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Involvement of citrate and malate metabolisms in internal ph homeostasis in oenococcus oeni

Decarboxylation of metabolites is a desirable step in dairy, meat and wine industries, but the conversion of amino acids in biogenic amines results in the production of unwanted toxic products. The pathways consist of a membrane transporter and a cytosolic decarboxylase. The benefit of the pathway for the bacteria could be the generation of metabolic energy and/or homeostasis of intracellular pH. Regulation of the intracellular pH is a crucial parameter for the cellular functioning in low pH conditions. We aim to understand the mechanisms behind the energy generation and resistances provided by decarboxylation pathway. Consequently, due to their participation in the homoeostasis of intracellular pH, the decarboxylation pathways of malic and citric acids are of interest.

In these study, we analysed the effects of L-malic acid and citric acid on tolerance to acidic conditions and on regulation of intracellular pH in Oenococcus oeni. Bacterial cultures growth with addition of various organic acids and pH conditions revealed that L-malate enhanced on the growth at pH equal or below 4.5. On the other hand, the presence of citrate in the medium led to complete inhibition of the growth at pH 3.2.

Metabolism of hexoses, but not of organic acids was impaired by low pH. Regulation of intracellular pH revealed that both L-malic and citric acids participated in the enhancement of the transmembrane pH gradient. For growth performed at pH 5.3 or 4.5, maintenance of a pH potential above 1 unit due to L-malate metabolism was dependent on the presence of either L-malate or citrate during growth. Interestingly, at pH 3.2, only cells grown with L-malate produced a pH potential over 1.5 unit whatever the buffer used.

Finally, study of the expression of genes involved in the metabolism of organic acids showed that the presence of L-malate in the medium increased the amount of mleP mRNA at pH 4.5 and 3.2, whereas the expression of citrate genes was neither affected by pH nor by the presence of organic acids.

A 20 bp chromosomal deletion within the mae gene was achieved in L. lactis through a two step homologous recombination process. Growth of parental and mutant strains was performed with or without citrate. The mutant remained able to metabolise the citrate. Acetate and diaceyl/acetoin production observed during growth showed that the mutant was still able to convert citrate into oxaloacetate and acetate. Results suggest that oxaloacetate was decarboxylated to pyruvate at least for a part, although the deletion mutant lacks oxaloacetate decarboxylase encoded by mae. In contrast, a delay in the lag phase was observed in M17 medium during growth of the mutant with citrate and its growth rate was at least 20% lower compared to the growth rate of the parental strain.

This suggested that generation of ATP was affected in the mutant strain. One hypothesis would be that the deletion of mae would hinder the ability to generate the delta-pH in the presence of citrate. This hypothesis was analysed by comparing the capacity of parental and mutant cells to generated transmembrane proton gradient in presence of citrate. The behaviour of the parental and mutant strains was different. Our result revealed that the intracellular pH of the mutant was identical in the presence or absence of citrate. These results suggest that the decarboxylation reaction play a role by consuming a proton in the cytoplasm, which results in a proton gradient over the cytoplasmic membrane.

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Laboratoire de Microbiologie UMR 1232 UB-INRA, Universite de Bourgogne
1 esplande Erasme
21000 Dijon
France
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