CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

Abstract

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.Lactococcus lactis is widely used as a starter in the cheese industry, and consequently, all the enzymatic activities involved in its growth in milk and in the formation of aroma compounds are of major interest. Several studies have shown that the proteolytic system of L. lactis, especially the extracellular proteinase (PrtP) and the oligopeptide transport system (Opp), is essential for rapid growth of L. lactis in milk (6,18,21). Indeed, dairy strains are auxotrophic for a number of amino acids, generally including leucine, valine, isoleucine, histidine, and methionine, and the free amino acids in milk are not sufficient to support the growth of L. lactis at high cell densities. However, the proteolytic system does not provide all the amino acids necessary for growth in milk, and biosynthesis of certain amino acids, such as aspartic acid, is also necessary (8,34,35).Amino acid transamination, catalyzed by aminotransferases, is a reversible reaction and is therefore responsible for both the first step of amino acid catabolism and the last step of amino acid biosynthesis. Consequently, aminotransferases may be involved in the growth of L. lactis in milk either by providing the amino acids released at low concentrations by the proteolytic system or by degrading some amino acids, such as branchedchain amino acids, that accumulate in cells and mediate the repression of major components of the L. lactis proteolytic system (17). In L. lactis, repression of the proteolytic system occurs via the pleiotropic transcriptional repressor CodY, which se...

The Lactococcus lactis CodY Regulon

“…The bcaT gene is repressed by CodY in a chemically defined medium supplemented with casitone, a complex source of nitrogen, and in that respect comparable with the GM17 used in this work (13,33). Although expression of bcaT was significantly altered (p Ͻ 0.001) in our experiments as well, the extent of derepression measured on the DNA microarrays was somewhat low compared with that observed by Chambellon and Yvon (54). Changes in mRNA levels were lower for all the genes examined here for which CodY repression ratios have already been determined, yet the trends are comparable (13,(15)(16)(17).…”

“…Expression of the gene encoding the aminotransferase BcaT has been shown recently to be controlled by CodY (54). The bcaT gene is repressed by CodY in a chemically defined medium supplemented with casitone, a complex source of nitrogen, and in that respect comparable with the GM17 used in this work (13,33).…”

“…Thus, although L. lactis MG1363 like many other lactococcal dairy strains is auxotrophic for these amino acids, several genes of the BCAA biosynthetic operon are present on the chromosome and are actively transcribed (1,53). The gene encoding the aminotransferase BcaT, catalyzing either the first step of aromatic or branched-chain amino acid catabolism or the last step of their biosynthesis (54,55), was also significantly derepressed.…”

Branched chain aldehydes: production and breakdown pathways and relevance for flavour in foods

“…Extracellular molecules can influence the regulation of several reactions. As stated earlier, isoleucine is able to regulate the amino acid metabolism via CodY (Guedon et al 2001;Chambellon and Yvon 2003). Adding isoleucine to the cheese matrix should theoretically lead to lower 3-methyl butanal levels by high-3-methyl butanal producing strains.…”

Overall control of nitrogen metabolism in Lactococcus lactis by CodY, and possible models for CodY regulation in Firmicutes

“…Interestingly, studies in Lactococcus lactis have shown that in this bacterium, CodY responds to BCAA, but not GTP, suggesting that the activity of CodY is modulated differently between these two bacteria (den Hengst et al, 2005;Guedon et al, 2001b;Petranovic et al, 2004). In L. lactis, CodY has been found to repress genes involved in the utilization of proteins in the medium, including the cell-wall proteinase (prtP), peptide transporters (opp, opt), peptidases (pepC, pepN) and aminopeptidases (araT, bcaT) (Chambellon & Yvon, 2003;Guedon et al, 2001a, b;Sanz et al, 2001). CodY thus appears to be involved in external nitrogen supply, and its activity may be modulated by an intracellular pool of BCAA that would constitute an indicator of the nitrogen status of the cell (Guedon et al, 2001b;Petranovic et al, 2004).…”