Strains Of Lactic Streptococci Research Articles

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene

A recombinant Streptococcus lactis strain which carries the structural gene for a surface protein antigen (PAc) of 190,000 daltons from Streptococcus mutans serotype c was constructed for development of an oral vaccine against dental caries. The gene from S. mutans MT8148 joined to shuttle vector pSA3 was successfully transformed into S. lactis IL1403. A small amount of PAc was detected in the cell homogenate and cytoplasmic fraction of the recombinant S. lactis, but not in the culture supernatant of the recombinant, by Western immunoblotting and dot immunoblotting. The level of PAc-specific mRNA in the recombinant strain was lower than that in S. mutans MT8148. However, significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.

Bacteriocin-Like Inhibitors of Streptococcus lactis Against Listeria monocytogenes

Culture extracts containing inhibitory substances produced by seven Streptococcus lactis strains and different concentrations of nisin were tested on six strains of Listeria monocytogenes. The antimicrobial effect was revealed by the presence of zones of inhibition after spotting of 10μl of neutralized, filter sterilized, and catalase treated culture extracts onto agar lawns of L. monocytogenes. Culture extracts demonstrated a bactericidal action without cell lysis of pathogenic organisms. Furthermore, a wide sensitivity of inhibitor substances to proteolytic enzymes was observed. Thus the antimicrobial compounds could be defined as bacteriocin-like substances.

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32°C in an unbuffered medium (M17−) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17− broth followed by extended incubation at 32°C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.

Proportion of phage-insensitive and phage-sensitive cells within pure strains of lactic streptococci, and the influence of calcium

It is of industrial importance to investigate the interaction ofStreptococcus lactiswith phages. Although it has been long recognized that in phage–bacterial relationships the phage-carrier state can occur (Hunter, 1947), relatively little study has been done on this subject. The terras ‘phage-carrier state’ and ‘pseudolysogeny’ have been used synonymously to describe bacterial cultures which are persistently infected with a virus (Barksdale & Arden, 1974, Lawrenceet al.1976). The phagecarrier state differs from lysogenesis in that the bacteria are easily separated from the bacteriophage by a simple plating and re-isolation procedure (Grahamet al.1952). Süssmuth & Tayran (1986) showed that after lysis of one single strain, phage and phage-insensitive bacteria coexist. This work investigates the proportion of phage-insensitive bacteria remaining after lysis of otherStr. lactisstrains, the effect of calcium on this proportion, and the number of generations required to return to a normal sensitive population.

Effect of antibiotic resistance on the flavour profile of lactic acid bacteria

The major problem encountered in the dairy industry is due to the failure of starter in the manufacture of fermented dairy products such as cheese, Yogurt buttermilk and also the Indian fermented milk products srikhand and dahi, in terms of low acid production and absence of flavour and aroma. Usually, in such cases the presence of antibiotics is detected. Little work has been done on the effect of antibiotic resistance in the starter on flavour production, though many workers have investigated acid production and proteolytic activity (Kondratenko et al. 1978 Ottogalli et al. 1975; Shahani & Harper 1958; Wilkowske & Krienke, 1951). The present investigation has been undertaken to develop antibiotic-resistant strains of lactic streptococci, i.e. Streptococcus lactis biovar. diacetylactis and Lactobacillus delbrueckii subsp. bulgaricus which could be used for the preparation of fermanted milk products.

Development and use of a defined strain starter system for Cheddar cheese

Out of 210 strains of Streptococcus cremoris and Streptococcus lactis screened, 35 were suitable for inclusion in multiple strain starters for Cheddar cheese. The use of various combinations of these 35 strains and bacteriophage‐insensitive mutants isolated from some of them as cheese starters over six seasons is described. All cheese graded well for body, texture and flavour, with <1% being downgraded due to starter failure. The system is now being used in all Cheddar cheese plants in Ireland producing ˜60,000 tonnes a year. The problems encountered in phage monitoring are also discussed.

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.

Mutagenesis in Streptococcus lactis exposed to UV irradiation and alkylating agents.

