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 (PTS(Man)), 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 IIA(Man) [EIIA(Man)]- and EIIB(Gat)-like). We demonstrate here that in Listeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIA(Man)-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTS(Mpo) and that EIIB(Mpo) plays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIB(Mpo) interacts with the two C-terminal domains of ManR (EIIB(Gat)-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIB(Mpo) (P∼EIIB(Mpo)) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIB(Mpo) and P∼PRD2 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.

Importance: Listeria monocytogenes is a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported into L. monocytogenes mainly via a mannose/glucose-specific phosphotransferase system (PTS(Man)). Expression of the man operon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIB(Mpo) component plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.