Absence of Malolactic Activity Is a Characteristic of H+-ATPase-Deficient Mutants of the Lactic Acid BacteriumOenococcus oeni

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Abstract
The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni. Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR. No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found. The effect of a reduction in H+-ATPase activity on l-malate metabolism was then investigated by using other malolactic bacteria. Spontaneous H+-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance. Two mutants were selected. These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L. lactis and L. mesenteroides parental strains, respectively. These mutants were also acid sensitive. However, in contrast to the ATPase-deficient mutants of O. oeni, activation of l-malate metabolism was observed with the L. lactis and L. mesenteroides mutants under optimal or acidic growth conditions. These data support the suggestion that expression of the genes encoding malolactic enzymes in O. oeni is regulated by the mleR product, as it is in L. lactis. Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O. oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria.