Lactis C2 Research Articles

Metagenomic Analysis of Dairy Bacteriophages: Extraction Method and Pilot Study on Whey Samples Derived from Using Undefined and Defined Mesophilic Starter Cultures.

Despite being potentially highly useful for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows the removal of the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus lactis 936 (now Sk1virus), P335, c2 (now C2virus) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain cultures containing Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species (i.e., DL starter cultures) and defined cultures, respectively, were assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales, family Siphoviridae) in dairies using undefined DL starter cultures and Lc. lactis c2 phages (order Caudovirales, family Siphoviridae) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible coinduction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed.IMPORTANCE The method optimized in this study could provide an important basis for understanding the dynamics of the phage community (abundance, development, diversity, evolution, etc.) in dairies with different sizes, locations, and production strategies. It may also enable the discovery of previously unknown phages, which is crucial for the development of rapid molecular biology-based methods for phage burden surveillance systems. The dominance of only a few phage groups in the dairy environment signifies the depth of knowledge gained over the past decades, which served as the basis for designing current phage control strategies. The presence of a correlation between phages and the type of starter cultures being used in dairies might help to improve the selection and/or design of suitable, custom, and cost-efficient phage control strategies.

Heat tolerance of dairy lactococcal c2 phages

Nine Lactococcus lactis c2 phages propagated on different hosts were screened for thermal resistance in skimmed milk. Pronounced variations in thermal resistance were found. Three phages displayed high sensitivity towards heat resulting in >8 log reductions after 70 °C for 5 min, whereas the most thermal resistant phages required 80 °C for 5 min to obtain the same reduction. Inactivation kinetics were determined for a thermo-sensitive and a thermo-resistant phage at 60–70 °C and 65–78 °C, respectively, using a submerged-coil system with extremely short heating-up times. Inactivation followed first-order kinetics with correlation coefficients of 0.96–0.99. D 70-values of 12 s and 16.6 min were calculated for the most sensitive and resistant phage, respectively. Release of phage DNA from capsids, and disintegration of phage heads and tails were among the first morphological changes observed for moderately thermal inactivated lysates (15% phage inactivation) of the heat tolerant phage P635.

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10 5 CFU mL −1 of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL −1) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10 6–10 7 CFU mL −1 by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa. Industrial relevance Continuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.

Mutational and biochemical analyses of the endolysin Lys gaY encoded by the Lactobacillus gasseri JCM 1131 T phage φgaY

The Lys gaY of Lactobacillus gasseri JCM 1131 T phage φgaY endolysin was purified to homogeneity using the Escherichia coli/His·Tag system. Zymographic and spectrophotometric assays showed that Lys gaY lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37°C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys gaY were involved in cell-lytic activity: in the β/α gaY domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b gaY domain, Y272 and W284. In addition, deletion analysis demonstrated that the β/α gaY domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys gaY. These mutational experiments suggested that β/α gaY (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b gaY promotes β/α gaY possibly through cell-wall binding function. The purified His-tagged SH3b gaY domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys gaY, (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131 T, L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys gaY, and (iv) impeded autolysis of L. gasseri JCM 1131 T in buffer systems. A truncated protein HΔSH3b gaY lacking in N-terminal 21 residues (from S217 to E237) of SH3b gaY and an amino-acid substituted protein HSH3b gaYG (W284G) lost the activities of HSH3b gaY, showing that the N-terminal region and W284 probably play important roles in the SH3b gaY function(s).

Identification of a Gene Cluster Encoding Krebs Cycle Oxidative Enzymes Linked to the Pyruvate Carboxylase Gene in Lactococcus lactis ssp. lactis C21,2

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Membrane Receptor for Prolate Phages is Not Required for Infection of Lactococcus lactis by Small or Large Isometric Phages

