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Isolation and Characterizationof Plasmids from Strains ofStreptococcus ThermophilusBulgarian Yogurt StartersP. Petrovaa, S. Danovaa, G. Stoyanchevaa & V.Mitevaa

a Institute of Microbiology, Bulgarian Academy ofSciences, Sofia, BulgariaPublished online: 15 Apr 2014.

To cite this article: P. Petrova, S. Danova, G. Stoyancheva & V. Miteva (2003)Isolation and Characterization of Plasmids from Strains of Streptococcus ThermophilusBulgarian Yogurt Starters, Biotechnology & Biotechnological Equipment, 17:1,105-113, DOI: 10.1080/13102818.2003.10819203

To link to this article: http://dx.doi.org/10.1080/13102818.2003.10819203

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ISOLATION AND CHARACTERIZATION OF

PLASMIDS FROM STRAINS OF STREPTOCOCCUS

THERMOPHILUS BULGARIAN YOGURT STARTERS

P. Petrova, S. Danova, G. Stoyancheva, V. Miteva Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

ABSTRACT Twenty Bulgarian Streptococcus thermophil us strains used in industrial milk fermentation were analyzed for their plasmid content. Six of them were found to contain one or two plasmids. The newly isolated plasmids were divided into three groups based on DNA ho­mology. Six plasmids, sharing homology with pt38 and p2992, were classified within group/, two plasmids were classified into group II and one plasmid, p299/, possessing homology only with itself, was classified in Group III. Hybridization experiments revealed low homology between streptococcal and Lactobacillus plasmids. Using plus-origin­specific (dso) DIG-labeled probes we determined that two of the plasmids (pt38 and p2992) belong to the pC194 plasmid family, that uses the rolling circle mode of replication. One plasmid from group I, pt38 was further characterized. We constructed a restriction map and identified the restriction fragment bearing the dso of replication. pt38 appears to be an excellent candidate for creation of a shuttle vector for gene expression in Escherichia coli and the Streptococcus thermophilus because it is small in size, has five unique restriction sites and known location ofthe plus-origin of replication.

Introduction Streptococcus salivarius subsp. thermoplzi­lus (S. thermophilus) belongs to a diverse group of industrial microbes known as lac­tic acid bacteria (LAB), which includes streptococci, lactococci and lactobacilli. S. thermophilus is a common symbiotic partner of Lactobacillus bulgaricus, Lacto­bacillus helveticus, Lactococcus lactis or Propionibacterium sp. and is responsible for the development of flavor and physical characteristics of different fermented dairy products.

Despite the centuries-long use of S. thermophilus in fermentation, progress in our understanding of its plasmid content has only been made in the last fifteen years. Plasmids determine important industrial phenotypes, including carbohydrate fer­mentation, bacteriocin and exopolysaccha-

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ride production, and phage resistance mechanisms (29) and thus play a critical role in all LAB. Streptococcus thermoplzi­lus is naturally poor of plasmids (18); a li­mited number of isolates have been repor­ted to contain single plasmids of relatively small size (2-15 kb) and in exceptional cases, two or three replicons were found. The majority of the studied plasmids have remained cryptic, probably due to their small molecular mass.

Many authors have tried to classify these plasmids using DNA-DNA homology. Herman and McKay (1 0) showed that cross-hybridization occurs between three single plasmids from different S. thermo­phi/us strains. Later, Girard (4) demon­strated the existence of 3 homology groups of plasmids within the species. Two other studies showed that S. thermophilus plas-

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mids might be divided into five distinct DNA homology groups (13, 26). The same authors used the 2.2 kb plasmid ER8 from S. thermophilus in searching for homology with plasmids from Lactobacillus and Lactococcus strains and detected none (27).

Presently, nine plasmids have been se­quenced, ranging in size from 2093 to 9500 bp (29, 24, 25, Solow and Somkuti, 2001, 28). The majority of them belong to the same DNA homology group, the pC 194 family (14). There are only two Strepto­coccus thermophilus plasmids, that are members of the pEJ94 group: pMVJ58 (pLS I - 5.5 kb, carrying a tet-resistance gene) from S. agalactiae (2), and pSMQ 172 from Streptococcus thermo-phi/us (29).

Sequence data and some experimental evidence suggest those small plasmids from S. tlzermoplzilus replicate by the roll­ing circle (RC) mode. This mechanism in­volves the generation of a site-specific nick by the plasmid encoded initiator RepA protein at the leading strand dso (double stranded origin), followed by extension of the 3'-hydroxyl end by DNA polymerase and appearance of single-stranded (ss) forms of the plasmid molecule (5). A spe­cific sequence motif, known as the minus origin, enables the conversion of ss-DNA intermediates into ds-DNA molecules.

