Timothy Randall Hoover

Research in my lab focuses on flagellar biogenesis in Helicobacter pylori, a significant human pathogen that is the causative agent of several gastric diseases including chronic gastritis, peptic ulcer disease and gastric cancer. Flagella are used for motility in H. pylori, which is needed for colonization of the gastric mucosa by the bacterium. Flagellar biosynthesis in H. pylori requires the coordinated regulation of over 40 genes through a transcriptional hierarchy that involves all three of the sigma factors found in this bacterium - sigma80, sigma54 (RpoN), and sigma28 (FliA). The sigma80-dependent flagellar genes encode components of the basal body and flagellar protein export apparatus, and are expressed early in the transcriptional hierarchy. The RpoN-dependent flagellar genes encode hook and filament proteins and are expressed later in the hierarchy. Transcription of these genes requires the activator FlgR and its cognate sensor kinase FlgS. The FliA-dependent genes encode components of the flagellar filament and are the last set of flagellar genes to be expressed. Expression of the FliA regulon is inhibited by the anti-sigma factor FlgM. Results from my lab, as well as work from other labs, suggest that transcription of the RpoN and FliA regulons is coupled to flagellar assembly through the activity of the flagellar protein export apparatus. Current goals of my lab include: (1) identify components of the flagellar protein export apparatus responsible for controlling the RpoN regulon; (2) determine if FlgS activity is controlled through interactions with the export apparatus; (3) identify other factors that influence function of RpoN or FliA; and (4) identify a potential master regulator that initiates flagellar biosynthesis in H. pylori.

A second project in the lab examines acetone metabolism in H. pylori and its role in colonization of the stomach. H. pylori possesses a cluster of genes that encode proteins that are predicted to convert acetone to acetyl-CoA. The first enzyme of the predicted pathway is acetone carboxylase, which catalyzes the conversion of acetone to acetoacetate. Inactivation of the operon that encodes acetone carboxylase interferes with the ability of H. pylori to colonize the mouse stomach. Current goals are: (1) determine the reason disruption of acetone carboxylase affects colonization; (2) verify the proposed pathway for acetone utilization in H. pylori; and (3) examine the distribution of the acetone metabolism genes in other helicobacter species.

Additional work in my lab has focused on the mechanism of transcriptional activation with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme). Unlike other forms of RNA polymerase holoenzyme, sigma54-holoenzyme requires an activator protein to initiate transcription. Sigma54-holoenzyme binds to the promoter to form a stable closed complex. Activators of sigma54-holoenzyme stimulate isomerization of the closed complex to an open complex that is able to initiate transcription. This isomerization is coupled to ATP hydrolysis by the activator. Previous work from my lab related to the mechanism of transcriptional activation with sigma54-holoenzyme has examined: 1) interactions between the activator and sigma54; 2) structure-function relationships in sigma54-dependent activators; 3) sigma54-promoter interactions; and, 4) factors that influence sigma54 activity.

Work from my lab identified a novel Helicobacter pylori protein (HP0958) that is needed to prevent the rapid turnover of sigma54. Plans for future work include: 1) identifying the mechanism by which HP0958 protects sigma54 from proteolysis in H. pylori ; and 2) determining if HP0958 homologs in other bacteria have similar roles in protecting sigma54 from proteolysis.

A variety of important bacterial processes require sigma54 for expression of the genes involved in these biological activities. These microbial processes are significant in health, agriculture and the environment, and include motility, nitrogen fixation, hydrogen metabolism, solute transport and utilization of different carbon sources. Understanding how bacteria regulate expression of these genes is important for identifying new potential targets for controlling harmful bacterial processes and enhancing beneficial processes.