Characterization of a theta-replicating plasmid from Streptococcus thermophilus

Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.     

Electrotransformation of industrial strains of Streptococcus thermophilus

A standard electroporation procedure was utilized to introduce a range of Gram-positive plasmid vectors into nine industrial strains of Streptococcus thermophilus. All the strains were transformable with at least two of the plasmids assessed, but electrotransformation frequencies depended on both the strain and the nature of transforming DNA. In general, small rolling circle (RC) plasmids could be electroporated at high frequency into a wide range of strains with efficiencies of 10(2)-10(5) transformants microgram-1 of transforming DNA. The presence of these plasmids did not influence doubling times during growth in broth, and they were generally extremely stable in slow milk acidifying strains, with 85-100% of transformants retaining the selective markers over 105 generations. Vectors were less stable in fast-growing cultures. Of the three theta-type plasmids assessed, only one, pIL253, could be electroporated at low frequency into some slow growing strains. The presence of this plasmid caused a 40% increase in doubling time and it was lost from cells at a rate of 3% per generation. Attempts to alter the proteolytic status of slow acidifying strains of Strep. thermophilus by the introduction of heterologous proteinase genes are also described.     

Analysis of two theta-replicating plasmids of Streptococcus thermophilus

We report the characterization of two new theta-replicating plasmids of Streptococcus thermophilus (pSMQ-312b and pSMQ-316) as well as the further analysis of pSMQ-308. The nucleotide sequences of pSMQ-312b and pSMQ-316 were determined and both contained 6710 bp. In fact, the two sequences were identical, despite that the plasmids were isolated from two different S. thermophilus strains as demonstrated by pulsed-field gel electrophoresis. Comparative analyses indicated that the two plasmids were highly related to the previously characterized S. thermophilus plasmid pSMQ-308 (8144 bp). Plasmid stability tests showed that pSMQ-312b/316 was more stable in LM17 medium while pSMQ-308 was the most stable in milk. The presence of the plasmids did not modify the acidification profile of the S. thermophilus strains during growth in milk and under time-temperature conditions mimicking an industrial process. These theta-replicating plasmids are unique genetic material for the construction of stable cloning vectors for industrially relevant strains of S. thermophilus.     

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.     

Application of the shsp gene,encoding a small heat shock protein,as a food-grade selection marker for lactic acid bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

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.     

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.     

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.     

Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci?

A 32.5kb variable locus of the Streptococcus thermophilus CNRZ368 chromosome, the eps locus, contains 25 ORF and seven insertion sequences (IS). The putative products of 17 ORF are related to proteins involved in the synthesis of polysaccharides in various bacteria. The two distal regions and a small central region of the eps locus are constant and present in all or almost all of the S. thermophilus strains tested. The other regions are variable and present in only some S. thermophilus strains tested, particularly in the closely related strains CNRZ368 and A054. A 13.6kb variable region of the eps locus of S. thermophilus CNRZ368 contains two ORF that are almost identical to epsL and orfY of the eps locus of Lactococcus lactis NIZOB40 and seven IS belonging to four different families, ISS1, IS981, IS1193 and IS1194. Five of these sequences were probably acquired by horizontal transfer from L. lactis (Bourgoin, F., et al., 1996. Gene 178, 15-23). Three probes of this 13.6kb region hybridized with the DNA of several L. lactis strains tested. A specific probe for another sequence within the S. thermophilus eps locus, epsF, hybridized with the DNA of one of the L. lactis strains tested. Sequence comparisons also suggest that five ORF of the eps locus have a mosaic structure and probably result from recombinations between sequences that are 10 to 50% divergent. The chimeric structure of the eps locus suggests a very complex evolution. This evolution probably involves both the acquisition of the 13.6kb region from L. lactis by horizontal transfer and exchanges within the S. thermophilus species.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Narrow host range of some streptococcal R plasmids

Nine R plasmids originally harbored by Streptococcus faecalis (pIP614, pIP655, pIP685, pIP686, pIP 1075, pIP1017),S. faecium (pIP716, pIP991), and group B Streptococcus (pMV120) wild-type hosts were transferred by conjugation into various recipients in order to study the extent of their intraspecies, interspecies, and intergeneric host range. Recipients were streptococci of groups A, B, C, D (S. faecalis, S. faecium, S. durans, S. bovis), and G, S. sanguis, two S. pneumoniae strains (encapsulated and nonencapsulated), and two strains of different genera, Staphylococcus aureus and Listeria inocua. The plasmids carried different antibiotic resistance markers: tetracycline, high levels of gentamicin and kanamycin or of streptomycin and kanamycin, and chloramphenicol. These R plasmids displayed narrow host ranges. They transferred into S. faecalis recipients and plasmid DNA could be detected in these transconjugants. Occasionally, the R plasmids also transferred into one or more other recipients, but no detectable plasmid DNA could be demonstrated in the new hosts.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates

T. WATANABE, H. KUMATA, M. SASAMOTO AND M. SHIMIZU-KADOTA. 1992. Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates.

Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.     

Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.     

Distribution and analysis of plasmids inStreptococcus thermophilus

Summary In a survey of 35 strains ofStreptococcus thermophilus, 13 strains were found to harbor plasmid DNA. Most of these strains contained plasmid species varying in size from 2.2 to 7.15 kilobases. Only three strains had more than one plasmid species. Each of the nine distinct types of plasmid DNAs identified had two or more unique recognition sites for restriction endonucleases. The characteristics of the indigenous cryptic plasmids ofS. thermophilus may allow their development as cloning vectors useful in the genetic engineering of this species and other streptococci that are important in food production     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.     

Molecular genetics of Streptococcus thermophilus

Abstract The metabolism and genetics of Streptococcus thermophilus (presently Streptococcus salivarius ssp. thermophilus ) have only been investigated recently despite its widespread use in milk fermentation processes. The development of recombinant DNA technology has allowed impressive progress to be made in the knowledge of thermophilic dairy streptococci. In particular, it has permitted a careful analysis of phenotypically altered variants which were derived from a mother strain by plasmid or chromosomal DNA reorganization. While natural phage defense mechanisms of S. thermophilus remain poorly documented, information on the bacteriophages responsible for fermentation failures has accumulated. The lysogenic state of two S. thermophilus strains has also been demonstrated for the first time. Gene transfer techniques for this species have been established and improved to the point that targeted manipulation of their chromosomal determinants is now feasible. Cloning and expression vectors have been constructed, and a few heterologous genes were successfully expressed in S. thermophilus . The first homologous genes, involved in carbohydrate utilization, have been cloned and sequenced, shedding some light on the molecular organization of key metabolic steps.