Microcin PDI regulation and proteolytic cleavage are unique among known microcins.

Microcin PDI inhibits a diversity of pathogenic Escherichia coli through the action of an effector protein, McpM. In this study we demonstrated that expression of the inhibitory phenotype is induced under low osmolarity conditions and expression is primarily controlled by the EnvZ/OmpR two-component regulatory system. Functional, mutagenesis and complementation experiments were used to empirically demonstrate that EnvZ is required for the inhibitory phenotype and that regulation of mcpM is dependent on binding of the phosphorylated OmpR to the mcpM promoter region. The phosphorylated OmpR may recognize three different binding sites within this promoter region. Site-directed mutagenesis revealed that the McpM precursor peptide includes two leader peptides that undergo sequential cleavage at positions G17/G18 and G35/A36 during export through the type I secretion system. Competition assays showed that both cleaved products are required for the PDI phenotype although we could not distinguish loss of function from loss of secretion in these assays. McpM has four cysteines within the mature peptide and site-directed mutagenesis experiments demonstrated that the first two cysteines are necessary for McpM to inhibit susceptible cells. Together these data combined with previous work indicate that MccPDI is unique amongst the microcins that have been described to date.