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The Lactobacillus casei ptsHI47T Mutation Causes Overexpression of a LevR-Regulated but RpoN-Independent Operon Encoding a Mannose Class Phosphotransferase System

Alain Mazé,^1,2, Grégory Boël,^1,2 Sandrine Poncet,¹ Ivan Mijakovic,¹ Yoann Le Breton,² Abdellah Benachour,² Vicente Monedero,³ Josef Deutscher,^1* and Axel Hartke²

Laboratoire de Microbiologie et Génétique Moléculaire, INRA-INAPG-CNRS, F-78850 Thiverval-Grignon,¹ Laboratoire de Microbiologie de l'Environnement (EA956 USC INRA), IRBA, Université de Caen, F-14032 Caen, France,² Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos, IATA-CSIC, 46100 Burjassot, Valencia, Spain³

Received 11 February 2004/ Accepted 7 April 2004

A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (⁵⁴)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No "–12, –24" promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by PHis-HPr stimulates its activity, while phosphorylation by PEIIB^Lev inhibits it. Disruption of the EIIB^Lev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both PEIIB^Lev and PHis-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant PHis-HPr to EIIA^Lev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.

Present address: Alimentary Pharmabiotic Centre, Department of Microbiology, National University of Ireland, Cork, Cork, Ireland.

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