Regulation and localization of flp pili: an under-investigated system (Year 4) - PROJECT SUMMARY Pili are bacterial surface structures that are used in many ways. They can be used for motility, genetic exchange, and surface attachment, and are often critical virulence factors for pathogens. A particular type of pilus, the flp pilus, is found in many bacterial pathogens, including Vibrio vulnificus and Aggregatibacter actinomycetemcomitans, and is used by some members of the human microbiome to establish permanence in the intestinal tract. Despite its importance, the flp pilus is not nearly studied to the depth of other pilus systems, and has many unexplored facets and curiosities. In this proposal we outline experiments to explore the flp pilus using a model bacterium that provides many technical benefits for studying pili: Caulobacter crescentus. A common characteristic of the flp system is that the pilin gene is regulated separately from the rest of the pilus genes. In C. crescentus, the transcriptional activator CtrA binds to four sites in the pilin promoter. We have shown that one site induces expression, while the remaining sites inhibit and delay transcription from their upstream positions, a phenomenon not previously reported in bacteria. The inhibitory sites increase the affinity of CtrA for the promoter. We have also shown that relief of upstream inhibition leads to premature pilus production which results in increased susceptibility to a bacterial virus that uses the pilus to infect. Regulation of the pilin promoter will be explored by mapping CtrA binding over a concentration range in different promoter variations to identify which sites become occupied first. The spacing between binding sites and within the sites themselves will be investigated by altering spacing and testing for inhibitory activity through a variety of means. Lastly, we will explore the real-world consequences of premature pilus production by examining cell survival rates under persistent phage threat in a microfluidics chamber (Aim 1). Our previous work has identified an uncharacterized predicted RNase that appears to associate with the pilA promoter that inhibits transcription in some constructs but promotes expression in others. We will investigate the role of this RNase by examining its DNA binding ability, its role in RNA stability, and the proteins it interacts with (Aim 2). Lastly, we have shown that several different lineages of bacterial viruses infecting diverse Alphaproteobacteria convergently evolved usage of the host CtrA to control virus gene expression. We hypothesize that this is a Trojan horse mechanism to increase virus spread. We will investigate this hypothesis by characterizing the timing of virus gene expression during infection, disrupting CtrA regulation of virus genes by mutating the virus using novel methodology, and determining the impact of these mutations on gene expression (Aim 3). These studies will provide insight into unusual features of flp pili systems, and thus provide insight into a structure important for many human pathogenic and commensal organisms.
