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

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 ATPasedeficient 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.

Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars

“…Similar to mutants in other studies (23,24,52), these mutants had slower growth at pH 4.5 (results not shown). In addition, the ATPase activities of mutant cells were lower than those of the parent strain, and the four mutant strains selected for further study had 52 to 62% less F 0 F 1 -ATPase activity than the parent strain, which corresponded to activities of 2.9 to 3.6 mol P i min Ϫ1 mg Ϫ1 (Fig.…”

Rapid assessment of Oenococcus oeni activity by measuring intracellular pH and membrane potential by flow cytometry, and its application to the more effective control of malolactic fermentation

“…Due to the simplicity and lower time consumption of FCM associated with fluorescent probes, we simultaneously measured pH in and ΔΨ throughout MLF, both in synthetic medium and in wine, and determined their changes. Moreover, with the vitality descriptor ΔFI 15 , we confirmed ATP synthesis, as suggested by (Galland et al, 2003). The kinetics of physiologic cellular changes of O. oeni are illustrated in Fig.…”

“…The role of ATPase in relation to acid tolerance has been demonstrated with ATPasedeficient mutants (Tourdot-Maréchal et al, 1999). Since all the ATPase-deficient mutants lack malolactic activity, a link has been suggested between ATPase and malolactic activity in O. oeni (Galland et al, 2003), and, moreover, it has been observed that ATPase activity was maximal in the exponential growth phase of O. oeni (Carrete et al, 2002). It has been demonstrated (Salema et al, 1996b) that MLF generates both a transmembrane pH gradient and an electrical potential gradient ΔΨ in O. oeni, and that the latter controls the rate of fermentation.…”

Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni

“…Radiolabeling with [ 35 S]methionine has been used to study protein profiles in this bacterium under stress conditions (14,15). Immunoblotting has been used to study the expression of specific proteins, e.g., malolactic enzyme (12,23), [H ϩ ]ATPase (43), and Lo18 (16,18,43).…”