the accumulation of an abnormal large ribosomal subunit that migrates at 45S in a sucrose gradient. Two ribosomal proteins, L16 and L27, are missing from the 45S complex. RbgA specifically interacts with the 45S intermediate, indicating that RbgA plays a direct role in the assembly of the 45S intermediate. We have formulated a model in which RbgA facilitates the assembly of the 45S intermediate by regulating the incorporation L16.

YphC and YsxC appear to also be involved late 50S ribosome assembly as mutations affecting the levels of these proteins cause a phenotype similar to that observed in RbgA-depleted cells, accumulation of an abnormal 45S large subunit and a reduction in the level of 70S ribosomes. Preliminary analysis of these 45S complexes from RbgA, YphC, and YsxC-depleted cells indicate they contain proteins unique to each complex, indicating they may be arrested at unique steps in the assembly process. Our results suggest that, as has been found in eukaryotic large subunit biogenesis, several GTPases participate in the assembly of the 50S subunit in bacteria.

Genomics of probiotic bacteria

Probiotic bacteria are defined as live microbes that when administered provide a health benefit to the host by influencing the intestinal microbiota. Interest in probiotics has increased in the United States in recent years as a possible alternative to antibiotics in both medicine and agriculture. Probiotics are being considered in the treatment of a number of human diseases including infectious diarrhea, antibiotic induced diarrhea, inflammatory bowel disease (IBD), and infant necrotizing enterocolitis. However, clinical trials aimed at assessing the effectiveness of probiotics have generally been mixed and therefore a significant amount of skepticism regarding the efficacy of probiotics remains. Until we understand the underlying mechanisms behind the beneficial effects exerted by probiotic bacteria, the use of probiotics in medicine to improve human health will be less than fully realized. Several possible mechanisms for the beneficial effects of probiotics have been proposed, including the ability to modulate the immune system, production of antimicrobial compounds to eliminate pathogens, and the alteration of the microbial flora.

Our lab is interested in elucidating both the mechanisms by which probiotic bacteria positively affect the health of the host and the mechanisms by which probiotic Lactobacillus reuteri can inhibit infectious diarrhea caused by enteropathogenic Escherichia coli (EPEC) and enterohemmoragic E. coli (EHEC). Towards the first aim, we are using DNA microarrays constructed from two different species of Lactobacillus to uncover the regulatory networks that govern acid and bile resistance and to understand how Lactobacillus responds to interactions with eukaryotic cells and intestinal pathogens. To assess the ability of Lactobacillus reuteri to prevent or ameliorate pathogenic E. coli infection, we are working to elucidate the mechanism by which reuterin, an anti-microbial compound produced by L.reuteri, is produced and accumulated by L.reuteri and by which it exerts its bacteriocidal effects. We are doing this by exploring the interaction of reuterin with EPEC and EHEC strains and by using microarrays to examine gene expression changes in pathogenic E. coli upon exposure to reuterin. In addition, we are establishing tissue culture and animal models of EHEC and EPEC infection.