title: In vivo effect of mutations in the antiterminator LacT in Lactobacillus casei.
abstract: The antiterminator LacT regulates the expression of the lactose operon in Lactobacillus casei and its activity is controlled by EII(Lac) and common PTS elements. LacT shows the two conserved domains (PRD-I and PRD-II) characteristic of the BglG antiterminator family that are implicated in the regulation of their activity, possibly by phosphorylation of conserved histidines. By site-directed mutagenesis of LacT, four histidines (His-101, His-159 in PRD-I and His-210, His-273 in PRD-II) were replaced by alanine or aspartate, mimicking non-phosphorylated and phosphorylated forms, respectively. These constructions were used to complement DeltalacT and DeltaccpA mutants. L. casei strains (DeltalacT) carrying the replacement of His-101 or His-159 by Ala showed phospho-beta-galactosidase activity in absence of the inducer (lactose), indicating that these amino acids, located in PRD-I, are essential for EII-dependent induction of the lac operon, possibly by dephosphorylation. Interestingly, these mutations rendered LacT thermosensitive. Moreover, expression of H210A and H273A (PRD-II) mutations in L. casei DeltaccpA showed that these two histidyl residues could have a role in LacT-dependent carbon catabolite repression (CCR) of this system. Overexpression of LacT in a ccpA background rendered the lac operon insensitive to CCR, but it was still sensitive to lactose induction. This suggests that the transfer of phosphate groups from PTS elements, which controls these two regulatory processes (CCR and substrate induction), could have different affinity for PRD-I and PRD-II histidines.
authors.affiliation: Departamento de Biotecnologı́a, Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Polı́gono de la Coma s/n, Apartado de correos (PO Box) 73, 46100-Burjassot, Valencia, Spain1.
authors.forename: Marı A J
authors.initials: MAJ
authors.lastname: Gosalbes
authors.affiliation: Departamento de Biotecnologı́a, Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Polı́gono de la Coma s/n, Apartado de correos (PO Box) 73, 46100-Burjassot, Valencia, Spain1.
authors.forename: Carlos D
authors.initials: CD
authors.lastname: Esteban
authors.affiliation: Departamento de Biotecnologı́a, Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Polı́gono de la Coma s/n, Apartado de correos (PO Box) 73, 46100-Burjassot, Valencia, Spain1.
authors.forename: Gaspar
authors.initials: G
authors.lastname: Pérez-Martı Nez
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D007780:Lacticaseibacillus casei / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria; D007785:Lactose