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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↗
PubMed2006

Glutamate dehydrogenase activity can be transmitted naturally to Lactococcus lactis strains to stimulate amino acid conversion to aroma compounds.

Amino acid conversion to aroma compounds by Lactococcus lactis is limited by the low production of alpha-ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to alpha-ketoglutarate was detected in L. lactis strains isolated from a vegetal source, and the gene responsible for the activity in L. lactis NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study, gdh gene inactivation and overexpression confirmed the direct impact of GDH activity of L. lactis on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying gdh was naturally transmitted to another L. lactis strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.

Amino Acids↗
PubMed2006

Complementation of the Lactococcus lactis secretion machinery with Bacillus subtilis SecDF improves secretion of staphylococcal nuclease.

Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30 degrees C) and was even greater at 15 degrees C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.

Bacillus subtilis↗
PubMed2006

Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4.

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity.

Amino Acid Sequence↗
PubMed2001

Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin.

The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.

Administration, Intranasal↗
PubMed1998

Binding properties of Streptococcus gordonii SspA and SspB (antigen I/II family) polypeptides expressed on the cell surface of Lactococcus lactis MG1363.

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii.

Adhesins, Bacterial↗
PubMed1998

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine.

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.

Administration, Intranasal↗
PubMed2000

Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis.

In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogen Streptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter(-1)) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 x 10(6) CFU of live recombinant lactococci expressing a total of approximately 0.5 microgram of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 microgram of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

Animals↗
PubMed2000

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Bacterial Adhesion↗
PubMed2003

Intranasal immunization with recombinant Lactococcus lactis secreting murine interleukin-12 enhances antigen-specific Th1 cytokine production.

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-gamma) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-gamma production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-gamma) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

Administration, Intranasal↗
PubMed2004

Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C.

The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.

Animals↗
PubMed2004

Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes.

A novel vaccine (LL-CRR) made from live, nonpathogenic Lactococcus lactis that expresses the conserved C-repeat region (CRR) of M protein from Streptococcus pyogenes serotype 6 was tested in mice. Nasally vaccinated mice produced CRR-specific salivary immunoglobulin A (IgA) and serum IgG. Subcutaneously vaccinated mice produced CRR-specific serum IgG but not salivary IgA. A combined regimen produced responses similar to the salivary IgA of nasally vaccinated mice and serum IgG of subcutaneously vaccinated mice. Mice vaccinated nasally or with the combined regimen were significantly protected against pharyngeal infection following a nasal challenge with S. pyogenes M serotype 14. Mice vaccinated subcutaneously were not protected against pharyngeal infection. Mice in all three LL-CRR vaccination groups were significantly protected against the lethal effects of S. pyogenes. Only 1 of 77 challenged mice that were vaccinated with LL-CRR died, whereas 60 of 118 challenged mice that were vaccinated with a control strain or phosphate-buffered saline died. In conclusion, mucosal vaccination with LL-CRR produced CRR-specific salivary IgA and serum IgG, prevented pharyngeal infection with S. pyogenes, and promoted survival.

Administration, Intranasal↗
PubMed2006

GlnR-mediated regulation of nitrogen metabolism in Lactococcus lactis.

We show that the nitrogen regulatory protein GlnR of Lactococcus lactis represses transcription of the amtB-glnK, glnRA, and glnPQ operons. This likely occurs through a conserved DNA motif, 5'-TGTNA-7N-TNACAT-3', and takes place in response to extracellular glutamine and ammonium. GlnR-independent repression of amtB-glnK is mediated by the pleiotropic nitrogen regulator CodY.

Bacterial Proteins↗
PubMed2006

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.

Base Sequence↗
PubMed2006

Genome sequence and global gene expression of Q54, a new phage species linking the 936 and c2 phage species of Lactococcus lactis.

The lytic lactococcal phage Q54 was previously isolated from a failed sour cream production. Its complete genomic sequence (26,537 bp) is reported here, and the analysis indicated that it represents a new Lactococcus lactis phage species. A striking feature of phage Q54 is the low level of similarity of its proteome (47 open reading frames) with proteins in databases. A global gene expression study confirmed the presence of two early gene modules in Q54. The unusual configuration of these modules, combined with results of comparative analysis with other lactococcal phage genomes, suggests that one of these modules was acquired through recombination events between c2- and 936-like phages. Proteolytic cleavage and cross-linking of the major capsid protein were demonstrated through structural protein analyses. A programmed translational frameshift between the major tail protein (MTP) and the receptor-binding protein (RBP) was also discovered. A "shifty stop" signal followed by putative secondary structures is likely involved in frameshifting. To our knowledge, this is only the second report of translational frameshifting (+1) in double-stranded DNA bacteriophages and the first case of translational coupling between an MTP and an RBP. Thus, phage Q54 represents a fascinating member of a new species with unusual characteristics that brings new insights into lactococcal phage evolution.

Amino Acid Sequence↗
PubMed2006

Time-resolved determination of the CcpA regulon of Lactococcus lactis subsp. cremoris MG1363.

Carbon catabolite control protein A (CcpA) is the main regulator involved in carbon catabolite repression in gram-positive bacteria. Time series gene expression analyses of Lactococcus lactis MG1363 and L. lactis MG1363DeltaccpA using DNA microarrays were used to define the CcpA regulon of L. lactis. Based on a comparison of the transcriptome data with putative CcpA binding motifs (cre sites) in promoter sequences in the genome of L. lactis, 82 direct targets of CcpA were predicted. The main differences in time-dependent expression of CcpA-regulated genes were differences between the exponential and transition growth phases. Large effects were observed for carbon and nitrogen metabolic genes in the exponential growth phase. Effects on nucleotide metabolism genes were observed primarily in the transition phase. Analysis of the positions of putative cre sites revealed that there is a link between either repression or activation and the location of the cre site within the promoter region. Activation was observed when putative cre sites were located upstream of the hexameric -35 sequence at an average position of -56.5 or further upstream with decrements of 10.5 bp. Repression was observed when the cre site was located in or downstream of putative -35 and -10 sequences. The highest level of repression was observed when the cre site was present at a defined side of the DNA helix relative to the canonical -10 sequence. Gel retardation experiments, Northern blotting, and enzyme assays showed that CcpA represses its own expression and activates the expression of the divergently oriented prolidase-encoding pepQ gene, which constitutes a link between regulation of carbon metabolism and regulation of nitrogen metabolism.

Bacterial Proteins↗
PubMed1989

Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis.

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.

Amino Acids↗
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↗