RC plasmids are classified according to the dso-origin sequence motif~. The aim of thi<; study was tn examine the plasmid contt:nt or different S. thermophilus yogmt strains anu classify the discovered plasmids using DNA homology criteria. One of the discovered plasmids, pt38, previously iso­lated in our laboratory ( 19) was further characterized as a possible shuttle vector.

Materials and Methods Bacterial strains, media and reagents. All bacterial strains and plasmids used in this study are listed in Table 1. S. tlzermo­plzilus strains are Bulgarian natural yogurt isolates, starter cultures, kindly supplied by ELBY- Center for Research and Develop-

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ment and Production of milk products. The referent staphylococcal plasmids pE 194, pC 194 and pT 181 are maintained in our laboratory collection in Bacillus subtilis as a host. The pA 1-6 (3.5 kb, with ely-resis­tance gene) was kindly provided by Prof. Topisirovic from the Institute of Molecular Genetics and Genetic Engineering, Bel­grade), (30). The hybridization analysis included the following plasmids from the genus Lactobacillus: pLBL4 (2.6 kb, (3)), plasmids from L. lzelveticus A TCC 15009 (22 kb; 6 kb and 3.5 kb; (21)); pb8 (5.2 kb) - a plasmid from L. bulgaricus, discovered previously in our laboratory (unpublished).

S. tlzermoplzilus strains were grown at 42°C in M 17 broth (Difco Laboratories, Detroit, Mich.), supplemented with I% beef L'Xtract (Difco) and 1,9% B-glycero­pho:-.l~h.tte (Sigma), (17). Lactobacillus strains were grown in MRS medium (Difco) at 37°C, and E. coli in Luria broth at 37°C (10% bactotryptone, 5% yeast ex­tract, 5% NaCl, Sambrook et al., 1989). All media were solidified with 1,5 % agar (Difco), when used as agar plates. Ampi­cillin, chloramphenicol and erythromycin were added in LB medium at final concen­trations respectively of I OOmg/1, 25mg/l and 300mg/l. All antibiotics were purcha­sed from Sigma-Ah.lrich. DNA manipulations and DIG labeling. The piasmid DNA from E. coli and Bacil­ltts subtilis was isolated by the Bimboim and Dolly (l) alkaline lysis method. The procedure of Somkuti and Steinberg (26) was used for the extraction of plasmids from streptococci and the method of Klaenhammer (16) - for Lactobacillus plasmid DNA. Plasmids were analyzed in 0,8 % agarose gels in TBE buffer, pHS.

Random priming with the DNA Labeling and Detection System of Boehringer Mannheim was performed for DIG labeling of the plasmid probes. Additional steps of purification and extraction of the super­coiled plasmid molecules from agarose gel were included. The 5'-DIG labeled oligo-

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TABLE I Bacterial strains and plasmids used in this study

Bacterial Plasmid Size (kb) Relevant characteristics Source Reference

strain

ST2783 pt38 2.9 kb S. thermophi/us, yogurt starter ELBY Miteva eta/., 1991

ST2973 No S. thermophilus, yogurt starter ELBY This work

ST2974 No S. thermophilus, yogurt starter ELBY This work

ST2975 No S. thermophil us, yogurt starter ELBY This work

ST2976 No S. thermophilus, yogurt starter ELBY This work

ST2977 p2977 S. thermophilus, yogurt starter ELBY This work

ST2978 No S. thermophilus, yogurt starter ELBY This work

ST2979 No S. thermophilus, yogurt starter ELBY This work

ST2980 No S. thennophilus, yogurt starter ELBY This work

ST2981 No S. thermophilus, yogurt starter ELBY This work

ST2982 No S. thermoplzilus, yogurt starter ELBY This work

ST2983 p2983A, p2983B S. thermophil us, yogurt starter ELBY This work

ST2984 No S. thermophilus, yogurt starter ELBY This work

ST2985 No S. thermophilus, yogurt starter ELBY This work

ST2986 No S. thermophilus, yogurt starter ELBY This work

ST2987 No S. thennophilus, yogurt starter ELBY This work

ST2988 No S. thermophilus, yogurt starter ELBY This work

ST2990 p2990 S. thermophilus, yogurt starter ELBY This work

ST2991 p2991 S. thermophilus, yogurt starter ELBY This work

ST2992 p2992 S. thermophilus, yogurt starter ELBY This work

ST2993 No S. thermophilus, yogurt starter ELBY This work

15009 pLHI 22 kb L. helveticus ATCC Pridmore eta/., 1993 pLH2 6 kb pLH3 3.5 kb

B8 pbS 5.2 kb L. bulgaricus, yogurt starter ELBY Boudakov I., PhD Thesis, 1999, BAS

