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PubMed2006

Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization.

The present work reports the use of non-living non-recombinant bacteria as a delivery system for mucosal vaccination. Antigens are bound to the cell-wall of pretreated Lactococcus lactis, designated as Gram-positive enhancer matrix (GEM), by means of a peptidoglycan binding domain. The influence of the GEM particles on the antigen-specific serum antibody response was studied. Following nasal immunization with the GEM-based vaccines, antibody responses were induced at systemic and local levels. Furthermore, different GEM-based vaccines could be used consecutively in the same mice without adverse effects or loss of activity. Taken together, the results evidence the adjuvant properties of the GEM particles and indicate that GEM-based vaccines can be used repeatedly and are particularly suitable for nasal immunization purposes.

Administration, Intranasal↗
PubMed2006

Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.

In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels.

Amino Acid Sequence↗
PubMed2006

Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae.

A comparative immunoproteomic study was carried out to investigate the immunogenicity of capsulate (KG9408) and non-capsulate (NSS9310) strains of Lactococcus garvieae. Immunoblot assays, following two-dimensional gel electrophoresis (2-DE) for L. garvieae strains, revealed a significant difference between anti-capsulate and anti-non-capsulate rabbit sera with respect to the number and antigenicity of antigenic spots. Anti-capsulate and anti-non-capsulate rabbit sera reacted with an average of 72 and 127 antigenic spots, respectively. The strong reaction of anti-non-capsulate sera with elongation factor (EF)-G and -Tu, and GMP synthase, of the L. garvieae strains identifies these as specific major antigens. This study clearly demonstrates the differences in 2-DE immunoblot profiles between the capsulate and non-capsulate strains of L. garvieae. These differences may be the reason for variations in immunogenicity between capsulate and non-capsulate strains. Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, arginine deaminase and ornithine carbamoyltransferase were identified from the 2-DE immunoblot profiles of both strains. Therefore, these common antigens are potential markers for the development of vaccines against L. garvieae, irrespective of strain. Immunoproteomics, a powerful tool for studying antigens at the proteomic level, allowed a comparative investigation of the immunogenicity of capsulate and non-capsulate strains of L. garvieae for vaccine development.

Animals↗
PubMed2004

Complete genome sequence of the Lactococcus lactis temperate phage phiLC3: comparative analysis of phiLC3 and its relatives in lactococci and streptococci.

Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage phiLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. phiLC3 contained a unique replication module and orf201 was identified as the putative replication initiator protein-encoding gene. phiLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. phiLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.

Base Sequence↗
PubMed2004

Multivitamin production in Lactococcus lactis using metabolic engineering.

The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).

Folic Acid↗
PubMed2006

Natural sweetening of food products by engineering Lactococcus lactis for glucose production.

We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for glucokinase (glk), EII(man/glc) (ptnABCD), and the newly discovered glucose-PTS EII(cel) (ptcBAC). After introducing the lactose metabolic genes, the deletion strain could solely ferment the galactose moiety of lactose, while the glucose moiety accumulated extracellularly. Additionally, less lactose remained in the medium after fermentation. The resulting strain can be used for in situ production of glucose, circumventing the need to add sweeteners as additional ingredients to dairy products. Moreover, the enhanced removal of lactose achieved by this strain could be very useful in the manufacture of products for lactose intolerant individuals.

Escherichia coli Proteins↗
PubMed2006

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host.

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.

Biotechnology↗
PubMed2003

Substrate inhibition of Lactococcus lactis cytidine 5'-triphosphate synthase by ammonium chloride is enhanced by salt-dependent tetramer dissociation.

Cytidine 5(')-triphosphate (CTP) synthase (EC 6.4.3.2) catalyzes the transfer of an amino group to the 4 position of uridine 5(')-triphosphate (UTP) to yield CTP. The reaction proceeds by activation of the base moiety of UTP by adenosine 5(')-triphosphate (ATP)-dependent phosphorylation. The activated intermediate reacts with NH(3) in the solution or is obtained by hydrolysis of glutamine. The Lactococcus lactis CTP synthase shows significant differences from the enzymes from Escherichia coli, yeast, and mammals. One is the apparent stability of the L. lactis CTP synthase tetramer in the absence of the nucleotides ATP and UTP. This condition causes the E. coli, yeast, and mammal enzymes to dissociate into dimers. However, the L. lactis CTP synthase shows substrate inhibition by NH(4)Cl that coincides with the range of NH(4)Cl concentrations that apparently dissociates tetrameric enzyme into dimers. Even though regular substrate inhibition was observed with NH(4)Cl when the ionic strength was held constant, a significant part of the inhibition could be shown to be due to the increase in ionic strength with increasing substrate concentration. Since the substrate inhibition by NH(4)Cl was relieved by increasing the equimolar ATP and UTP concentrations, it appeared that the substrate nucleotides stabilized the tetramer in a manner similar to that found in the absence of salt for other CTP synthases. In contrast to the suggested hydrophobic nature of the tetramer interactions in E. coli CTP synthase, the dissociation of the L. lactis CTP synthase tetramer in response to an increase in ionic strength suggests that the tetramer is stabilized by ionic interactions.

