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PubMed1992

Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763.

1. The specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763 towards caseins has been submitted to a statistical study. Positive and negative relations have been evidenced between several amino acids and positions P6 to P'2 of the cleaved bonds. 2. Fragment 1-23 of alpha s1 and oxidized B chain of insulin are well cleaved by the proteinase while CMP (fragment 106-169 of kappa-casein) is a poor substrate. 3. Comparison with other cell envelope-located proteinase has been done. The enzyme of the strain 763 hydrolyses alpha s1-casein and fragment 1-23 of alpha s1-casein as the enzyme of the strain Sk11 and beta-casein as the enzyme of the strain Wg2. 4. The specificity of these proteinases and the comparison of their amino acid sequences let us postulate a more complex substrate binding area for these lactococcal proteinases than for the subtilisin.

Amino Acid Sequence↗

PubMed1989

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007.

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007 was examined. A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa. Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88. The ability to metabolize lactose (Lac+) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic. When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec-), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac+ transconjugants. DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88. These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate. A Rec- strain containing pKB32 and pJS88 mediated Lac+ conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event. Resolution of the pKB32::pJS88 cointegrate was observed in both Rec- and Rec+ hosts. Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family.

Bacteriocins↗

PubMed1989

Characterization of the genetic element coding for lactose metabolism in Lactococcus lactis subsp. lactis KP3.

The Lactococcus lactis subsp. lactis KP3 Lac genetic element was investigated. KP3 is a lactose-positive (Lac+) transconjugant which contains no detectable plasmid DNA. The KP3 Lac genetic element was self-transmissible (Tra+) and encoded a reduced bacteriophage sensitivity (Rbs+) phenotype. Matings of KP3 with a recombination-deficient (Rec-) recipient resulted in Lac+ transconjugants which were phenotypically indistinguishable from KP3 and contained a 96-MDa plasmid (pJS96). Phenotypic and physical analyses of pJS96 indicated that it was a deletion derivative of a putative pKB32::pJS88 Lac+ Tra+ cointegrate. pKB32 is the Lac plasmid and pJS88 is the Tra+ Rbs+ plasmid in L. lactis subsp. lactis 11007, the donor used in obtaining KP3. The results presented suggest that pJS96 is an episome, since it appeared to replicate both as a plasmid and as an integrated part of the chromosome. Conjugal transfer of chromosomal DNA mediated by pJS96 was not observed. Conjugal transfer of pJS96 resulted in Lac+ transconjugants containing plasmids ranging in size from 21 to 90 MDa. Only in Rec+ recipients were transconjugants isolated which appeared to contain pJS96 integrated into the host chromosome. Restriction analysis of several plasmids in the 21 to 90 MDa range suggested the deletions were due to intramolecular transposition of a transposable element on pJS96. This report suggests that a self-transmissible episome exists in KP3 and provides an explanation of how plasmids which vary in size yet encode similar phenotypes may be formed and disseminated.

Bacteriophages↗

PubMed1990

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis.

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.

Chromosomes, Bacterial↗

PubMed1990

Stacking of three different restriction and modification systems in Lactococcus lactis by cotransformation.

Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2. The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events. Analysis of cotransformants showed that three different R/M plasmids could be combined in L. lactis MG1363. The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased. Some plasmid combinations were unstable suggesting replicon incompatibility.

Chloramphenicol Resistance↗

PubMed1991

Heat shock induces thermotolerance and inhibition of lysis in a lysogenic strain of Lactococcus lactis.

In this preliminary work, the heat shock response of lactic acid bacteria was investigated and characterized. Log-phase Lactococcus lactis cells pre-incubated at 40 degrees C before heat challenge at 52 degrees C for 30 min demonstrated increased thermotolerance as compared with cells pre-incubated at 30 degrees C. The response persisted for at least 60 min. Additionally, we demonstrated that: (i) the physiological expression of the heat shock response is temperature dependent; (ii) ethanol 4.0% (v/v) caused, to a lesser extent, a response similar to the heat shock; and (iii) hydrogen peroxide failed to induce a detectable response. Furthermore, we suggest that the induction of the heat shock response increases the resistance of a lysogenic strain of L. lactis, treated by mitomycin C (1.25 micrograms/ml), to lysis by the bacteriophage.

Ethanol↗

PubMed1991

Capsular polysaccharide of a slime-forming Lactococcus lactis ssp. cremoris LAPT 3001 isolated from Swedish fermented milk 'långfil'.

