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PubMed2006

Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: an FT-IR study.

The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in (2)H(2)O or (2)H(2)O with 1. 5 M sucrose in the range of 6-75 degrees Celsius. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The (1)H/(2)H exchange in proteins, and secondary structure elements were detected by the analysis of amide I', amide II and amide II' bands. A reduced (1)H/(2)H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the (1)H/(2)H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.

Amides↗
PubMed2006

A novel phenotype based on esterase electrophoretic polymorphism for the differentiation of Lactococcus lactis ssp. lactis and cremoris.

AIMS: To evaluate the esterase phenotype in Lactococcus lactis strains isolated from traditional Tunisian dairy products. METHODS AND RESULTS: A collection of 55 L. lactis strains isolated from traditional fermented milk products and three reference strains were identified at species and subspecies level using molecular methods targeted to the 16S rRNA gene and to the histidine operon. The genotypic data obtained allowed the identification of the strains as L. lactis ssp. lactis and L. lactis ssp. cremoris with the prevalence of the ssp. lactis. The phenotypic identification based on arginine hydrolysis, the growth at 40 degrees C and in presence of 4% NaCl showed several discrepancy with the identification data based on genotypic analysis. Additional experiments carried out evaluating the esterase electrophoretic patterns revealed four classes of esterases identified on the basis of their electrophoretic mobility and specific activity on alpha- and beta-naphthyl ester of acetate and propionate. Esterase profiles discriminated the strains in two main groups corresponding to the subspecies cremoris and lactis according to a DNA-based identification. CONCLUSIONS: The evaluation of esterase activity represents a novel phenotype for the taxonomic discrimination of the L. lactis ssp. lactis and cremoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Besides the DNA-based techniques that allow the rapid and accurate species/subspecies identification, the electrophoretic esterase profiles of L. lactis strains represents: (i) a new phenotypic tool to understand the physiology and the ecology of this species; and (ii) a new test for the potential selection of flavour producing strains.

Esterases↗
PubMed2003

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.

Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

Amino Acid Sequence↗
PubMed1996

Splicing of a group II intron in a functional transfer gene of Lactococcus lactis.

A chromosomally located sex factor that controls conjugation in Lactococcus lactis 712 has been cloned and sequenced, leading to the discovery of an open reading frame with homology to the maturases of group II self-splicing introns. Reverse transcriptase polymerase chain reaction amplification was used to demonstrate that the intron was spliced out of mRNA in vivo, and sequence analysis revealed the site of splicing. The intron was inserted within a sex-factor gene which encodes a protein with homology to proteins involved in rolling-circle DNA replication. Gene-disruption experiments were used to demonstrate that this mobA gene was essential for sex-factor transfer and this suggests that intron splicing is a necessary part of the conjugation process. The sequence of the intron was modelled to produce a secondary structure that exhibited several features characteristic of the IIA subgroup. Here we report the characterization of a new group II intron in the Gram-positive bacterium L. lactis and demonstrate for the first time in bacteria both splicing in vivo and an active role for the gene carrying the intron.

Amino Acid Sequence↗
PubMed1996

Analysis of heat shock gene expression in Lactococcus lactis MG1363.

The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnaJ and groEL homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnaJ and groELS after transfer from 30 degrees C to 43 degrees C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, orf1 and grpE, resembled the pattern observed for the above genes, although maximum induction was observed earlier for orf1 and grpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for ftsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock.

Bacterial Proteins↗
PubMed1999

Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis.

A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal theta-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 degrees C and 37 degrees C, whereas derivatives that lack cisE were highly unstable at 37 degrees C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal theta-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 degrees C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 degrees C.

Base Sequence↗
PubMed1995

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

Amino Acid Sequence↗
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↗
PubMed1992

Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis.

The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl beta-D-gluco-pyranoside and reconstituted into proteoliposomes. Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids. Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step. Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L. lactis. Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes (Driessen, A.J.M., Zheng, T., In't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation.

Amino Acids, Branched-Chain↗
PubMed1997

Nisin production by Lactococcus lactis using two-phase batch culture.

Nisin production by Lactococcus lactis subsp. lactis C2SmPrt-(TnNip) was investigated using two-phase batch culture. A solvent (phenyl-methyl silicone oil) was introduced in addition to the aqueous phase. The partition coefficient of this solvent in aqueous nisin varied as the cultivation medium changed, with a minimum value being 1.04. The two-phase batch culture supported a 21% increase in growth and a 24% increase in the level of nisin produced compared to the single-phase batch culture.

Bacteriological Techniques↗
PubMed1989

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.

Bacteriophages↗
PubMed1996

Purification of tributyrin esterase from Lactococcus lactis subsp. cremoris E8.

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at -20 degrees C, and had a half life of 1 h at 50 degrees C.

Amino Acid Sequence↗
PubMed1996

Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis.

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

Base Sequence↗
PubMed1995

Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp. lactis.

Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system. The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium. Sequence analysis revealed correct processing at the first amino acid of the mature protein. A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source.

Amino Acid Sequence↗
PubMed1996

Induction of IFN-gamma and IL-1 alpha production in macrophages stimulated with phosphopolysaccharide produced by Lactococcus lactis ssp. cremoris.

The induction of interferon (IFN) and interleukin-1 (IL-1) production in murine macrophages by a phosphopolysaccharide, produced by a dairy lactic acid bacteria, Lactococcus lactis ssp. cremoris, was investigated. When the phosphopolysaccharide was added into macrophage cultures at concentrations from 1 to 200 micrograms/ml, substantial IFN titers (6.2-79.2 IU/ml) were detected. Using the reverse transcription-polymerase chain reaction (RT-PCR), the expression of mRNA encoding IFN-gamma was verified in spleen macrophage cultures. Macrophages stimulated with the phosphopolysaccharide also produced IL-1 alpha at a concentration of 50 micrograms/ml. This study showed for the first time that phosphopolysaccharide derived from a dairy lactic acid bacterium can induce IFN-gamma and IL-1 alpha production in macrophages. These findings strongly suggest that the phosphopolysaccharide is a type of 'biological response modifier' and the fermented dairy foods containing Lactococcus lactis ssp. cremoris can be designated as a physiologically functional food.

Animals↗
PubMed1996

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.

Bacteriocins↗
PubMed1998

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation.

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate-gamma-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Amino Acid Sequence↗
PubMed2006

DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14.

A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.

Amino Acid Sequence↗