Lactococcus lactis contains a chromosomal gene (pip) for a membrane protein that serves as a receptor for the prolate bacteriophage c2 and other phages of the c2 species. A mutated allele of this receptor gene was used to replace the wild-type allele in L. lactis strains MM210, NCK 203, and C2. Allele replacement was confirmed by the presence of a restriction site marker in a polymerase chain reaction product from the mutated allele. The mutated pip derivative of strain C2 was completely resistant to phages of the c2 species but was fully sensitive to the small isometric phage sk1 of the 936 species, as expected. The mutated derivatives of MM210 and NCK203 were fully sensitive to the small isometric phages mm210b and 31 (p335 species) and to the large isometric phage 949 (949 species). These results show that pip is not required for infection by phages of species 936, p335, or 949. The resultant mutants grew as well as the parental strains in liquid media. The mutated derivatives of MM210 and C2 acidified and clotted milk as readily as the wild-type strains. These results show that phage receptor replacement in a commercial strain of L. lactis does not affect growth and acid production in milk.

Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions.

A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0 while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1 beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1 beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

Acid Sensitivity of a Mutant ofLactococcus lactissubsp.lactisC2 with Reduced Membrane-bound ATPase Activity

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp. lactis C2 as spontaneous neomycin-resistant mutants. Characteristics of the representative mutant, No. 1016–51, were compared with the parental strain in cultures using a jar fermentor with the pH controlled at various values. At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical. At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24 h decreased to 60, 36, and 60% of the parental strain, respectively. During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80 h. On the other hand, at pH 4.0, the viable count of the strain No. 1016–51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability. Therefore, it was concluded that t...

Inactivation of Lacto Coccus Lactis Ssp. Lactis C2 and Alkaline Phosphatase During Spray Drying

ABSTRACT Bacteria dispersed in condensed skim milk were spray dried using five different outlet-air temperatures. Powder samples were analyzed for survival and lactic acid production. Inactivation resulted from dehydration, and exposure to heat during drying. A mathematical model of thermal inactivation of alkaline phosphatase was used to analyze thermal inactivation of the bacteria during drying. Most thermal inactivation was predicted to occur in a critical period of 10 to 20 ms and 70% to 85% solids content. In the absence of relevant experimental data, a procedure was proposed for an order-of-magnitude estimate of overall inactivation to candidate bacteria during spray drying. It utilized predictions of thermal inactivation from the model, pasteurization data from the iterature, and estimates of dehydration inactivation from survival after freezing and freeze drying

Lactococcus lactis ssp. lactis C2 Bacteriophage sk1 Receptor involving Rhamnose and Glucose Moieties in the Cell Wall

Adsorption of bacteriophage sk1 to Lactococcus lactis ssp. lactis C2 was reduced 64, 26, 26, 5, and 8% by addition of rhamnose, glucose, galactose, N-acetylglucosamine, and fucose, respectively, to the adsorption assay. Lectins that were specific for N-acetylglucoseamine and galactose did not prevent phage adsorption. However, three lectins specific for glucose, but different in size, reduced phage adsorption from 0 to 50%. Another lectin, which was specific for rhamnose, reduced phage adsorption 80%. Rhamnose, but not glucose, partially inhibited phage infection when added to a growing culture. The data suggest that a glucose moiety is near the adsorption site or is an important conformational determinant of it.

Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.

Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii P126.

The genus Propionibacterium includes cutaneous species typically found on human skin and the dairy or classical species (Propionibacterium freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici) used industrially for the production of Swiss cheese and propionic acid. Grinstead (1989, M.S. thesis, Iowa State University, Ames) has previously observed that some dairy propionibacteria inhibit other species in the classical grouping. We further investigated the inhibitor(s) produced by P. jensenii P126 (ATCC 4872). An antagonist(s) from anaerobic agar cultures of P126 strongly inhibited two closely related strains of propionibacteria, P. acidipropionici P5 and P. jensenii P54, and Lactobacillus bulgaricus NCDO 1489, Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lactococcus cremoris NCDO 799, and Lactococcus lactis subsp. lactis C2. The inhibitor, designated jenseniin G, was active at pH 7.0; inactivated by treatment with pronase E, proteinase K, and type 14 protease; insensitive to catalase; and stable to freezing, cold storage (4 degrees C, 3 days), and heat (100 degrees C, 15 min). Classification of the inhibitor as a bacteriocin is supported by its proteinaceous nature and its bactericidal activity against L. delbrueckii subsp. lactis ATCC 4797. The lack of detectable plasmids suggests a chromosomal location for the determinant(s) of jenseniin G.

