Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.     

Growth and Energy Generation by Lactococcus lactis subsp. lactis biovar diacetylactis during Citrate Metabolism

Growth of Lactococcus lactis subsp. lactis biovar diacetylactis was observed on media with citrate as the only energy source. At pH 5.6, steady state was achieved in a chemostat on a citrate-containing medium in the absence of a carbohydrate. Under these conditions, pyruvate, acetate, and some acetoin and butanediol were the main fermentation products. This indicated that energy was conserved in L. lactis subsp. lactis biovar diacetylactis during citrate metabolism and presumably during the conversion of citrate into pyruvate. The presumed energy-conserving step, decarboxylation of oxaloacetate, was studied in detail. Oxaloacetate decarboxylase was purified to homogeneity and characterized. The enzyme has a native molecular mass of approximately 300 kDa and consists of three subunits of 52, 34, and 12 kDa. The enzyme is apparently not sodium dependent and does not contain a biotin moiety, and it seems to be different from the energy-generating oxaloacetate decarboxylase from Klebsiella pneumoniae. Energy-depleted L. lactis subsp. lactis biovar diacetylactis cells generated a membrane potential and a pH gradient immediately upon addition of citrate, whereas ATP formation was slow and limited. In contrast, lactose energization resulted in rapid ATP formation and gradual generation of a proton motive force. These data were confirmed during studies on amino acid uptake. α-Aminoisobutyrate uptake was rapid but glutamate uptake was slow in citrate-energized cells, whereas lactose-energized cells showed the reverse tendency. These data suggest that, in L. lactis subsp. lactis bv. diacetylactis, a proton motive force could be generated during citrate metabolism as a result of electrogenic citrate uptake or citrate/product exchange together with proton consumption by the intracellular oxaloacetate decarboxylase.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.     

Citrate can partially replace carbon dioxide required for growth of Lactococcus lactis subsp. lactis biovar diacetylactis

Lactococcus lactis subsp. lactis biovar diacetylactis was grown as batch cultures on a chemically defined medium. No growth was observed when the cultures were sparged with pure nitrogen (1.3 l l-1 min-1) whereas the cultures displayed exponential growth in the presence of minute amounts of carbon dioxide (0.035 mol-% of the inlet gas). However, in the former case, the addition of citrate restored growth. This suggested that oxaloacetate required for aspartate biosynthesis can be formed by the carboxylation of pyruvate or by citrate catabolism. When the cultures were heavily sparged with nitrogen (2.6 l l-1 min-1), no growth was observed even in the presence of citrate. This indicated that growth in these conditions was repressed by the absence of carbon dioxide required in some other biosynthetic reaction than in the carboxylation of pyruvate leading to oxaloacetate/aspartate biosynthesis.     

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.     

Genetic organization and expression of citrate permease in lactic acid bacteria

Citrate is present in many natural substrates, such as milk, vegetables and fruits, and its metabolism by lactic acid bacteria (LAB) plays an important role in food fermentation. The industrial importance of LAB stems mainly from their ability to convert carbohydrates into lactic acid and, in some species, like Lactococcus lactis and Leuconostoc mesenteroides, to produce C4 flavor compounds (diacetyl, acetoin) through citrate metabolism. Three types of genetic organization and gene locations, involving citrate metabolism, have been found in LAB. Citrate uptake is mediated by a citrate permease, which leads to a membrane potential upon electrogenic exchange of divalent citrate and monovalent lactate. The internal citrate is cleaved into acetate and oxaloacetate by a citrate lyase, and oxaloacetate is decarboxylated into pyruvate by an oxaloacetate decarboxylase, yielding a pH gradient through the consumption of scalar protons.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Formation of diacetyl and acetoin by Lactococcus lactis via aspartate catabolism

Aims:  To verify whether diacetyl can be produced by Lactococcus lactis via amino acid catabolism, and to investigate the impact of the pH on the conversion.
Methods and Results:  Resting cells of L. lactis were incubated in reaction media at different pH values, containing l -aspartic acid or l -alanine as a substrate. After incubation, the amino acid and metabolites were analysed by HPLC and GC/MS. At pH 5 about 75% of aspartic acid and only 40% of alanine was degraded to pyruvate via a transamination step that requires the presence of α-ketoglutarate in the medium, but diacetyl was only produced from aspartic acid. Three per cent of pyruvate was transformed to acetolactate of which 50% was converted into diacetyl. At pH 5·5 and above the pyruvate conversion into acetolactate was less efficient than at pH 5, and acetolactate was mainly decarboxylated to acetoin.
Conclusions:  Acetoin and diacetyl can be formed as a result of aspartate or alanine catabolism by L. lactis in the presence of α-ketoglutarate in the medium.
Significance and Impact of the Study:  Lactic acid bacteria exhibiting both glutamate dehydrogenase activity and high aspartate aminotransferase activity are expected to be good diacetyl producers during cheese ripening at pH close to 5.     

Molecular characterization of plasmids pS7a and pS7b from Lactococcus lactis subsp. lactis bv. diacetylactis S50 as a base for the construction of mobilizable cloning vectors

Aims:  Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar.
Methods and Results:  Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four Eco RI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells.
Conclusions:  Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids.
Significance and Impact of the Study:  Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.     

Contribution of Citrate Metabolism to the Growth of Lactococcus lactis CRL264 at Low pH

Lactococcus 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.     

