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PubMed2006

Differential expression of proteins and genes in the lag phase of Lactococcus lactis subsp. lactis grown in synthetic medium and reconstituted skim milk.

We investigated protein and gene expression in the lag phase of Lactococcus lactis subsp. lactis CNRZ 157 and compared it to the exponential and stationary phases. By means of two-dimensional polyacrylamide gel electrophoresis, 28 highly expressed lag-phase proteins, implicated in nucleotide metabolism, glycolysis, stress response, translation, transcription, cell division, amino acid metabolism, and coenzyme synthesis, were identified. Among the identified proteins, >2-fold induction and down-regulation in the lag phase were determined for 12 proteins in respect to the exponential phase and for 18 proteins in respect to the stationary phase. Transcriptional changes of the lag-phase proteins in L. lactis were studied by oligonucleotide microarrays. Good correlation between protein and gene expression studies was demonstrated for several differentially expressed proteins, including nucleotide biosynthetic enzymes, adenylosuccinate synthase (PurA), IMP dehydrogenase (GuaB), and aspartate carbamoyl transferase (PyrB); heat-shock protein DnaK; serine hydroxymethyl transferase (GlyA); carbon catabolite control protein (CcpA); elongation factor G (FusA); and cell division protein (FtsZ).

Animals↗
PubMed1998

The contribution of caseins to the amino acid supply for Lactococcus lactis depends on the type of cell envelope proteinase.

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing alphas1-, beta-, and kappa-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124-2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

Amino Acid Sequence↗
PubMed1989

Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein.

The chlorated dipeptide L-alanyl-beta-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant of L. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di- and tripeptide transport. Uptake of di- and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.

Amino Acids↗
PubMed2002

Production of bacteriocins from Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 using mussel-processing wastes.

The growth and bacteriocin production by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 were investigated on mussel-processing wastes. Both bacteriocin productions were satisfactorily modelled using a modified form of the Luedeking and Piret expression, which includes a term for the influence of the pH reduction rate. Experimental data from cultures buffered at different initial concentrations (0, 0.03, 0.10 and 0.25 M) of both bacteria were used to fit and verify the model. The influence of total sugars, nitrogen, phosphorus and buffer concentration on nisin and pediocin production was also studied using response-surface methodology and empirical modelling. Enhanced nisin production (33 BU/ml) was achieved in media buffered with 0.10 M potassium hydrogen phthalate/NaOH. However, the highest levels of pediocin (368 BU/ml) were obtained in the non-buffered media.

Animals↗
PubMed1994

Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

Acetolactate Synthase↗
PubMed1992

B-cell mitogenic activity of slime products produced from slime-forming, encapsulated Lactococcus lactis ssp. cremoris.

The mitogenic activities of whole cell lyophylized preparations, cell-wall components, and slime products obtained from Lactococcus lactis ssp. cremoris KVS20 were examined on murine spleen cells. Whole cell lyophylized preparations and slime products significantly (P < .05) stimulated mitogenic responses of the cells. The highest activity was induced by slime products in which the optimal concentration was 116 microg/ml. The significant (P < .05) increase of mitogenic activity induced by slime products occurred at 24 h, and the peak response was obtained 48 h after the stimulation. The activity was much higher in the fraction enriched with B cells than in the fraction enriched with T cells. In addition, slime products induced mitogenic activity to spleen cells of athymic nu/nu mice. The chemical analysis of lipopolysaccharide and the minimal concentration for mitogenic response eliminated the possibility that the activity of slime products may be due to the contamination of lipopolysaccharide. The data demonstrate that slime products are a potent B-cell-dependent mitogen.

Animals↗
PubMed1995

Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.

Aerobiosis↗
PubMed2006

Transfer of nisin gene cluster from Lactococcus lactis ATCC 11454 into the chromosome of Bacillus subtilis 168.

Nisin is an antimicrobial peptide produced by certain strains of Lactococcus lactis. It is a gene-encoded peptide that contains unusual amino acid residues. These novel residues are introduced by posttranslational modification machinery and confer unique chemical and physical properties that are not attainable by regular amino acid residues. To study the modification mechanisms and to create structural analogs with superior properties, it would be advantageous to insert the nisin genes into a bacterial strain that is amenable to genetic manipulation. In this study, we report the cloning and integration of the complete and intact nisin gene cluster into the Bacillus subtilis 168 chromosome. Furthermore, we demonstrate that the nisin genes are transcriptionally active. These results should greatly facilitate the studies of the genes and proteins involved in nisin expression, as well as provide a standard system for the manipulation and expression of genes involved in other members of the lantibiotic family of antimicrobial peptides.

Bacillus subtilis↗
PubMed1996

AbiG, a genotypically novel abortive infection mechanism encoded by plasmid pCI750 of Lactococcus lactis subsp. cremoris UC653.

