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PubMed2003

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection.

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.

Adsorption↗
PubMed1993

Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22.

pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non-transcribed region, the replication origin, followed by a gene encoding a putative 388-amino-acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co-resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co-existence of numerous related replicons in lactococcal strains.

Amino Acid Sequence↗
PubMed2000

Short communication: salt extends the upper temperature limit for growth of Lactococcus lactis ssp. cremoris on solid M17 medium.

We have determined conditions for plating of the Lactococcus lactis ssp. cremoris laboratory strain MG1363 on solid M17 broth at 38 degrees C, which is required for the optimal use of the pGhost plasmids. The addition of 1% NaCl (or KCl, potassium acetate, or sucrose at 170 mM) to M17 agar plates results in extension of the upper temperature limit for growth from 37 to 40 degrees C; no decrease in plating efficiency was detected from 30 to 39 degrees C.

Culture Media↗
PubMed2001

Expression of clpX, an ATPase subunit of the Clp protease, is heat and cold shock inducible in Lactococcus lactis.

In this study, the clpX gene and surrounding sequences were cloned and sequenced from Lactococcus lactis. The putative clpX gene encodes a 411 amino acid polypeptide with a predicted molecular weight of 45.8 kDa. Analysis of the relative levels of clpX transcript revealed that in addition to a role in proteolysis of heat damaged proteins, ClpX may also be involved in cryoprotection.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Sequence of a Lactococcus lactis DNA fragment homologous to the recF gene of Bacillus subtilis.

The recF gene of Lactococcus lactis ATCC 7962 is located 3 kb downstream from the lacZ gene and is transcribed in the opposite orientation. The recF gene is immediately preceded by a 121-codon ORF, and both recF and orf121 may be transcribed from the same promoter. The deduced RecF amino-acid sequence shows high homology to that of the Bacillus subtilis and Streptococcus pyogenes RecF proteins.

Amino Acid Sequence↗
PubMed2000

Expression of the Staphylococcus hyicus lipase in Lactococcus lactis.

The extracellular Staphylococcus hyicus lipase was expressed under the control of different promoters in Lactococcus lactis and Bacillus subtilis. Its expression at high and moderate levels is toxic for the former and the latter hosts, respectively. In L. lactis, the lipase was expressed at a high level, up to 30% of the total cellular proteins, under the control of the inducible promoter PnisA. About 80% of the lipase remained associated with the cells. Close to half of this amount remained associated with the inner side of the cytoplasmic membrane as unprocessed pre-pro-lipase. The other half was trapped by the cell wall and partially degraded at the N-terminal end. This result suggests that extracellular proteases degrade the lipase. Surprisingly, the kinetics and the pattern of lipase degradation were different in the two L. lactis subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis. The extracellular proteolytic systems that degrade lipase are thus different in these closely related subspecies. The incorrect export of the lipase is not due to an inappropriate leader peptide but may be due to an inefficiency of several steps of lipase secretion. We propose that (i) the S. hyicus lipase may require a special accessory system to be correctly exported or (ii) the kinetics of lipase synthesis may be a critical factor for proper folding.

Bacillus subtilis↗
PubMed2004

Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC.

The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.

Acetylglucosaminidase↗
PubMed2006

Phenotypic and molecular characterization of Lactococcus lactis from milk and plants.

AIMS: The aim of this study was to obtain new Lactococcus lactis strains from nondairy materials for use as milk fermentation starters. The genetic and phenotypic traits of the obtained strains were characterized and compared with those of L. lactis strains derived from milk. It was confirmed that the plant-derived bacteria could be used as milk fermentation starters. METHODS AND RESULTS: About 2600 lactic acid bacteria were subjected to screening for L. lactis with species-specific PCR. Specific DNA amplification was observed in 106 isolates. Forty-one strains were selected, including 30 strains of milk-derived and 11 of plant-derived, and their phenotypic traits and genetic profiles were determined. The plant-derived strains showed tolerance for high salt concentration and high pH value, and fermented many more kinds of carbohydrates than the milk-derived strains. There were no remarkable differences in the profiles of enzymes, such as lipases, peptidases and phosphatases. Isolates were investigated by cluster analysis based on randomly amplified polymorphic DNA profiles. There were no significant differences between isolates from milk and those from plant. The L. lactis subsp. cremoris strains were clustered into two distinct groups, one composed of the strains having the typical cremoris phenotype and the other composed of strains having a phenotype similar to subsp. lactis. Fermented milk manufactured using the plant-derived strains were not inferior in flavour to that manufactured using the milk-derived strains. CONCLUSIONS: Plant-derived L. lactis strains are genetically close to milk-derived strains but have various additional capabilities, such as the ability to ferment many additional kinds of carbohydrates and greater stress-tolerance compared with the milk-derived strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The lactic acid bacteria obtained from plants in this study may be applicable for use in the dairy product industry.

Animals↗
PubMed2000

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis.

