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.     

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.     

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac⁻) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.     

Plasmid Distribution and Evidence for a Proteinase Plasmid in Streptococcus lactis C2

Five plasmids, distinguishable by their molecular weights (10⁶, 2 × 10⁶, 5 × 10⁶, 10⁷, 3 × 10⁷, respectively) were isolated from Streptococcus lactis C2. A spontaneous proteinase-negative derivative of this strain lacked the 10⁷ plasmid.     

On the relationship between glycerophosphoglycolipids and lipoteichoic acids of gram-positive bacteria. III. Di(glycerophospho)-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis NCDO 712.

1. Streptococcus lactis NCDO 712 contains at lease three unusually polar glycerophosphoglycolipids. One of them was composed of D-glucose, glycerol, fatty acid ester, and phosphorus in the molar ratio of approx. 2 : 3 : 3 : 2. The structure was established as 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho-3-sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]-glycerol. 2. The second glycerophosphoglycolipid was shown to have the same core structure but is lacking the carbohydrate-linked fatty acid. The third glycero-phosphoglycolipid is a glycosylated derivative of the first one bearing an alpha-galactosyl residue at position 2 of the inner glycolipid-linked glycerophosphate moiety. 3. These novel phosphoglycolipids are considered to be the so far missing link between simple glycerophosphoglycolipids and lipoteichoic acids of Gram-positive bacteria.     

Chondroitin sulfate-depolymerizing activity in Streptococcus intermedius and other streptococci

The type strain (ATCC 27335) and 18 human oral isolates of Streptococcus intermedius and some other related streptococcal species were tested for chondroitin sulfate C-depolymerizing activity employing a modified screening plate method of Smith and Willett. As the results, S. intermedius strains except for ATCC 31412 strain were found to possess this activity. Propionibacterium acnes ATCC 11828 used as a positive control strain demonstrated strong activity, whereas S. intermedius strains showed only slightly detectable activity. This finding might be interesting in view of the classification of this species as well as its pathogenicity.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Specificity of a cell wall proteinase from Streptococcus lactis NCDO763 towards bovine β-casein

Abstract A cell wall proteinase from Streptococcus lactis liberated, with a molar ratio of enzyme over substrate of approx. 10⁻⁵, at least 5 peptides from the C-terminal part of β-casein. The substrate specificity of the enzyme appears to be low. The occurrence of a hydrophobic residue at position P3 seems to be the only feature common to 4 of the 5 susceptible peptide bonds. The released fragments have features found in peptides producing a bitter taste.     

Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis.

Streptococcus lactis subsp. diacetylactis strain WM4 transferred lactose-fermenting and bacteriocin-producing (Bac+) abilities to S. lactis LM2301, a lactose-negative, streptomycin-resistant (Lac- Strr), plasmid-cured derivative of S. lactis C2. Three types of transconjugants were obtained: Lac+ Bac+, Lac+ Bac-, and Lac-Bac+.S. diacetylactis WM4 possessed plasmids of 88, 33, 30, 5.5, 4.8, and 3.8 megadaltons (Mdal). In Lac+ Bac+ transconjugants, lactose-fermenting ability was linked to the 33-Mdal plasmid and bacteriocin-producing ability to the 88-Mdal plasmid. Curing the 33-Mdal plasmid from Lac+ Bac+ transconjugants resulted in loss of lactose-fermenting ability but not bacteriocin-producing ability (Lac- Bac+). These strains retained the 88-Mdal plasmid. Curing of both plasmids resulted in a Lac- Bac- phenotype. The Lac+ Bac- transconjugant phenotype was associated with a recombinant plasmid of 55 or 65 Mdal. When these transconjugants were used as donors in subsequent matings, the frequency of Lac transfer was about 2.0 X 10(-2) per recipient plated, whereas when Lac+ Bac+ transconjugants served as donors, the frequency of Lac transfer was about 2.0 X 10(-5) per recipient plated. Also, Lac- Bac+ transconjugants were found to contain the 88-Mdal plasmid. The data indicate that the ability of WM4 to produce bacteriocin is linked to an 88-Mdal conjugative plasmid and that lactose-fermenting ability resides on a 33-Mdal plasmid.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Lactose Hydrolysing Enzymes in Streptococcus lactis and Streptococcus cremoris and also in some other Species of Streptococci

Two types of Streptococcus lactis could be identified: cheese starter strains, which contain β-phosphogalactosidase and ferment lactose rapidly to lactate, and non-dairy strains, which contain both β-galactosidase and β-phosphogalactosidase and ferment lactose slowly to a variety of end products. All strains had homolactic glucose fermentations and heterolactic galactose fermentations. Other species of streptococci were examined for lactose hydrolysing enzymes and found to contain β-phosphogalactosidase, except Strep, thermophilus and Strep. faecium which had high levels of β-galactosidase. Discrepancies were found in the lactose hydrolysing enzymes content when the cells were treated in different ways.