Lactis C2 Research Articles

In vivo gene transfer systems and transposons

The continued development of our understanding and application of the in vivo gene transfer systems, transduction and conjugation, and the more recent use of transposons in lactic acid bacteria is reviewed. The discovery of transduction and its use as a tool for genetic analysis is described. The history of the use of conjugation in lactic acid bacteria is outlined, but more detailed discussion is reserved for in-depth analysis of the conjugation system associated with the lactose plasmids of Streptococcus lactis strains ML3 and C2. This system is notable for an unusual cell aggregation phenotype associated with variants of lactose plasmids capable of high frequency transfer and the complex DNA interactions associated with this property. Recent advances in the use of wide host range conjugation systems, such as that of plasmid pAMß1 are described, including the mobilisation of vectors by cointegrate formation and subsequent segregation after transfer. The successful exploitation of conjugation for the construction of bacteriophage-resistant starter cultures is highlighted. A description of transposable genetic elements in the lactic acid bacteria, both insertion sequences and transposons, puts emphasis on the elegant analysis of insertion sequence ISL 1 in Lactobacillus casei and on the exploitation of the conjugative transposons Tn 916 and Tn 919. The latter is especially important for providing a technology to initiate analysis of the bacterial chromosome of the lactic acid bacteria.

Spontaneous Release of Temperate Phage by Relysogenized Lactose-Positive Transductants of Streptococcus lactis C2

Transduction for lactose-fermenting ability between the lactose-positive lysogen Streptococcus lactis LM0221 and plasmid-cured, prophage-cured, lactose-negative S. lactis LM2301 resulted in the appearance of lactose-positive transductants surrounded by a zone of clearing in the lactose-negative cell lawn. By using plaque assay procedures, the zones were shown to contain bacteriophage particles, and both spontaneous release and UV induction of prophage from these transductants were demonstrated. The DNA hybridization confirmed that LM2301 did not contain the prophage in its chromosome and that the zone-producing transductant KZ1 was relysogenized by the temperate bacteriophage. Further, a 4.35 Kb EcoRI digestion fragment appeared to contain the DNA sequences for integration into the chromosome and may provide a means for stabilizing cloned DNA by effecting chromosomal insertion in LM2301 derivatives. The selection of zone-producing lactose-positive transductants of LM2301 provided a means for detecting strains relysogenized by the temperate phage induced from LM0221.

Isolation of Streptococcus lactis C2 Mutants Selected for Temperature Sensitivity and Potential Use in Cheese Manufacture

In an attempt to select streptococcal strains that could be used to accelerate the cheese ripening process, we used ethyl methanesulfonate treatment to isolate Streptococcus lactis C2 temperature-sensitive mutants exhibiting normal growth patterns at 30 to 32°C but not at the highest cooking temperatures used in Cheddar cheese manufacture. Three classes of mutants isolated included: 1) mutants unable to grow and produce acid at 38 to 40°C; 2) mutants unable to grow at 38 to 40°C but that continued to produce acid at a rate similar to temperature-sensitive strains of S. cremoris; and 3) mutants that underwent lysis when the cells were shifted to 38 to 40°C, as evidenced by a decrease in optical density of the cell suspension and the concomitant release of the intracellular enzyme, phospho-β-galactosidase. The thermolytic variants could possibly be used to accelerate cheese ripening either by releasing their intracellular enzymes into the curd at an early stage in the cheese making process or by serving as a “delivery system” of ripening enzymes, the genes of which could be cloned into the thermolytic strains.

Calcium Alginate-Immobilized Cultures of Lactic Streptococci are Protected from Bacteriophages

Calcium alginate-immobilized cultures of lactic streptococci were grown in milk and assessed for their sensitivity to homologous bacteriophage, proteolytic activity, and acid production. Immobilized cultures of Streptococcus lactis C2 and Streptococcus cremoris HP were protected from attack by bacteriophage due to the exclusion of phage particles from the gel matrix. These cultures were also functionally proteinase-deficient when immobilized in calcium alginate beads and grown in milk. Acid was produced by immobilized cultures at a lower rate than cells freely suspended in milk due in part to the inability of the immobilized cells to hydrolyze milk proteins. Agitation of immobilized cultures slightly increased acid production, suggesting that diffusional limitations of substrate into the beads contributed to decreased acid production. Use of immobilized cultures of lactic streptococci in certain dairy fermentations may be advantageous due to the protection of these cultures from attack by virulent bacteriophage.

Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Conjugation between lactose-fermenting (Lac+)Streptococcus lactis C2 and Lac−Leuconostoc cremoris CAF7 was performed. The frequency of Lac+ transfer was 1.5 · 10−2 per donor cell. Lac+Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac+ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 μg/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac+ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.

Evaluation of Inorganic Phosphate on Growth and Lactose Metabolism of Lactic Streptococci in Batch and Continuous Culture

Addition of 1.9% inorganic phosphates (K2HPO4, 1.33% + KH2PO4, 0.57% wt/vol) in place of 1.9% disodium (β-glycerophosphate (GP) in M17 medium (M17P) resulted in increased buffering capacity. Even at equimolar concentration (88 mM), Na2HPO4 (M17P1) or K2HPO4 (M17P2) showed higher buffering than GP(M17). Cultures consistently showed lower cell density in M17P2 than in other buffered media after 7 h of growth at 32°C, suggesting that buffering media with Na2HPO4 is better than buffering with K2HPO4 for cultivation of lactic streptococci. The effect of buffering media with Na2HPO4 on culture growth and appearance of lactose-negative (Lac−) variants was also tested under continuous culture growth conditions in a chemostat. Growth of Streptococcus lactis C2 continuously at pH 6.8 for 168 h in M17P1, and M17 broths at 32°C failed to yield any lac− strains. Results showed that successful propagation of lactic streptococci in continuous culture growth can be achieved without enriching for cells with undesirable metabolic characteristics.

Partial purification and some properties of phi C2(W) lysin, a lytic enzyme produced by phage-infected cells of Streptococcus lactis C2.

The lytic enzyme present in phi C2(W) lysates was isolated by means of ion-exchange chromatography and further purified by gel filtration and ultrafiltration. The phage enzyme had an apparent pH optimum of 6.5-6.9 and was rapidly inactivated at temperatures greater than 47 degrees C. The apparent temperature optimum was 37 degrees C and Q10 and Ea values over the range 22-32 degrees C were 2.5 and 69.2 kJ/mol respectively. Monovalent and divalent cations activated the enzyme. Reduced -SH groups on the enzyme were required for lytic activity. Gel filtration revealed a mol. wt of approximately 46000. Strain-dependent differences in sensitivity of group N lactic streptococci to lysin were found. Group D streptococci were also lysed. Strains of three species of Leuconostoc, two species of Lactobacillus, one strain of Escherichia coli and of Micrococcus lysodeikticus were apparently resistant. Analysis of cell wall degradation products gave results which were consistent with the lysin having the specificity of an N-acetylmuramidase.

Isolation and Partial Characterization of Lactose-Negative Mutants from Streptococcus lactis C2 Defective in the Phosphotransferase System

Development of strains of dairy streptococci that improve or control rate of lactic acid production would be of value in dairy fermentations. However, improvement of the ability of dairy starter cultures to metabolize lactose has been limited because of inability to examine and characterize these plasmidlinked genes. A study was initiated to define further the genetic and enzymatic basis of lactose utilization of Streptococcus lactis C2. Lactose-negative mutants were isolated by ultraviolet irradiation or ethyl methanesulfonate mutagenesis from a C2 strain in which genes for lactose utilization had become integrated into the chromosome. Mutants were examined for phospho-β-galactosidase activity and for their ability to accumulate [carbon-14] thiomethyl-β-D-galactoside. Selected mutants were tested for enzyme complementation by Staphylococcus aureus mutants with known specific defects in lactose-utilizing enzymes. Genetic complementation via transduction between lactose-negative derivatives to yield lactose-positive recombinants also was obtained. Lactose-negative mutants missing only the lactose-specific Enzyme IIlac (EIIlac) or Factor IIIlac (FIIIlac) were identified. A mutant that retained the lactose-negative phenotype, even though it possessed EIIlac, FIIIlac, and phospho-β-galactosidase activities, was isolated. Single site mutations in these lactose-negative variants were supported by the observation that full lactose-fermenting revertants were obtainable from each of the three types.