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.

Genetic characterization of partial lactose-fermenting revertants from lactose-negative mutants of lactococci

Partial lactose-positive (Lac+) revertants, designated RS0121 and RS033, were isolated from lactose-negative (Lac−) mutants of Lactococcus lactis subsp. lactis C2 and ML3, respectively. These revertants were found to coagulate 10% reconstituted skim milk, but the rate of lactose fermentation was much slower than that of their parental Lac+ strains. Such partial Lac+ revertants were also isolated from Lac− mutants of L. lactis subsp. Lactis SL712, SH4109, and SL763 and of L. lactis subsp. cremoris ML1 and SC607, showing a generalized phenomenon. The Lac+ characteristic of the revertant strains was conjugally transferred to Lac− variants. When the plasmid profiles of the Lac− variants and their partial Lac+ revertants and transconjugants were analyzed, there were no detectable differences in the plasmid bands. A strain with no plasmids, MG1363, also yielded partial Lac+ revertants, which were devoid of all plasmids. In addition, the Lac+ phenotype of one such revertant, designated RS101, was transferable to other Lac− strains by conjugation. The results indicate that the genetic information for slow lactose metabolism in such partial Lac+ revertants is mediated through chromosomal gene(s) and is under the influence of some suppressor. These observations also suggest the possibility of utilizing this method for isolating slow acid producing variants, which could be used as adjuncts with regular cheese starter cultures for accelerated cheese ripening. Key words: lactococci, lactose-negative mutants, plasmid DNA, lactose-fermenting revertants.

DNA fingerprinting of lactococci and streptococci used in dairy fermentations

A DNA fingerprinting procedure was developed for strains of Lactococcus lactis subsps. lactis and cremoris, biovar. diacetylactis, and Streptococcus salivarius subsp. thermophilus, used in dairy fermentations. Total cellular DNA was extracted and digested with restriction endonucleases, HindIII or HaeIII, followed by separation of the fragments using agarose gel electrophoresis. L. lactis C2 was used as a representative strain for examining the effect of growth phase and cell concentration, cell washing conditions prior to lysis, type and concentration of the enzyme used to digest the cell wall, composition of the lysis buffer, and gel electrophoresis conditions. Following optimization of the fingerprinting procedure, electrophoretic migration of fragments from 23 strains produced reproducible gel patterns. L. lactis subsp. lactis strains ML3 and C2 appeared to be identical when restrricted with either Hind III or HaeIII. Similarly, S. salivarius subsp. thermophilus strains 19987 and 19258, and L. lactis subsp. cremoris strains 134 and C3, appeared to have identical DNA fingerprints following digestion with HindIII. To determine the usefulness of this technique for monitoring population changes during fermentation, various ratios of two closely related strains were inoculated into milk and allowed to grow for 16 h at 32° C. The initial inoculum ratios were determined by standard plate counts, and the final ratio was deterimined by DNA fingerprinting. DNA fingerprinting will be useful in the identification, characterization, and comparison of food fermentation microorganisms.

Effect of Growth Media and Extended Incubation on the Appearance of Lactose - Nonfermenting Variants in Lactococci

Growth and storage were investigated for the development of Lac− (lactose-nonfermenting) variants of Lactococcus lactis subsp. lactis C2, ML3 and L. lactis subsp. cremoris ML1, SC607 under different conditions, in an unbuffered medium (M17−), and in media buffered with inorganic or organic phosphates. Strains were grown overnight (16–18 h) at 32°C and subsequently held at 32 and 22°C. The cell survival was much higher at 22°C than at 32°C after storage for 96 h. Most of the survivors in M17− broth were of the Lac− phenotype. Lac− variants were also observed when the cultures were grown in skim milk at 32°C and then held at that temperature for 96 h. These results showed that the lactose-fermenting ability of lactococcus in general is lost when overnight cultures in M17− broth are kept either at room temperature (22°C) or at 32°C for an extended period. However, the cultures in buffered media under similar conditions showed little or no loss of lactose-fermenting ability, suggesting that phosphate in the media had a stabilizing effect on plasmid-encoded lactose-fermenting gene(s). These observations indicate the possibility of utilizing this method as a simple technique for isolating mutants deficient in plasmid-linked genetic traits in lactococci.