AbiG is an abortive infection (Abi) mechanism encoded by the conjugative plasmid pCI750 originally isolated from Lactococcus lactis subsp. cremoris UC653. Insensitivity conferred by this Abi manifested itself as complete resistance to phi 712 (936 phage species) with only partial resistance to phi c2 (c2 species). The mechanism did not inhibit phage DNA replication. The smallest subclone of pCI750 which expressed the Abi phenotype contained a 3.5-kb insert which encoded two potential open reading frames. abiGi (750 bp) and abiGii (1,194 bp) were separated by 2 bp and appeared to share a single promoter upstream of abiGi. These open reading frames showed no significant homology to sequences of either the DNA or protein databases; however, they did exhibit the typical low G+C content (29 and 27%, respectively) characteristic of lactococcal abi genes. In fact, the G+C content of a 7.0-kb fragment incorporating the abiG locus was 30%, which may suggest horizontal gene transfer from a species of low G+C content. In this context, it is notable that remnants of IS elements were observed throughout this 7.0-kb region.

Amino Acid Sequence↗
PubMed2003

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp cremoris P8-2-47.

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered (copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.

Amino Acid Sequence↗
PubMed1996

Identical transcriptional control of the divergently transcribed prtP and prtM genes that are required for proteinase production in lactococcus lactis SK11.

We have investigated transcriptional regulation of the divergently transcribed genes required for proteinase production (prtP and prtM) of Lactococcus lactis SK11. Their promoters partially overlap and are arranged in a face-to-face configuration. The medium-dependent activities of both prtP and prtM promoters were analyzed by quantitative primer extension studies and beta-glucuronidase assays with L. lactis MG1363 cells harboring transcriptional gene fusions of each promoter with the promoterless beta-glucuronidase gene (gusA) from Escherichia coli. High-level production of prtP- or prtM-specific mRNAs was found after the growth of cells in media with low peptide concentrations, while increases in peptide concentrations resulted in an approximately eightfold decrease in mRNA production. Furthermore, prtP and prtM promoters exhibited similar efficiencies under different growth conditions. Deletion analysis of the prt promoter region showed that all the information needed for full activity and regulation of the prtP and prtM promoters is retained within a 90-bp region which includes both transcription initiation sites. An inverted repeat sequence positioned around the prtP and prtM transcription initiation sites was disrupted by either deletion or insertion of a small DNA sequence to analyze their effects on the activities of both prtP and prtM promoters. The mutations affected the activities of these promoters only marginally at low peptide concentrations but resulted in 1.5- to 5-fold derepression at high peptide concentrations. These results indicate that the expression of both prtM and prtP genes is controlled in an identical manner via a control mechanism capable of repressing transcription initiation at high peptide concentrations.

Bacterial Proteins↗
PubMed2001

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis.

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.

Amino Acid Sequence↗
PubMed2000

Six putative two-component regulatory systems isolated from Lactococcus lactis subsp. cremoris MG1363.

The genetic elements specifying six putative two-component regulatory systems (2CSs) were identified on the chromosome of Lactococcus lactis MG1363. These 2CSs appear to represent distinct loci, each containing a histidine kinase and response-regulator-encoding gene pair. Transcriptional analysis of the six 2CSs was performed either by generating transcriptional fusions to a reporter gene or by primer extension. Two of the systems appeared to be expressed constitutively at a high level, whilst the remaining four exhibited growth-phase-dependent expression. Insertional mutagenesis established that the two constitutively expressed 2CSs are necessary for normal cell growth and/or survival. Mutational analysis of the remaining four systems revealed that they are implicated in susceptibility to extreme pH, osmotic or oxidative conditions, or the regulation of phosphatase activity in L. lactis.

Amino Acid Sequence↗
PubMed2002

Oral treatment with Lactococcus lactis expressing Staphylococcus hyicus lipase enhances lipid digestion in pigs with induced pancreatic insufficiency.

The Staphylococcus hyicus lip gene was cloned in Lactococcus lactis. Pancreatic insufficiency was induced by ligation of the pancreatic duct in pigs. In pigs who had undergone pancreatic ligation, the coefficient of fat absorption was higher after consumption of lipase-expressing L. lactis (91.9% +/- 3.7%) than that after consumption of the inactive control strain (78.4% +/- 2.4%).

Animals↗
PubMed1991

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis.

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.

Cloning, Molecular↗
PubMed1998

Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory apparatus.

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions -1 and -2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an inframe fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.

Anti-Bacterial Agents↗
PubMed1989

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.

Amino Acids, Diamino↗
PubMed2001

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29.

Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene ( gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated P (prtP) and P (prtM), respectively). beta-Glucuronidase assays and Northern blot analysis showed that the activities of P (prtP) and P (prtM) are controlled by casitone at the transcriptional level.

Amino Acid Sequence↗