Novel characteristic for distinguishing Lactococcus lactis subsp. lactis from subsp. cremoris.
Lactococcus lactis strains were examined for their ability to produce gamma-aminobutyric acid (GABA). Results showed that strains of L. lactis subsp. lactis were able to produce this acid, whereas L. lactis subsp. cremoris were not. GABA production thus represents another effective characteristic for distinguishing L. lactis subsp. lactis from L. lactis subsp. cremoris. (+info)
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. (+info)
A general method for selection of alpha-acetolactate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation.
The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the alpha-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains. (+info)
Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.
The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription. (+info)
Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation.
Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane. (+info)
Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2.
Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 126.96.36.199) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 188.8.131.52) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 184.108.40.206) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues. (+info)
Regulation of expression of the Lactococcus lactis histidine operon.
In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L. lactis. Analysis of the variable positions supports the folding model of the central part of the his leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the L. lactis operon by histidine. (+info)
Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria.
In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress. (+info)