Genetic organization of the mle locus and identification of a mleR-like gene from Leuconostoc oenos

Abstract

Characterization of the mle locus harboring the malolactic enzyme gene mleA and malate permease gene mleP from Leuconostoc oenos was completed in this study by mRNA analysis. Northern (RNA) blot experiments revealed a 2.6-kb transcript, suggesting an operon structure harboring mleA and mleP genes. Primer extension analysis showed that the mle operon has a single transcription start site located 17 nucleotides upstream of the ATG translation start site for the mleA gene. We found sequences, TTGACT and TATGAT (which are separated by 18 bp), that are closely related to the gram-positive and Escherichia coli consensus promoter sequences. Upstream of the mleA gene, an 894-bp open reading frame that transcribed divergently from the mle operon was found. Sequence analysis and expression in E. coli minicells suggest that this open reading frame encodes a polypeptide with an apparent molecular mass of 34 kDa belonging to the LysR-type regulatory protein family. Protein comparisons showed the highest level of identity with the MleR regulatory protein from Lactococcus lactis, which is involved in the expression of the malolactic genes in the presence of L-malate. However, the MleR-like protein of L. oenos seems different from the protein of Lactococcus lactis, since no regulation of the malolactic enzyme by L-malate was effective under our experimental conditions.