LBL4 pLBL4 2.6 kb L. he!l•eticus, cheese starter ELBY Gancheva eta/., 2000

B. subtilis pE194 3.7 kb Referent plasmid Lab. collection Gryczan, T. et al., 1980

B. subtilis pC194 2.9 kb Referent plasmid Lab. collection Horinouchi S. &Weis-blum, B., 1982a

B. subtilis pTI81 4.4 kb Referent plasmid Lab. collection Khan&Novick, 1983

E. coli pAI-6 3.5 kb Referent plasmid IMGGE- Vujcic&Topisirovic, Belgrade 1993

E. coli pY517 Standard plasmids strain Lab. collection Macrina et al., 1978

B. subtilis pHV33 6.9 kb Referent plasmid Lab. collection Primrose&Ehrlich,I981

E. coli Ml3 phage 6.4 kb Referent DNA Boehringer Ehrlich eta/., 1991 Mannheim

ELBY- Bulgarian Center of Research, Development and Production of Milk Products ATCC- American Type Culture Collection IMGGE- Institute of Molecular Genetics and Gene Engineering BAS- Bulgarian Academy of Sciences

nucleotide probes were synthesized by MWG, Germany. Their sequences and ac­cession numbers in NCBI are as follows:

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pC 194 dso-specific probe: DIG -5' TTC TTT CTT ATC TTG AT 3' (NC 002013, (II));

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pE194 dso-specific probe: DIG -S' GGG GGG TAC TAC GAC CT 3' (NC 001386, (6), (12));

pT181 dso-specific probe: DIG-S' AAA ACC GGC TAC TCT AAT 3' (NC 001393, (IS)).

The optimized temperatures for the 18h hybridization with the different probes are as follows:

- for the pC 194 dso- DIG probe - 3S°C, - for the pE 194 dso- DIG probe - 43 °C, - for the pT181 dso- DIG probe - 40°C. Hybridization at gradually decreasing

temperatures gave better results. The hy­bridization started at a temperature so higher than the Tm of the probe. It was decreased by so every 30 minutes until reaching 20°C. The starting temperatures for the different probes were as follows: 42°C for the pC194 dso-DIG probe, S2°C for the pE 194 dso-DIG probe, and sooc for the pT181 dso-DIG probe. Southern blotting and hybridization. The transfer of plasmid DNA from the agarose gel to nylon filter (Boehringer Mannheim GmbH) was performed with a vacuum blotter (BioRad) and hybridization with each probe was carried out at the condi­tions specified above. The hybridization solution was 20xSSC, 10% SDS, 10% SLS, 10% blocking reagent. Post-hybridi­zation washes and detection were per­formed according to the manufacturer's in­structions. Restriction mapping of pt38. Restriction enzymes, supplied by Boehringer Mann­heim GmbH, were used according to manu­facturer's instructions.

Results and Discussion Plasmid content and size of plasmid DNA in S. tlzermophilus strains A total of 6 out of the 20 S. thennophilus strains analyzed were found to contain plasmid DNA. Fourteen strains were plas­mid free. The agarose gel electrophoretic profiles of the different types of plasmid DNAs and the calculated size of each

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plasmid are shown in Fig. 1A and Table 2, respectively. Seven different in size plas­mids were detected in the S. thermophilus Bulgarian dairy cultures, ranging in size from 2.9 kb to 8.2 kb. Only one strain, ST2983, harbored two plasmids. All other plasmids were found in different strains. Identification of the plasmid homology groups DNA-DNA hybridization experiments were used to explore the homology of new S. thermophilus plasmids and to separate them in several groups.

First, we used the smallest plasmid pt38 as probe. Six plasmids hybridized with pt38-DIG and were included in group I: pt38, p2977, p2983A, p2983B, p2990 and p2992 (Fig. 1, B). These results were con­firmed with a second probe (p2992-DIG,), another small plasmid from group I, who showed hybridization profile identical to that of pt38-DIG (data not shown).

One strain of this group (ST2983) con­tained two plasmids p2983A and p2983B, both of which hybridized to pt38-DIG and p2992-DIG and needed additional charac­terization. For this purpose we used a third probe p2990, labeled with DIG. It showed a high level of homology with p2983B, and pt38 (Fig. 1, D). Plasmids, which were homologous to pt38 and p2992, but not to p2990, formed group II: p2977, p2983A. Group III contained p2991, not similar to any one of the probes and hybridizing only with itself (data not shown).

Plasmids of groups II and I may be con­sidered compatible, since strain ST2983 carried plasmids, which belonged to diffe­rent groups. These results are consistent with previous investigations of Somkuti and Steinberg (26) and Turgeon and Moineau (29) that distributed 13 and 1S S. thermophilus plasmids within five and four· groups, respectively and proved the com­patibility of plasmids of different groups in one strain.