Slime-forming Lactococcus lactis ssp. cremoris strain LAPT 3001 isolated from Swedish ropy sour milk 'långfil' was investigated for the chemical nature of its capsule. The capsular material purified by gel filtration chromatography and ion-exchange chromatography consisted of rhamnose, glucose, galactose, glycerol and phosphorus. It is most likely a deacylated lipoteichoic acid.

Chromatography, Gel↗

PubMed1992

Is thermotolerance correlated to heat-shock protein synthesis in Lactococcus lactis subsp. lactis?

Exposure of Lactococcus lactis subsp. lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min). This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps). Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis. These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L. lactis subsp. lactis.

Adaptation, Physiological↗

PubMed1988

Utilization of dipeptides by Lactococcus lactis ssp. cremoris.

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Identification of a RecA-like protein in Lactococcus lactis.

We have identified in Lactococcus lactis, an analogue of Escherichia coli RecA protein. Physiological responses such as ultraviolet (UV) and chemical mutagenesis and induction of prophage have been characterized and suggest the existence of RecA-like functions in this commercially important species. The putative RecA protein was detected at the position of an apparent molecular weight of 39 kDa by Western blot analysis by using antiserum against E coli RecA protein. In addition, the protein level is significantly increased after UV irradiation in a wild-type strain compared to the recombination deficient mutant strain.

Blotting, Western↗

PubMed1991

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator.

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.

Calibration↗

PubMed1989

Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 has been constructed in the broad host range plasmid pLAFR1 in Escherichia coli LE392. Three lactococcal genes from the bank were identified by heterologous complementation of specific mutations in strains of E. coli. A cosmid clone encoding a putative lactose transport gene was identified by complementing an E. coli lacY mutant. The complemented clone supported the uptake of 14C lactose in transport assays. The DNA fragment responsible was subcloned and localised to a 1.28 kb fragment of the lactococcal chromosome.

Biological Transport↗

PubMed1989

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus.

Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis. No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.

Conjugation, Genetic↗

PubMed1990

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions.

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.

Bacteriophage lambda↗

PubMed1990

Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages.

Conjugal matings were performed between Lactococcus lactis DRC1 (a lactose-fermenting (Lac+), bacteriocin-producing (Bac+) strain) and L. lactis HID113 (Lac- and Bac-). Transconjugant derivatives of HID113 were identified on the basis of lactose fermentation, resistance to the DRC1 bacteriocin (dricin) or reduced sensitivity to phage sk1. Regardless of how they were identified, all transconjugants gave fewer and smaller plaques with phages c2 and sk1 than did HID113. All but one of 275 transconjugants tested also produced dricin, suggesting some functional relationship or close genetic linkage between the reduced phage sensitivity and dricin production and resistance. Some transconjugants were also Lac+, but this property was unstable.

Bacteriocins↗

PubMed1991

A transposon-like element on the lactose plasmid of Lactococcus lactis subsp. lactis Z270.

An inverted repeat previously called IR was identified on the lactose plasmid of Lactococcus lactis subsp. lactis Z270 by self-annealing; it was now named IS1076. The two sequences were 3.3 kb apart. Both copies were cloned in E. coli, sequenced and found to be identical, except for an additional 44 bp direct repeat at the 5' end of the right-hand copy; they were thus respectively 1296 bp (IS1076R) and 1252 bp (IS1076L) long. Both elements end in near-perfect 39 bp inverted repeats, similar to the IS904 termini. Promoter consensus sequences and a RBS site precede an ORF1 of 384 amino acids. Subclones of IS1076R and IS1076L produced a new 44 kDa protein corresponding to the size of the ORF1. The distal part of the ORF1 coding region is very similar to the IS3 ORFI sequence and the IS904 ORF sequence, and the proximal part shows some homologies with IS3 ORFII. A three-base target is present as a direct repeat flanking the 5.9 kb genetic block including IS1076L, IS1076R and the internal region, resulting in a structure similar of that of a transposon.

Amino Acid Sequence↗

PubMed1991

Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 was probed for the presence of 16S rRNA genes, using 32P 5' end-labelled 16S rRNA fragments. Cosmid DNA from positive clones responsible for hybridisation was subcloned into a high copy number vector and a restriction map was constructed. The location of the 16S, 23S and 5S rRNA genes was determined on this map. Transcriptional promoter activity was identified upstream of the 5' end of the 16S rRNA gene. By probing L. lactis 712 chromosomal DNA cut with a range of restriction endonucleases, with a conserved oligonucleotide to the 5' end of the 16S rRNA gene, 6 copies of rRNA genes were identified.

Base Sequence↗

PubMed1992

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis.

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.

Bacteriophages↗