AEM Accepts, published online ahead of print on 6 November 2009

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Appl. Environ. Microbiol. doi:10.1128/AEM.02145-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The Lactobacillus casei MaeKR two component system is required for L-malic acid utilization through a malic enzyme pathway.

José María Landete, Luisa García-Haro, Amalia Blasco, Paloma Manzanares, Carmen Berbegal, Vicente Monedero, and Manuel Zúñiga^*

Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC) PO Box 73, 46100 Burjassot, Valencia, Spain; Departamento de Microbiología y Ecología, Facultad de Biología, ENOLAB-Laboratorio de Microbiología Enológica, Universidad de Valencia, Burjassot, Valencia, Spain

^* To whom correspondence should be addressed. Email: btcman{at}iata.csic.es.

Lactobacillus casei can metabolize L-malic acid via malolactic enzyme (MLF) or malic enzyme (ME). Whereas utilization of L-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on L-malic acid. In this work we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), a ME (maeE) and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus uberis. Our results show that ME is essential for L-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on L-malic acid thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of L-malic acid and repressed by glucose whereas the TC system expression was induced by L-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats (5'-TTATT(A/T)AA-3') in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.