PTS-Mediated Regulation of the Transcription Activator MtlR from Different Species: Surprising Differences despite Strong Sequence Conservation

Abstract

The hexitol <smlcap>D</smlcap>-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the <smlcap>D</smlcap>-mannitol-specific PTS components and <smlcap>D</smlcap>-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIB^Gat- and an EIIA^Mtl-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from <i>Lactobacillus casei</i>, <i>Bacillus subtilis</i> and <i>Geobacillus stearothermophilus</i> are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, <i>L. casei</i> MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from <i>B. subtilis</i> and <i>G. stearothermophilus</i> was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, <i>L. casei</i> MtlR functions without phosphorylation in PRD2 because in a <i>ptsI</i> (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTS^Mtl components, which inactivate MtlR by phosphorylating its EIIB^Gat- or EIIA^Mtl-like domain. This explains the constitutive phenotype of the <i>ptsI</i> mutant. The absence of EIIB^Mtl-mediated phosphorylation leads to induction of the <i>L. casei</i><i>mtl </i>operon. This mechanism resembles <i>mtlARFD</i> induction in <i>G. stearothermophilus</i>, but differs from EIIA^Mtl-mediated induction in <i>B. subtilis</i>. In contrast to <i>B. subtilis</i> MtlR, <i>L. casei</i> MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIB^Mtl domain.