Isolation of chromosomal mutations of Lactococcus lactis subsp. lactis biovar. diacetylactis 18-16 after introduction of Tn919

Abstract: The conjugative transposon Tn 919 was introduced at high frequency to L. lactis subsp. lactis biovar. diacetylactis 18-16 and transconjugants were screened for mutations in two chromosomally located genotypes; citrate metabolism and maltose utilization. A citrate negative mutant, lacking citritase activity, was isolated at a frequency of 1.18 × 10⁻⁴. The mutant, 18-16C5, contained a single copy of Tn 919 in a chromosomal location. A junction fragment of Tn 919 ::18-16C5 chromosomal DNA was cloned in Escherichia coli . Mutations in maltose metabolism were detected at a frequency of 4.0 × 10⁻⁴. No mutants were detected when Tn 919 was not introduced. Reversion to a Mal⁺ phenotype occurred at high frequency, but was not due to Tn 919 transposition.     

The citrate metabolic pathway in Leuconostoc mesenteroides: expression, amino acid synthesis, and alpha-ketocarboxylate transport.

Citrate metabolism in Leuconostoc mesenteroides subspecies mesenteroides is associated with the generation of a proton motive force by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178-2185, 1996). The pathway consists of four steps: (i) uptake of citrate, (ii) splitting of citrate into acetate and oxaloacetate, (iii) pyruvate formation by decarboxylation of oxaloacetate, and (iv) reduction of pyruvate to lactate. Studies of citrate uptake and metabolism in resting cells of L. mesenteroides grown in the presence or absence of citrate show that the citrate transporter CitP and citrate lyase are constitutively expressed. On the other hand, oxaloacetate decarboxylase is under stringent control of the citrate in the medium and is not expressed in its absence, thereby blocking the pathway at the level of oxaloacetate. Under those conditions, the pathway is completely directed towards the formation of aspartate, which is formed from oxaloacetate by transaminase activity. The data indicate a role for citrate metabolism in amino acid biosynthesis. Internalized radiolabeled aspartate produced from citrate metabolism could be chased from the cells by addition of the amino acid precursors oxaloacetate, pyruvate, alpha-ketoglutarate, and alpha-ketoisocaproate to the cells, indicating a broad specificity of the transamination reaction. The alpha-ketocarboxylates are readily transported across the cytoplasmic membrane. alpha-Ketoglutarate uptake in resting cells of L. mesenteroides was dependent upon the presence of an energy source and was inhibited by inhibition of the proton motive force generating F(0)F(1) ATPase and by selective dissipation of the membrane potential and the transmembrane pH gradient. It is concluded that in L. mesenteroides alpha-ketoglutarate is transported via a secondary transporter that may be a general alpha-ketocarboxylate carrier.     

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish

Aims:  We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture.
Methods and Results:  In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l -arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate.
Conclusions:  Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described.
Significance and Impact of the Study:  The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.     

Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NF-kappaB induced transcription

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the K_m value for oxaloacetate was 0.52 ± 0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.     

天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。     

Inhibition of Listeria monocytogenes by acetate, benzoate and sorbate: weak acid tolerance is not influenced by the glutamate decarboxylase system

Aims:  Weak acids are widely used by the food industry to prevent spoilage and to inhibit the growth of pathogenic micro-organisms. In this study the inhibitory effects of three commonly used weak acids, acetic acid, benzoic acid and sorbic acid, on the growth of Listeria monocytogenes were investigated.
Methods and Results:  In a chemically defined medium at pH 6·4 benzoic acid had the greatest inhibitory effect (50% inhibition of growth at 4 mmol l⁻¹), while acetate was the least inhibitory (50% inhibition of growth at 50 mmol l⁻¹). Mutants lacking either sigmaB (Δ sigB ) or two of the glutamate decarboxylase systems (Δ gadAB ) were used to investigate the contribution these systems make to weak acid tolerance in L. monocytogenes .
Conclusions:  The stress-inducible sigma factor sigmaB (σ^B) was not required for protection against acetate and played only a minor role in tolerating benzoate and sorbate. The glutamate decarboxylase system, which plays an important role in tolerating inorganic acids, played no significant role in the ability of L. monocytogenes to tolerate these weak acids, and neither did the presence of glutamate in the growth medium.
Significance and Impact of the Study:  These results suggest that the effectiveness of weak acid preservatives in food will not be compromised by the presence of glutamate, at least under mildly acidic conditions.     

Transposition of IS10R in Lactococcus lactis

Aims:  To characterize the transposition mechanism of the IS-element IS 10 R and study how this element is involved in gene disruption in Lactococcus lactis .
Methods and Results:  The gene flciA confers immunity against lactococcin A in lactococci. However, the immunity function was lost when flciA was co-expressed with the regulator gene nisR on a plasmid in L. lactis NZ9000. By PCR and DNA sequencing, it was revealed that flciA in immune-negative transformants was disrupted by the IS-element IS 10 R. Such gene disruption did not occur when flciA was expressed alone nor when the plasmid-located nisR was mutated, suggesting that nisR is directly involved in the transposition. The sequence 5'-CACTTAACC-3', which was found in flciA and at both ends of the inserted IS 10 R, was identified as target site by site-directed mutagenesis.
Conclusions:  IS 10 R transposes in L. lactis NZ9000 in a nisR -dependent fashion and employs the sequence 5'-CACTTAACC-3' as integration site.
Significance and Impact of the Study:  To our knowledge, this is the first time IS 10 R and aspects of its transposition are described in the industrial important bacterium L. lactis . The highly controllable insertion of IS 10 R into a target site might present a great potential as a gene disruption system.