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage.

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.

Rapid Test for Detecting Lactic Streptococcal Agglutinins in Cheese Milk

A Rose bengal-stained antigen plate testing procedure was developed for detecting lactic streptococcal agglutinins in milk. Colostrum samples were used as sources for immunoglobulins G and M. Results were positive from 2% packed cell volumes of heat-killed Rose bengal-stained cells suspended in .5 molar Tris maleate buffer (pH 7.0). Lactic streptococcal antigens were susceptible to immunoglobulins G and M. Streptococcus lactis strain C2 was not an agglutination resistant organism but among 15 strains tested was the least susceptible when exposed to low concentrations of agglutinins.Stained antigens reacted with equal effectiveness when tested against colostrum whey (pH 7.0) or whey samples prepared from cheese milk to detect agglutinins. The indirect immunoassay revealed the specificity of immunoglobulin G to clump starter cells in contrast to immunoglobulin M. For example, Streptococcus lactis C2 was agglutinated by G but did not conjugate well with M. The immunoassay testing also revealed a potential procedure for detecting lactic streptococcal agglutinins. Autoclaving milk prior to adding agglutinin-containing whey dramatically reduced agglutination of starter strains.

Influence of Incubation Time and Culturing Conditions on Alcohol Dehydrogenase Activity of Streptococcus lactis

Effects of incubation temperatures of 21 and 32°C, incubation time, and pH control at 6.0 on alcohol dehydrogenase (E.C.1.1.1.1., alcohol: nicotinanide dinucleotide alcohol dehydrogenase oxidoreductase) activity were evaluated for batch cultures of Streptococcus lactis 60 and C2 in Elliker broth. Without pH control, alcohol dehydrogenase was maximal in both strains after 6h incubation and declined with increasing incubation of stationary phase cells. Increasing growth temperature increased optical density and plate count data at 6h incubation for both strains. Alcohol dehydrogenase activity of 6h cells of Streptococcus lactis 60 was higher with 32°C than with 21°C incubation. For Streptococcus lactis C2, increasing incubation temperature resulted in lower detectable alcohol dehydrogenase activity at 6h incubation. Controlling growth pH at 6.0 increased growth and increased alcohol dehydrogenase activity. Alcohol dehydrogenase activity of Streptococcus lactis grown with pH control did not decline as dramatically during increased incubation time as that of Streptococcus lactis grown without pH control.

Spectrophotometric Assay Using o-Phthaldialdehyde for Determination of Proteolysis in Milk and Isolated Milk Proteins

A rapid, sensitive, and convenient Spectrophotometric assay was developed and characterized for measurement of proteolysis of milk proteins in buffered solutions or in milk. α-Amino groups released by hydrolysis react with ο-phthaldialdehyde and (β-mercaptoethanol to form an adduct that absorbs strongly at 340nm. The absorptivity (ɛ = 6000M−1 cm−1) is similar for all α-amino groups. Moreover, the absorptivity of the adduct with both α- and ɛ-amino groups of proteins is also similar and unaffected by local environment when proteins are denatured in sodium dodecyl sulfate. Thus, background is constant for a particular sample, and α-amino groups released by proteolysis can be quantitated accurately. Inclusion of sodium dodecyl sulfate in the assay provides a convenient way to terminate proteolysis and to insure full exposure and complete reaction of amino groups. Because all hydrolytic products are assayed, the method is more accurate than procedures that depend upon properties of aromatic residues (Hull and Lowry methods). Furthermore, the &ogr;-phthaldialdehyde Spectrophotometric assay is more rapid and convenient than methods using ninhydrin, 2,4,6-trinitrobenzenesulfonic acid, or fluorescamine. The assay proved to be especially useful for measuring proteolysis in milk from microbial culture organisms such as Streptococcus lactis C2. Because trichloroacetic acid filtrates are used, the method should be adaptable to other dairy products.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.

