Interaction with Enzyme IIBMpo(EIIBMpo) and Phosphorylation by Phosphorylated EIIBMpoExert Antagonistic Effects on the Transcriptional Activator ManR of Listeria monocytogenes

Abstract

ABSTRACTListeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTSMan), whose three components are encoded by themanLMNgenes. 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 inListeria 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 PTSMpoand that EIIBMpoplays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIBMpointeracts 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(P∼EIIBMpo) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIBMpoand P∼PRD2 of ManR, which together lead to the induction of themanLMNoperon. Complementation of a ΔmanRmutant with variousmanRalleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion ofmanRprevented not only the expression of themanLMNoperon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression ofmanLMNinListeria innocuawas reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, theL. monocytogeneshomologue, Lmo0095, is not required formanLMNexpression; its absence rather stimulatesmanexpression.IMPORTANCEListeria monocytogenesis 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 intoL. monocytogenesmainly via a mannose/glucose-specific phosphotransferase system (PTSMan). Expression of themanoperon 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 EIIBMpocomponent 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.