Adenosine Triphosphate↗
PubMed2002

Multidrug transporters and antibiotic resistance in Lactococcus lactis.

The Gram-positive bacterium Lactococcus lactis produces two distinct multidrug transporters, designated LmrA and LmrP, that both confer resistance to a wide variety of cationic lipophilic cytotoxic compounds as well as to many clinically relevant antibiotics. While LmrP is a proton/drug antiporter that belongs to the major facilitator superfamily of secondary transporters, LmrA is an ATP-dependent primary transporter that belongs to the ATP-binding cassette superfamily of transport proteins. Both LmrA and LmrP function as "hydrophobic vacuum cleaners" by excreting lipophilic cationic compounds from the inner leaflet of the membrane directly into the external water phase. LmrA is both functionally and structurally homologous to the human multidrug transporter P-glycoprotein. LmrA is a half ABC transporter that is functional as a homodimer, consistent with the general four-domain organization of ABC transporters, and is proposed to mediate drug transport by an alternating two-site transport mechanism.

ATP-Binding Cassette Transporters↗
PubMed2003

Lactococcus lactis as host for overproduction of functional membrane proteins.

Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.

Animals↗
PubMed1999

Structure-function analysis of multidrug transporters in Lactococcus lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

ATP-Binding Cassette Transporters↗
PubMed2000

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B891.

Lactococcus lactis subsp. cremoris B891 grown on whey permeate produced an exopolysaccharide containing D-Gal and D-Glc in a molar ratio of 2:3. The polysaccharide was partially O-acetylated. By means of HF solvolysis, O-deacetylation, enzymic modification, sugar linkage analysis and ID/2D NMR studies the exopolysaccharide was shown to be composed of repeating units with the following structure: [structure: see text].

Acetylation↗
PubMed2000

Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp. cremoris B39 and B891.

Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp. cremoris B39 and B891. The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate. The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures. The enzyme degraded O-deacetylated EPS B891 faster than EPS B39. Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.

Acetylation↗
PubMed1999

Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp. cremoris B40.

EPS B40 from Lactococcus lactis subsp. cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-[alpha-L-Rhap-(1 -->2)][alpha-D-Galp-1-PO4-3]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment. Purified endoV from T. viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed. After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers. Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV [J.-P. Vincken, G. Beldman, A.G.J. Voragen, Carbohydr. Res., 298 (1997) 299-310].

Carbohydrate Sequence↗
PubMed2000

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B39.

Lactococcus lactis subsp. cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2. The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide. Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: [structure: see text].

Aspergillus↗
PubMed1999

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 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) 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.

Aminopeptidases↗
PubMed2003

Identification of the host determinant of two prolate-headed phages infecting Lactococcus lactis.

A gene responsible for host determination was identified in two prolate-headed bacteriophages of the c2 species infecting strains of Lactococcus lactis. The identification of the host determinant gene was based on low DNA sequence homology in a specific open reading frame (ORF) between prolate-headed phages with different host ranges. When a host carrying this ORF from one phage on a plasmid was infected with another phage, we obtained phages with an altered host range at a frequency of 10(-6) to 10(-7). Sequencing of phage DNA originating from 10 independent single plaques confirmed that a genetic recombination had taken place at different positions between the ORF on the plasmid and the infecting phage. The adsorption of the recombinant phages to their bacterial hosts had also changed to match the phage origin of the ORF. Consequently, it is concluded that this ORF codes for the host range determinant.

Amino Acid Sequence↗
PubMed2002

Functional study of Lactococcus lactis RNase III in Escherichia coli.

Endoribonuclease III (RNase III) is known to participate in a number of RNA maturation and decay pathways. Previous research carried out on citQRP mRNA processing had provided strong evidence that RNase III exists in Lactococcus lactis and acts as a major endoribonuclease in the control of citQRP mRNA stability. The DNA coding (rnc) for the putative lactococcal RNase III was cloned by PCR technology. In addition, we provide the transcriptional analysis of rnc gene and compared the lactococcal RNase III sequence to those of Gram-positive and Gram-negative organisms. This computer-assisted comparison showed an identity ranking from 37% to 54%, and the highest score was obtained with Streptococcus pneumoniae RNase III. Moreover, heterologous complementation indicated that lactococcal RNase III is able to complement the loss of Escherichia coli RNase III for both 30S RNA processing and lambda N posttranscriptional regulator.

Amino Acid Sequence↗