Genetic transfer systems in lactic acid bacteria.

Gene transfer processes (transduction, conjugation, protoplast fusion mediated exchange, transformation in protoplasts) in lactic acid bacteria are reviewed in this paper. Besides, the detailed molecular nature of lactose plasmids in the Streptococcus lactis C2, 712 and ML3 strain complex is discussed.

D-tagatose 1,6-diphosphate aldolase from lactic streptococci: purification, properties, and use in measuring intracellular tagatose 1,6-diphosphate.

Two D-ketohexose 1,6-diphosphate aldolases are present in Streptococcus cremoris E8 and S. lactis C10. One aldolase, which was induced by growth on either lactose or galactose, was active with both tagatose 1,6-diphosphate (TDP) and fructose 1,6-diphosphate (FDP), having a lower Km and a higher Vmax with TDP as the substrate. This enzyme, named TDP aldolase, had properties typical of a class I aldolase, being insensitive to EDTA and showing substrate-dependent inactivation by sodium borohydride. Sodium dodecyl sulfate-gel electrophoresis indicated a subunit molecular weight of 34,500. The amino acid composition of TDP aldolase is reported. When the enzyme was incubated with either triose phosphates or FDP, the equilibrium mixture contained an FDP/TDP ratio of 6.9:1. The other aldolase, which had properties typical of a class II aldolase, showed activity with FDP but not with TDP. The intracellular TDP concentration, measured with the purified TDP aldolase, was 0.4 to 4.0 mM in cells growing on lactose or galactose and was lower (0 to 1.0 mM) in cells growing on glucose. The intracellular concentration of FDP was always higher than that of TDP. The role of ketohexose diphosphates in the regulation of end product fermentation by lactic streptococci is discussed.

Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.

Scanning Electron Microscopy of Microbial Attachment to Milk Contact Surfaces

Milk contact surfaces were observed by scanning electron microscopy (SEM) techniques for possible microbial attachment. Cultures of Pseudomonas fragi 4973, Staphylococcus aureus JAL, Streptococcus lactis C2, Streptococcus cremoris and Lactobacillus bulgaricus RR inoculated onto glass coverslips or stainless steel chips were examined. Stainless steel surfaces displayed many possible harborages for microbial colonization. SEM examination of P. fragi 4973 showed development of fibrous material, with numerous stick-like projections extending from the cell to the glass or stainless steel surface. These apparent attachment appendages became more pronounced as contact time increased. S. aureus, S. lactis, S. cremoris and L. bulgaricus did not display such fibrous material.

Elevated Cyclic Adenosine 3′, 5′ Monophosphate Enhances Lactic Acid Production by Streptococcus lactis

Elevation of cyclic adenosine 3′, 5′ monophosphate levels induced by prostaglandin E1 enhanced growth of Streptococcus lactis C2 in a medium supplemented with lactose. The prostaglandin E1-induced enhancement of growth did not appear to result from stimulation of the phosphoenolpyruvate phosphotransferase system because o-nitrophenol-β-D-galactopyranoside phosphorylation was not elevated by prostaglandin E1, treatment. Measurement of the extracellular proteolytic activity revealed increased activity in prostaglandin E1-treated cultures suggesting that an increase in the organic nitrogen source contributed to increased growth rate. Moreover, elevations in intracellular cyclic adenosine 3’, 5’ monophosphate induced by prostaglandin E1 treatment increased lactic acid production at the cellular level.

Conjugal transfer of genetic information in group N streptococci.

Streptococcus lactis strains ML3 and C(2)O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM(4) were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac(-)) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac(+) Str(r)) recombinants were found when the Lac(+) Str(s) donor was mixed with Lac(-) Str(r) LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM(4), nor was reversion responsible for the high number of Lac(+) Str(r) recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C(2)O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM(4). In S. lactis C2 x LM0230 matings, the Str(r) marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.