REGULATION, FUNCTION AND POTENTIAL APPLICATIONS OF MICROCIN PDI FOR MEDICINE AND FOOD SAFTEY

Abstract

Microcin PDI (mccPDI) is a class IIa microcin that is encoded as a five-locus genetic unit that is borne by a conjugative plasmid. The microcin was first discovered in Escherichia coli strains 25 and 264 (cattle origin) and likely has a significant impact on the microbial composition of E. coli communities in livestock, even contributing to the persistence of antibiotic-resistant strains. This dissertation concerns the mechanism by which the mccPDI phenotype is regulated in the wild-type strains (Chapters 2), how the key effector protein (McpM) kills susceptible bacteria, and how the immunity protein (McpI) protects the mccPDI-producing bacteria (Chapter 3). Up-regulation of mccPDI is controlled by a two-component regulatory system (EnvZ/OmpR) that functions as an on/off switch. Low-salt conditions lead to upregulation of OmpR, which is a transcriptional regulator for both McpM and the cognate ligand on the susceptible cells, OmpF. Deletion of OmpR completely eliminates the mccPDI phenotype (Chapter 1). This on/off osmotic-sensing system is attenuated by the concentration of quorum sensing autoinducer-2 (AI-2) molecules in the culture media. Upregulation of mccPDI corresponds to increasing concentration of AI-2, while down regulation of the system corresponds to the decay of AI-2 as the bacterial population shifts from log-growth into stationary-growth phase. The kinetics of AI-2 expression correspond to the transcriptional pattern of mcpM, and deletion of the quorum sensing system eliminates upregulation of mcpM. It is very likely that sRNAs micC and micF also contribute to the mccPDI regulatory system (Chapter 2). A series of assays demonstrated that mccPDI inhibits the growth of susceptible bacteria when McpM permeabilizes the outer membrane, resulting in cell death. This phenotype can be blocked with the addition of low-molecular mass (200 – 600 Da) osmoprotectant to the culture media. The mechanism by which McpI protects mccPDI-producing cells is unknown, but experiments described in Chapter 3 show that McpI likely forms asymmetrical trimer structures that may serve to block pores that are formed by McpM.