CRP-dependent DNA anchoring and membrane sequestration define a dual mechanism for MtlR-mediated regulation.

Mannitol is a widely distributed sugar alcohol and a primary carbon source for diverse microbial communities, serving as a substrate for producing high-value metabolites. In Gammaproteobacteria, the conserved mtl operon is essential for mannitol metabolism, encoding the phosphotransferase system enzyme II (EIIMtl; mtlA), mannitol-1-phosphate dehydrogenase (mtlD), and the repressor MtlR (mtlR). While operon transcription is known to be subject to catabolite repression and dependent on cAMP receptor protein (CRP), the molecular mechanism of MtlR-mediated repression has remained unclear, as it lacks a canonical DNA-binding domain. Here, we demonstrate that MtlR represses transcription by utilizing CRP as a DNA-anchoring platform. In the absence of mannitol, MtlR forms a complex with CRP at the mtl operator, with recruitment specificity determined by the precise spacing between adjacent CRP-binding sites. With mannitol, the dephosphorylation of membrane-bound EIIMtl triggers the sequestration of MtlR at the membrane, thereby relieving repression and inducing operon expression. We reveal a dual regulatory mechanism: CRP spacing-dependent MtlR recruitment and EIIMtl-mediated MtlR sequestration. This dual-control strategy is selectively conserved across Gammaproteobacteria, with Enterobacterales retaining both functional modules, while other lineages exhibit distinct evolutionary divergence. This study highlights a non-canonical, broadly conserved strategy for integrating metabolic and environmental signals into bacterial gene control.