Construction of Plasmid Cloning Vectors for Lactic Streptococci

Applied and Environmental Microbiology, 1987

A chromosomal fragment of 6.7 megadaltons (MDa), apparently containing the genes for milk protein utilization by Streptococcus lactis subsp. lactis SSL135, was cloned in S. lactis subsp. lactis MG1614, a proteinase-negative strain. For the cloning, the chromosomal DNA of SSL135 was cleaved with restriction enzyme Bam HI and the resulting fragments were ligated to the single Bcl I site of pVS2, a 3.3-MDa chloramphenicol-erythromycin double-resistance plasmid constructed in this laboratory. S. lactis subsp. lactis MG1614 was transformed by using this ligation mixture and selecting for chloramphenicol resistance and growth in citrated milk medium. One clone containing a 10.0-MDa plasmid, subsequently designated as pVS6, was chosen for further studies. Despite the lack of homology with previously characterized proteinase genes of lactic streptococci, the cloned insert consistently conveyed the ability to grow in milk to proteinase-negative recipients in repeated transformation experimen...

Two plasmids determining resistance to tetracycline .(RIP500) and to chloramphenicol, erythromycin, lincomycin, and pristinamycin I (RIP501) were isolated from a strain of Streptococcus agalactiae. The frequency-of-resistance loss is very low for RIP500 (<3 x 104) but higher for RIP501 (the efficiency was dependent upon the curing agents and incubation temperature and varied between 0.5 and 96%). Derivatives susceptible to all drugs were also obtained. RIP500 and RIP501 have similar molecular weights (17.9 x 106 and 20 x 106, respectively) and represent different percentages of total deoxyribonucleic acid (0.4 and 4%, respectively). The number of copies of RIP500 and RIP501 per cell is different, and these plasmids are likely replicated under different kinds of control (stringent and/or relaxed). No plasmid deoxyribonucleic acid was found in a derivative of strain B96 susceptible to all drugs.

Applied and Environmental Microbiology, 1985

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned deter...

Journal of Bacteriology, 1984

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-tiegative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 x 10-2 to 2 x 10-1 per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.

Microbial Pathogenesis, 1987

Journal of Bacteriology, 1975

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Ben-), and those from strain JH3 had also lost resistance to the bacteriocin (Bnr-). The majority of Hly-, Ben- variants from JH1 retained bacteriocin resistance (Bnrplus). Strains JH1 and JH3 contained a plasmid deoxyribonucleic acid species of molecular weight 38 times 10-6 (plasmids pJH2 and pJH3, respectively), and strain JH1 also contained a 50 times 10-6 molecular weight plasmid (pJH1) which has previously been shown to carry the genes determining resistance to the antibiotics kanamycin, neomycin, streptomycin, erythromycin, and tetracycline. Hly-, Bcn-, Bnr- variants of strain JH3 had completely lost plasmid pJH3. Hly-, Bcn-, Bnr- variants of strain JH1 had completely lost plasmid pJH2 and retained plasmid ...

Journal of Bacteriology, 1974

Three plasmids designated α, β, and γ, distinguishable by their molecular weights (6, 17, and 34 million, respectively) were isolated from Streptococcus faecalis strain DS-5 (ATCC 14508). Derivatives of this strain “cured” for erythromycin resistance lacked the β-plasmid. In the parent strain the β-plasmid was estimated to be present to the extent of one to two copies per chromosomal genome equivalent whereas the α- and γ-plasmids were about nine and five copies, respectively.

Journal of Dairy Science, 1986

Examination of single colony isolates from a culture of Streptococcus crernoris M12R revealed a high degree of variability in plasmid deoxyribonucleic acid composition. Fifty percent of the M12R population displayed proteolytic activity and harbored a 13-Mdahon plasmid (pLR2013). This plasmid was not present in proteinase-deficient variants isolated from the culture, which provided correlative evidence for linkage of proteinase activity to pLR2013. Four percent of the M12R population demonstrated resistance to phage ml2r'M12. This resistance was identified by restriction and modification activities against ml2r-M12 phage, which was dependent on the presence of a 20-Md plasmid, pLR1020. Loss of restriction and modification activities was observed upon curing of pLR1020. In conjugal mating studies with Streptococcus lactis ME2, transfer frequency of lactose-fermenting ability to a restriction and modification-deficient variant of M12R was 102-fold higher than to a variant exhibiting restriction and modification activities. The data provided evidence for restriction and modification activities in select S. cremoris M12R variants that are linked to pLR1020 and restrict both the plaquing ability of phage and efficiency of plasmid transfer by conjugation.

Journal of dairy science, 1986

Examination of single colony isolates from a culture of Streptococcus cremoris M12R revealed a high degree of variability in plasmid deoxyribonucleic acid composition. Fifty percent of the M12R population displayed proteolytic activity and harbored a 13-Mdalton plasmid (pLR2013). This plasmid was not present in proteinase-deficient variants isolated from the culture, which provided correlative evidence for linkage of proteinase activity to pLR2013. Four percent of the M12R population demonstrated resistance to phage m12r X M12. This resistance was identified by restriction and modification activities against m12r X M12 phage, which was dependent on the presence of a 20-Md plasmid, pLR1020. Loss of restriction and modification activities was observed upon curing of pLR1020. In conjugal mating studies with Streptococcus lactis ME2, transfer frequency of lactose-fermenting ability to a restriction and modification-deficient variant of M12R was 10(2)-fold higher than to a variant exhibi...

Applied and environmental microbiology, 1988

An electroporation procedure for the plasmid-mediated genetic transformation of intact cells of Streptococcus cremoris and Streptococcus lactis was performed. Ten different strains were transformed. The method was simple and rapid and yielded transformant colonies in 14 to 24 h. The method was optimized for S. lactis LM0230, and transformation frequencies of between 1 x 10 and 5 x 10 transformants per mug of purified plasmid (pMU1328) were achieved routinely. The optimized procedure involved lysozyme treatment of cells. Transformation of LM0230 occurred at comparable frequencies with pLS1 (4.4 kilobase pair [kbp]), pMU1328 (7.4 kbp), and pAMbeta1 (26.5 kbp). Plasmid DNA isolated from transformants had not undergone detectable deletions or rearrangements. Transformation was possible with plasmid DNA which was religated after restriction endonuclease digestion. Phage DNA-dependent transfection of S. lactis LM0230 and S. lactis C6 was also achieved.