title: Membrane sequestration by the EIIB domain of the mannitol permease MtlA activates the Bacillus subtilis mtl operon regulator MtlR.
abstract: In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIB(Mtl)  domain of the mannitol permease MtlA (EIICB(Mtl) ). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3' part (EIIB(Mtl) ) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIB(Mtl)  domain with the two C-terminal domains of MtlR. Complementation of the Δ3'-mtlA ΔmtlF or ΔmtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3'-mtlA had no effect. Moreover, synthesis of EIIB(Mtl)  in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIB(Mtl)  fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIB(Mtl)  lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIB(Mtl) . Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICB(Mtl)  in order to be functional.
authors.affiliation: Institut de la Recherche Agronomique, UMR1319 Microbiologie de l'alimentation au service de la santé humaine Micalis, F-78350 Jouy-en-Josas, France.
authors.forename: Houda
authors.initials: H
authors.lastname: Bouraoui
authors.forename: Magali
authors.initials: M
authors.lastname: Ventroux
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Noirot-Gros
authors.forename: Josef
authors.initials: J
authors.lastname: Deutscher
authors.forename: Philippe
authors.initials: P
authors.lastname: Joyet
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008353:Mannitol / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009876:Operon; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; D008353:Mannitol