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Model for the role of citrate metabolism in the resistance of lactococcus lactis to acidic stress

Citrate present in milk is cometabolised with sugars by strains of lactic acid bacteria (LAB), including Lactococcus lactis biovar diacetylactis (L. diacetylactis). The breakdown of citrate results in production of carbon dioxide (responsible for the texture of some cheeses) and production of the flavour compound diacetyl, which is essential for the quality of dairy products such as butter, buttermilk, and cottage cheese. In addition, citrate metabolism results in synthesis of lactic acid, which has an important role in the hygienic quality of fermented food, since impairs the growth of pathogens. Therefore, a bacterial strain, able to produce aroma compounds and at the same time been acid producer and acid resitant, will have a very good potential as starter for dairy fermentations.

This work focused on the study of citrate metabolism by L. diacetylactis CRL264 isolated from artisanal argentinean cheese and its contribution to pH homeostasis and acid stress resistance. We used tightly controlled pH conditions and studied the effect of citrate metabolism on the growth parameters. This approach allowed assessing the cellular response to acid pH independently of the pH changes concomitant with the growth of batch cultures. The toxic effect of the organic lactic and acetic acids accumulated during the fermentation processes was also examined. The CRL264 strain harboring pCIT264 (which encodes the citrate permease P), and its derivatives CRL30 (CRL264 cured of pCIT264) and CRL30pCIT (CRL30 transformed with pCIT264) were studied.

The growth characteristics of these strains on glucose with/without citrate were compared. Under controlled pH, growth on glucose plus citrate is characterised by higher specific growth rate but lower biomass production; at the onset of citrate exhaustion, the specific growth rate decreased 7-fold. Acetic acid was the primary cause for this decrease upon citrate depletion and, also for the decrease in biomass when higher concentrations of citrate were catabolised. In vivo 13C-NMR confirm that during co-metabolism all acetate formed is derived from citrate, and glucose is converted to lactate. Addition of arginine combined with citrate led to a remarkable enhancement of the final biomass, showing the beneficial effect of the extra ATP derived from arginine to counteract the deleterious effects of weak acids on the magnitude of the proton motive force. In line with this hypothesis, lower ATP levels were measured by 31P-NMR in cells metabolizing glucose plus citrate as compared to glucose alone.

Given the crucial influence of citrate metabolism on the performance of CRL264 at low pH, its capacity for citrate uptake was analysed. Transport assays showed that in the parental strain CRL264 the citrate transport is 2.7 fold faster than in its derivative CRL30pCIT, correlating with a 2 fold higher copy number of pCIT264 than pCIT. This enhanced efficiency in the transport correlates with the better performance of CRL264 at acidic pH. Based on these results, we propose that increasing the transport capacity of CRL264 could be a useful strategy to improve furthers the performance of this strain under acidic conditions, a trait remarkably important in industrial fermentations.

NMR allows distinguishing between intra and extracellular lactate. In this work, we obtained in vivo 13C-NMR data that showed a decrease of the intracellular lactate pool when a pulse of citrate was given to the cells. These results suggest that the transport system operating in CRL264 strain is a citrate/lactate exchanger system, which provides the cells with an advantage against lactate toxicity.

The oxaloacetate decarboxylase activity results essential in the alcalinisation of the cytosol during citrate fermentation in L. diacetylactis. In this work, we demonstrated that citM gene from L. lactis CRL264 encodes an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes from other organisms. CitM was purified and its oxaloacetate decarboxylase activity demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the Km value for oxaloacetate was 0.52 +/- 0.03mM.

In addition, in this work the citM-CDEFXG genes encoding the subunits of the citrate lyase of CRL264 have been characterised. The genes are transcribed as a single polycistronic mRNA of 8.6kb, and their expression is induced at low pH. This response to acid stress takes place at the transcriptional level and result in an increase of enzyme levels, demonstrated by proteomic analysis, and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells more resistant to the inhibitory effects of the lactate fermentation product, which accumulates under these conditions.

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Instituto de Biologia Experimental e Tecnologica
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