The lethal and mutagenic effects of various mutagens on three strains of Streptococcus lactis were investigated. Lethality studies demonstrated that S. lactis was relatively sensitive to UV irradiation, methyl methanesulphonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and, to a lesser extent, to ethyl methanesulphonate (EMS). A spontaneous derivative Lac-, which has lost a 37-Md plasmid, was slightly more resistant and much less mutable than the wild-type after UV irradiation. Although the three strains were strongly mutated by EMS for the genetic marker assayed (Rifr), an increase in the mutation frequency was also observed after MMS and MNNG treatments.

Conjugative mobilization as an alternative vector delivery system for lactic streptococci.

Due to the current variability in applying polyethylene glycol-mediated protoplast transformation to lactic streptococci, a study was undertaken to assess the feasibility of conjugative mobilization as an alternative method for vector delivery. By using the broad-host-range conjugative plasmid pVA797, the partially homologous cloning vector pVA838 was successfully introduced into various strains of Streptococcus lactis, Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, Streptococcus thermophilus, and Streptococcus faecalis. Frequencies ranged from 10(-2) to 10(-6) transconjugants per recipient. Both pVA797 and pVA838 were acquired intact, without alteration in functionality. Also, the shuttle vector pSA3, which shares partial homology with pVA797, was mobilized via conjugation. The use of S. lactis LM2301 as the intermediate donor allowed the use of physiologic and metabolic characteristics for recipient differentiation. The construction of a vector containing a "DNA cassette" conferring mobilization and the resolution, segregation, and stability of the cointegrates, pVA797, pVA838, and pSA3, are also reported.

Effect of Oxygen on Lactose Metabolism in Lactic Streptococci

Three strains of Streptococcus lactis, two of Streptococcus cremoris, and one of Streptococcus thermophilus metabolized oxygen in the presence of added carbohydrate primarily via a closely coupled NADH oxidase/NADH peroxidase system. No buildup of the toxic intermediate H(2)O(2) was detected with the three S. lactis strains. All six strains contained significant superoxide dismutase activity and are clearly aerotolerant. Lactose- or glucose-driven oxygen consumption was biphasic, with a rapid initial rate followed by a slower secondary rate which correlated with factors affecting the in vivo activation of lactate dehydrogenase. The rate of oxygen consumption was rapid under conditions that led to a reduction in lactate dehydrogenase activity (low intracellular fructose 1,6-bisphosphate concentration). These conditions could be achieved with nongrowing cells by adding lactose at a constant but limiting rate. When the rate of lactose fermentation was limited to 5% of its maximum, nongrowing cells of S. lactis strains ML3 and ML8 carried out an essentially homoacetic fermentation under aerobic conditions. These same cells carried out the expected homolactic fermentation when presented with excess lactose under anaerobic conditions. Homoacetic fermentation leads to the generation of more energy, by substrate-level phosphorylation via acetate kinase, than the homolactic fermentation. However, it was not observed in growing cells and was restricted to slow fermentation rates with nongrowing cells.

Partial characterization and comparison of cell wall proteinases from 5 strains of Streptococcus lactis

Cell wall proteinases from 5 strains of Streptococcus lactis including one S. lactis subsp. diacetylactis were compared and partially characterized. They shared sorne common enzymatic features: only one activity optimum at 30-40°C, at pH 5.5-6 on casein and 4-4.5 on haemoglobin. Crude cell wall extracts from the S. lactis strains were fully inhibited by DFP, but this inhibition was only partial for the S. lactis subsp. diacetylactis strain. Electrophoregrams of as!, {3 and K-casein hydrolysates obtained with the cell-wall extracts of these strains were quite similar for each protein substrate. {3-caseinwas preferentially broken down; as! and K-caseins were hydrolysed to a lesser extent.

