Interaction with enzyme IIBMpo (EIIBMpo) and phosphorylation by phosphorylated EIIBMpo exert antagonistic effects on the transcriptional activator ManR of Listeria monocytogenes

Abstract

Listeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTSMan), whose three components are encoded by the manLMN genes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIAMan [EIIAMan]-and EIIBGat-like). We demonstrate here that in Listeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIAMan-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTSMpo and that EIIBMpo plays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIBMpo interacts with the two C-terminal domains of ManR (EIIBGat-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIBMpo (PEIIBMpo) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of PEIIBMpo and PPRD2 of ManR, which together lead to the induction of the manLMN operon. Complementation of a ΔmanR mutant with various manR alleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion of manR prevented not only the expression of the manLMN operon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression of manLMN in Listeria innocua was reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, the L. monocytogenes homologue, Lmo0095, is not required for manLMN expression; its absence rather stimulates man expression.