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The contribution of citrate metabolism to the growth of Lactococcus lactis CRL264 at low pH

AuthorsSánchez, Claudia; Rute Neves, Ana; Cavalheiro, J.; Moreira dos Santos, M.; García-Quintáns, Nieves; López, Paloma ; Santos, Helena
Issue DateFeb-2008
PublisherAmerican Society for Microbiology
CitationApplied and Environmental Microbiology 74(4): 1136-1144 (2008)
AbstractLactococcus lactis subsp. lactis biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo 13C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions
The industrial relevance of Lactococcus lactis is related to its ability to convert sugars almost exclusively into lactic acid. This trait has been invaluable in food manufacturing because the accompanying acidification prevents spoilage. In recent years, the number of potential applications beyond the food industry increased considerably. In particular, the use of different strains as probiotics or live vehicles for the oral delivery of heterologous proteins of vaccinal or therapeutic interest has been extensively investigated (38). L. lactis grows optimally at pH values in the range of 6.3 to 6.9, but the lower limit for growth is within the range of 4.0 to 5.0, depending on the strain and the medium composition (12, 14, 15, 31). In most industrial processes, it is the accumulation of lactic acid at low pH that causes growth arrest; hence, the ability to cope with acid stress is very important. Moreover, the efficacy of L. lactis as a live carrier of therapeutics or antigens depends ultimately on the fraction of cells that survive the harsh pH conditions in the upper gastrointestinal tract. Consequently, a deep understanding of the underlying mechanisms of resistance to low pH is of current significance and is essential for the success of strain design. While it is well known that organic acids inhibit cell growth at low pH, the molecular mechanisms for survival under acid stress conditions are still poorly understood (18, 40). The primary mechanism responsible for pH homeostasis in bacteria relies on the expulsion of protons from the cytoplasm by H+-ATPases (17). The glutamate decarboxylase γ-aminobutyrate antiporter, the arginine deaminase pathway, and the citrate-utilizing pathway are additional processes contributing to pH homeostasis in L. lactis (4, 18, 34, 37). L. lactis subsp. lactis biovar diacetylactis CRL264 utilizes citrate and is notable for its ability to thrive at pH values that would preclude growth of most L. lactis strains (20, 35). Citrate metabolism in CRL264 has been investigated at the biochemical as well as the genetic level (10, 20, 21, 23). Citrate is transported via a plasmid-encoded carrier (CitP); citrate lyase cleaves citrate into acetate and oxaloacetate, which is subsequently decarboxylated to pyruvate by the action of oxaloacetate decarboxylase. The genes encoding these enzymes are located in a large chromosomal operon; moreover, it has been shown that a low pH, but not citrate, is required for the induction of the catabolic genes as well as the plasmid-borne genes (transporter and regulator). Given the remarkable ability of strain CRL264 to grow under severe acid stress conditions (20, 22), we deemed it important to obtain further insight into the physiological basis of this trait. Therefore, the growth of CRL264 and isogenic mutant strains with different citrate-uptake capacities was thoroughly characterized under tightly controlled pH conditions. In this way, the cellular response to low pH was assessed independently of the pH changes associated with metabolism. Nuclear magnetic resonance (NMR) was used as a complementary technique to characterize biochemical parameters in living cells
Description9 páginas, 6 figuras, 3 tablas -- PAGS nros. 1136-1144
Publisher version (URL)http://dx.doi.org/10.1128/​AEM.01061-07
Appears in Collections:(CIB) Artículos
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