Enzyme activities of cell-free extracts from mutant strains of lactic streptococci subjected to sublethal heating or freeze-thawing

Two mutant lactose-negative (Lac −), proteinase-negative (Prt −) strains of lactic streptococci, Streptococcus lactis 25Sp and S. cremoris KHA2, and their parents, S. lactis C2 and S. cremoris KH Lac + Prt +, were grown in a suitable medium with the pH maintained at 6.5 by addition of NH 4OH. Cells were harvested by centrifugation, resuspended, and then heated sublethally at 54 or 69 °C for 15 sec. Cells also were frozen and stored for 1 week at −20 or −100 °C. Cell-free extracts of cells heated at 54 °C had more proteinase and aminopeptidase activities than did a similar extract of cells heated at 69 °C. The greatest enzyme activities occurred in the cell-free extracts prepared from cells frozen and stored at −100 °C. Specific activities of proteinase and dipeptidase generally decreased in extracts of freeze-shocked cells compared to those in extracts of untreated cells. Enzyme activity of extracts also decreased in the presence of 5% NaCl at pH 5.0. Cell-free extracts at pH values of 5 to 8 were heated at 69 °C for 1, 5, or 10 min. Heating them for 10 min caused a loss of dipeptidase activity which was most pronounced at pH 5.0 and least pronounced at pH 7.0.

Proteinase and Peptidase Activities of Cell-Free Extracts from Mutant Strains of Lactic Streptococci

Cell-free extracts prepared from two lactose-negative, proteinase-negative mutant strains of lactic streptococci, Streptococcus lactis (25Sp), and S. cremoris (KHA2), and the parent strains, S. lactis C2 and S. cremoris (KH), were evaluated for proteinase and peptidase activities. Of the mutant cultures tested, the extract from S. lactis (25Sp) had the highest aminopeptidase activity as determined with the substrates L-lysyl-para-nitroanilide, L-leucyl-para-nitroanilide, and L-prolyl-para-nitroanilide. Extract from the same strain also had the highest dipeptidase (DL-alanylglycine and DL-leucylglycine served as substrates) activity when cells were initially grown in a suitable medium with pH maintained at 6 or 6.5 by addition of NH4OH. The cell-free extracts from S. lactis (25Sp) and S. cremoris (KHA2) had higher enzyme activities (aminopeptidase, and dipeptidase) and had lower intracellular proteinase activity (N,N-dimethyl casein served as substrate) than did extracts of the parent strains when they were grown at pH 6.5. Electrophoretic patterns of cell-free extracts indicated different bands with various enzyme specifities. Both mutant strains had similar specificity for different aminoacyl-β-naphthylamides. Minor differences were noted for dipeptidase activities.

Heterologous expression and stability of the Escherichia coli β-galactosidase gene in Streptococcus lactis by translation fusion

An Escherichia coli/Streptococcus lactis shuttle vector for creating translation fusions to the E. coli galactosidase ( lacZ) has been constructed. Random S. lactis chromosomal DNA fragments inserted upstream of the lacZ gene and in frame promote the expression of active galactosidase in both E. coli and S. lactis. One fusion pSK216 has been characterized by Western blotting to reveal a fusion protein of 116 kd. S. lactis LM0230, a plasmidless lac − derivative of S. lactis C2 when carrying pSK216, exhibits a Lac + phenotype suggesting the presence of a chromosomally encoded lactose permease. Further evidence for this lactose permease system is provided by assay of lactose uptake by S. lactis LM0230. In the absence of selection pSK216 was less stable in S. lactis LM0230 than its derivative pSK41. The growth rate of S. lactis LM0230 carrying pSK216 is slower in comparison to the plasmid-free strain. Two genetic events were observed, the deletion of the lacZ gene and the loss of the entire plasmid. These results indicate that recombinant plasmids are unstable in S. lactis and imply that there is an obstacle in the genetic engineering of lactic acid bacteria.