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.

Bacterial Proteins↗
PubMed2000

The autoproteolysis of Lactococcus lactis lactocepin III affects its specificity towards beta-casein.

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards beta-casein was investigated. beta-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from beta-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.

Amino Acid Sequence↗
PubMed2005

A fuzzy logic-based model for the multistage high-pressure inactivation of Lactococcus lactis ssp. cremoris MG 1363.

The high-pressure inactivation (200 to 600 MPa) of Lactococcus lactis ssp. cremoris MG 1363 suspended in milk buffer was investigated with both experimental and theoretical methods. The inactivation kinetics were characterised by the determination of the viable cell counts, cell counts of undamaged cells, LmrP activity, membrane integrity, and metabolic activity. Pressures between 200 and 600 MPa were applied, and pressure holding times were varied between 0 and 120 min. Experiments were carried out in milk buffer at pH values ranging between 4.0 and 6.5, and the effect of the addition of molar concentrations of NaCl and sucrose was furthermore determined. The inactivation curves of L. lactis, as characterised by viable cell counts, exhibited typical sigmoid asymmetric shapes. Generally, inactivation of the membrane transport system LmrP was the most sensitive indicator of pressure-induced sublethal injury. Furthermore, the metabolic activity was inactivated concomitant with or prior to the loss of viability. Membrane integrity was lost concomitant with or later than cell death. For example, treatments at 200 MPa for 60 min in milk buffer did not inactivate L. lactis, but fully inactivated LmrP activity and reduced the metabolic activity by 50%. The membrane integrity was unaffected. Thus, the assay systems chosen are suitable to dissect the multistep high-pressure inactivation of L. lactis ssp. cremoris MG 1363. A fuzzy logic model accounting for the specific knowledge on the multistep pressure inactivation and allowing the prediction of the quantities of sublethally damaged cells was formulated. Furthermore, the fuzzy model could be used to accurately predict pressure inactivation of L. lactis using conditions not taken into account in model generation. It consists of 160 rules accounting for several dependent and independent variables. The rules were generated automatically with fuzzy clustering methods and rule-oriented statistical analysis. The set is open for the integration of further knowledge-based rules. A very good overall agreement between measured and predicted values was obtained. Single, deviating results have been identified and can be explained to be measurement errors or model intrinsic deficiencies.

Animals↗
PubMed2001

Nucleotide sequence and characterization of the cell envelope proteinase plasmid in Lactococcus lactis subsp. cremoris HP.

AIMS: The major cell envelope proteinase (lactocepin; EC 3.4.21.96) produced by Lactococcus lactis cheese starter bacteria is required for starter growth and acid production in milk. The aim of this study was to characterize a lactocepin plasmid from a L. lactis subsp. cremoris cheese starter strain. METHODS AND RESULTS: A restriction map of the lactocepin plasmid pHP003 from strain HP was constructed, fragments were cloned in Escherichia coli vectors, and the complete DNA sequence (13,433 bp) was determined. Among 120 industrial L. lactis starter strains screened, five contained the same specificity-type lactocepin as pHP003. The lactocepin gene in these strains was invariably linked with a partially-deleted abiB gene. CONCLUSION: The lactocepin specificity type of strain HP, conferred by a known configuration of key residues, is relatively uncommon. The gene is invariably linked with a partially deleted abiB gene on each lactocepin plasmid. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first complete sequence reported for a lactocepin plasmid, and provides the basis for better understanding, or manipulation, of lactocepin production.

Animals↗
PubMed1996

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Amino Acid Sequence↗
PubMed2006

Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis.

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.

Bacterial Proteins↗
PubMed2001

Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase.

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.

Base Sequence↗
PubMed2003

Growth kinetics of Lactococcus lactis ssp diacetylactis harboring different plasmid content.

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac(+)- and Prt(+)-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation.

Culture Media↗
PubMedDate not supplied

[Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116].

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.

Culture Media↗
PubMed2001

[Continuous production of nisin by calcium alginate-immobilized Lactococcus lactis subsp. lacits SM526].

The attempts were made to produce nisin by immobilized cells of Lactococcus lactis subsp. lactis SM526 entrapped in calcium alginate beads. The results showed that the immobilized cell beads were intact for 90 hr under 2% of calcium alginate in 10 mmol/L CaCl2 solution. The physical stability of beads was improved by removing phosphate, citrate and acetate salts supplied in SYS3 medium. Repeated-batch fermentation utilizing immobilized cells were performed in mSYS3 medium without agitation at 32 degrees C, the concentration of nisin produced during the third cycles increased to 850 IU/mL, and the beads were still intact with little cell leakage. The immobilized beads were loaded into a sterilized glass column to continuous fermentation, nisin concentration increased to 1150 IU/mL, as high as that obtained in free-cell fermentation, and the high productivity was stable for up to 70 h under a constant supplied of the fresh mSYS3 medium.

Alginates↗