Identification of lactose fermentation plasmids of streptococcal dairy starter strains by Southern hybridization

Twenty-two strains of Streptococcus cremoris, seven strains of Streptococcus lactis and three strains of Streptococcus lactis subsp. diacetilactis, each with a different plasmid complement, were isolated from a starter culture used in a Finnish dairy plant. By using DNA-DNA hybridization, with cloned 6-P-s-galactosidase gene of the Strep, lactis plasmid pLP712 as a probe, the lactose fermentation genes were located, in each strain, in the large ( 30 MD) plasmid.

Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis

We attempted to identify the genetic loci for sucrose-fermenting ability (Suc+), nisin-producing ability (Nip+), and nisin resistance (Nisr) in certain strains of Streptococcus lactis. To obtain genetic evidence linking the Suc+ Nip+ Nisr phenotype to a distinct plasmid, both conjugal transfer and transformation were attempted. A conjugation procedure modified to protect the recipients against the inhibitory action of nisin allowed the conjugal transfer of the Suc+ Nip+ Nisr marker from three Suc+ Nip+ Nisr donors to various recipients. The frequency of transfer ranged from 1.7 x 10(-4) to 5.6 x 10(-8) per input donor, depending on the mating pair. However, no additional plasmid DNA was apparent in these transconjugants. Transformation of S. lactis LM0230 to the Suc+ Nip+ Nisr phenotype by using the plasmid pool of S. lactis ATCC 11454 was not achieved, even though other plasmids present in the pool were successfully transferred. However, two results imply the involvement of plasmid DNA in coding for the Suc+ Nip+ Nisr phenotype. The Suc+ Nip+ Nisr marker was capable of conjugal transfer to a recipient deficient in host-mediated homologous recombination (Rec-), and the Suc+ Nip+ Nisr marker exhibited bilateral plasmid incompatibility with a number of lactose plasmids found in S. lactis. Although our results indicate that the Suc+ Nip+ Nisr phenotype is plasmid encoded, no physical evidence linking this phenotype to a distinct plasmid was obtained.

Characterization of streptococcal bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected.

Survey of antimicrobial resistance in lactic streptococci.

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.

Plasmid-mediated UV-protection inStreptococcus lactis

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments we showed that derivatives containing pIL7 (31 kb) were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

Plasmid-mediated UV-protection in Streptococcus lactis

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments we showed that derivatives containing pIL7 (31 kb) were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

Aminopeptidase Profiles of Lactic Streptococci

Aminopeptidase activities of cell free extract from 11 strains of Streptococcus lactis and Streptococcus cremoris, used as starter bacteria in cheese manufacture, were determined with 54 synthetic substrates. Based on these aminopeptidase profiles, 11 strains were divided into three groups by cluster analysis. Enzymes involved in representative strains of each cluster (Streptococcus lactis 527, Streptococcus cremoris ML-14, and Streptococcus cremoris nTR) were separated partly by diethylaminoethyl-cellulose chromatography and compared for each enzyme's optimum pH and the effect of various metal ions and chemicals. These strains have at least six aminopeptidases (dipeptidase specific for hydrophobic amino acid-x, dipeptidase for basic amino acid-x, prolinediaminopeptidase, aminopeptidase-P (or prolidase), general aminopeptidase, and tripeptidase), although contents of enzymes vary among strains.

Differentiation of Fast and Slow Milk-Coagulating Isolates in Strains of Lactic Streptococci

Differentiation of fast and slow milk-coagulating lactic streptococci was achieved on a modified glycerophosphate milk agar. The new medium contained .1% litmus, which improved colony visualization and provided a qualitative measure of acid production. Aerobic incubation of strains on this milk-based medium led to autoinhibition in many strains because of accumulation of hydrogen peroxide. This effect was minimized by addition of catalase or pyruvate. Incubated anaerobically at 30°C, this medium allowed recognition of fast milk-coagulating isolates in 28 single strains and 7 commercial frozen cheese starter concentrates and differentiation of both fast and slow isolates in strains containing both cell types. Genetically characterized slow isolates could be placed into one of two distinct groups by colony appearance. The first group fermented lactose but was proteinase negative, and the second group included lactose-negative colonies that were either proteinase positive or negative.