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Mol Microbiol. 2010 Jun 1;76(5):1279-94. doi: 10.1111/j.1365-2958.2010.07175.x. Epub 2010 May 4.

Control of Bacillus subtilis mtl operon expression by complex phosphorylation-dependent regulation of the transcriptional activator MtlR.

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1
Laboratoire de Microbiologie et Génétique Moléculaire, INRA-CNRS-AgroParisTech UMR2585, 78850 Thiverval-Grignon, France.

Abstract

Many bacteria transport mannitol via the mtlAF-encoded phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most firmicutes the transcriptional activator MtlR controls expression of the mtl operon. MtlR possesses an N-terminal DNA binding domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and EIIA(Mtl)-like domain. These four regulatory domains contain one or two potential PTS phosphorylation sites. Replacement of His-342 or His-399 in PRD2 with Ala prevented the phosphorylation of Bacillus subtilis MtlR by PEP, EI and HPr. These mutations as well as EI inactivation caused a loss of MtlR function in vivo. In contrast, phosphomimetic replacement of His-342 with Asp rendered MtlR constitutively active. The absence of phosphorylation in PRD2 serves as catabolite repression mechanism. When EIIA(Mtl) and the soluble EIIB(Mtl) domain of the EIICB(Mtl) permease were included in the phosphorylation mixture, His-599 in the EIIA-like domain of MtlR also became phosphorylated. Replacement of His-599 with Asp rendered MtlR inactive, while His599Ala replacement caused slightly constitutive, glucose-repressible MtlR activity. Doubly mutated His342Ala/His599Ala MtlR was still phosphorylated by EI, HPr and EIIA(Mtl) at Cys-419 in the EIIB(Gat)-like domain. Cys419Ala replacement and deletion of EIIA(Mtl) caused strong constitutive glucose-repressible MtlR activity. This is the first report that Cys phosphorylation controls PRD-containing transcriptional activators.

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