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In vivo effect of mutations in the antiterminator LacT in Lactobacillus casei

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Polígono de la Coma s/n, Apartado de correos (PO Box) 73, 46100-Burjassot, Valencia, Spain¹

Author for correspondence: Gaspar Pérez-Martínez. Tel: +34 96 3900022. Fax: +34 96 3636301. e-mail: gaspar.perez{at}iata.csic.es

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 lacT and ccpA mutants. L. casei strains (lacT) carrying the replacement of His-101 or His-159 by Ala showed phospho-ß-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 ccpA 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.

Keywords: L. casei, lactose induction, carbon catabolite repression, mutagenesis, phosphorylation

Abbreviations: CCR, carbon catabolite repression; P-ß-gal; phospho-ß-galactosidase; PRD, PTS regulation domain; PTS, phosphoenol-pyruvate-dependent phosphotransferase system; RAT, ribonucleic antiterminator

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