Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability.

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.     

Adaptative potential of the Lactococcus lactis IL594 strain encoded in its 7 plasmids

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways.     

Two plasmid-determined restriction and modification systems in Streptococcus lactis

Two restriction and modification systems were found in Streptococcus lactis strain IL594 which was found to contain 9 plasmids designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments, we showed that a restriction and modification system was related to the presence of pIL6 or pIL7. The pIL6-determined restriction and modification system was confirmed by cotransfer of the plasmid and of the restriction and modification system to a plasmid-free, nonrestricting, and nonmodifying derivative of S. lactis IL594.     

Thiol-stimulated secretion of riboflavin and porphyrins by Escherichia coli

Abstract 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.     

Molecular Properties of Streptococcus thermophilus Plasmid pER35 Encoding a Restriction Modification System

Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss. One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA. Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced. One of these plasmids, pER35, isolated from S. thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp. lactis and pND861 in Lactococcus lactis biovar diacetylactis. The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria. Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L. lactis subsp. lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S. thermophilus ST135. The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits. In addition, all three plasmids encoded proteins that are present on other S. thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp). The presence of a complete R-M system encoded on a plasmid in S. thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S. thermophilus from bacteriophage attack under dairy fermentation conditions.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

The effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1. Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1-2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.     

Multilocus enzyme typing of human and animal strains of Clostridium perfringens

Abstract The stability of plasmids introduced in Lactobacillus lactis IL1403 was followed in continuous cultures. Four strains with or without pIL205 and pIL252 or pIL253 (low or high copy number) were studied. pIL205 was remarkably stable even after 180 generations, suggesting the presence of a partitioning system which is still unidentified. In contrast, pIL252 and pIL253 were unstable, in the presence as well as in the absence of pIL205. The multicopy plasmid pIL253 was nevertheless more stable than pIL252 (90% loss after 180 and 60 generations, respectively). The instability was not related to an incompatibility between pIL205 and pIL252 (or pIL253). The segregational instability of pIL252 and pIL253 plasmids could be explained by the loss of the resolvase gene during their construction.     

pH influences growth and plasmid stability of recombinant Lactococcus lactis subsp. lactis

Continuous cultures of a recombinant Lactococcus lactis subsp. lactis strain were performed to display the effect of the fermentation pH on the specific growth rate and plasmid stability. The proportion of plasmid pIL252 bearing cells decreased exponentially with the number of generations. The influence of the pH on the rate of loss of plasmid pIL252 and on the specific growth rate of L. lactis IL2682 was described by second order polynomial equations. Optimal pH for the growth and plasmid stability were 6.39 and 6.41 respectively. © Rapid Science Ltd. 1998     

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.     

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).     

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA.

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).     

Plasmid content and bacteriocin production by five strains of Lactococcus lactis isolated from semi-hard homemade cheese

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

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-negative 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.     

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-ß-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.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Presence of Lactose Genes and Insertion Sequences in Plasmids of Minor Species of the Genus Lactococcus

The type strains of all known species and biovars of the Lactococcus genus were tested for the presence of plasmids, lactose genes, and insertion sequences cloned from the lactose plasmid of Lactococcus lactis subsp. lactis. Only the biovar xylosus of this subspecies is plasmid free. The lactose plasmid is present only in lactose-positive strains except in Lactococcus plantarum. The distribution of insertion sequences varies within the type strains of the Lactococcus genus.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Plasmid-Determined Systems for Restriction and Modification Activity and Abortive Infection in Streptococcus cremoris

Streptococcus cremoris strain IL964 possessed a restriction and modification (R/M) activity which resulted in a bacteriophage efficiency of plating of 5 × 10⁻⁶. Phage sensitivity of protoplast-induced plasmid-cured derivatives indicated that two plasmids called pIL103 (5.7 kilobases) and pIL107 (15.2 kilobases) were each coding for one R/M system. Plasmid pIL103-encoded R/M was ascertained by transfer into the plasmid-free, R⁻/M⁻ strain IL1403 of S. lactis, using protoplast cotransformation. This procedure failed for pIL107 because of some degree of incompatibility between pIL107 and the indicator plasmid pHV1301 used in cotransformation experiments. We also observed that plasmid pIL105 (8.7 kilobases) which showed no incidence on phage sensitivity in the parental strain IL964, mediated abortive infection in strain IL1403. In 97% of the infected cells, the phage infection was abortive, while in the remaining 3% phages were produced with a decreased burst size (50 instead of 180).