Interestingly, no homology was detected between streptococcal and several Lacto-

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A B

2 3 4 5 6 7 8 2 3 4 5 6 7 8

c D Fig.l. The plasmid profiles of Streptococcus thermophilus strains and plasmid grouping based on DNA-DNA homology. A. Streptococcal and referent plasmids separated in I% agarose gel. B. Hybridization with DIG labeled pt38 probe. No of lane and probe: I. ST2783, 2. ST2977, 3. ST2983, 4. ST2990, 5. ST2991, 6. ST2992, 7. B8, 8. LBL4, 9. ATCC15009, 10. pC194, II. pE194, 12. pT181, 13. pHV33, 14. pV517 (molecular weight marker). C. Streptococcal and referent plasmids separated in I o/o agarose gel. D. Streptococcal and ref­erent plasmids hybridized with DIG labeled p2990 plasmid probe. No of lane and probe: I. ST2990, 2. ST2990, 3. ST2977, 4. ST2977, 5. ST2983, 6. 2992, 7. pV 517 (molecular weight marker), 8. ST2783.

bacillus plasmids despite their symbiotic relationships in dairy fermentation and ex­isting evidence for exchange of genetic

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material among these species through the process of conjugation (7). None of the five small plasmids from lactobacilli (pLBL4,

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A B Fig.2. A. The plasmid profiles of Streptococcus thermophilus strains and referent plasmids separated in I% agarose gel. B. Hybridization with DIG-labeled pC 194-dso probe. No of lane and probe: I. ST2977, 2. ST2983, 3. ST2990, 4. ST2991, 5. ST2992, 6. ST2783, 7.B8. 8. LBL4, 9. ATCC15009, 10. pC194, II. pEI94, 12. pT181, 13. pHV33, 14 pAI-6, 15. pV 517 (molecular weight marker}, 16 Ml3 phage.

TABLE 2 Molecular weight of isolated plasmids and their host Streptococcus thermophilus strains

Host strain Plasmid Molecular weight

ST2977 p2977 6.9 kb

ST2983 p2983A 8.2 kb

ST2983 p2983B 5.0 kb

ST2990 p2990 3.5 kb

ST2991 p2991 5.2 kb

ST2992 p2992 3.8 kb

ST2783 pt38* 2.9 kb

*pt38 was previously isolated (19). Plasmid sizes arc determined from a standard curve of the log of mass (kb) of known plasmids of E. coli V517, versus their

respective RF values in I% agarose gel.

pLHl, pLH2, pLH3, pbS) showed homolo­gy to our streptococcal DIG-labeled probes pt38-DIG (Fig. I, B), p2992-DIG and p2990-DIG (data not shown). pt38 and p2992 are RCR-plasmids of pC194-type Usually small plasmids from Gram-positive bacteria, including the genus Streptococcus use the rolling-circle mode of replication. RCR plasrnids are classified according to the plus-origin (dso) sequence homologies. To determine the plus-origin group of the

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different plasrnids, Southern hybridization analyses were performed using Staphylo­coccus ss-generating plasmids pC 194, pE 194 and pT181 as reference plasrnids (Fig. 2, Table 3). Two of the established plasmids (pt38 and p2992) showed homol­ogy with pC 194 dso-DIG, hence it is most I ike! y that they are members of pC 194-dso group. The other two dso probes: pEI94dso-DIG and pT18ldso-DIG hy­bridized only with their corresponding ref­erence plasrnids (Table 3). Characterization of the plasmid pt38 with restriction endonucleases and con­struction of a physical map Plasmid pt38 was the smallest of the newly isolated plasrnids (2,9 kb). It was cleaved by only I 0 of 40 restriction endonucleases tested, and had single restriction sites for Hindiii, Haeiii, EcoRV, Muni and Cfoi (Fig. 3). Two linear fragments were gener­ated from this plasmid following digestions with BstEII (2.2 and 0.7 kb) and three with Rsal (1.5, 0.7, 0.6 kb) and Ddel (1.7, 0.9, 0.3 kb).

Using double and triple digests of pt38 and its constituent fragments we construc­ted a restriction map. This approach allow­ed the unambiguous alignment of all res-

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Fig.3. Restriction enzyme map of plasmid pt38 (2.9kb). The circular map is generated with the aid of computer program Clone Manager 4.0. The enzymes, which do not cut in pt38 are: A val, Avail, Asnl, BamHI, Bgll, Bglll, Clal, Cfrl, EcoRI, Kpnl, Neil, Nrul, Nsil, Mbol, Mspl, Pstl, Pvul, Sail, Sau3A, Smal, Xbal, Xhol.

TABLE3 Hybridization of isolated plasmids of Streptococ­cus thermophilus and reference plasmids with different dso-probes

dso-probes pCI94(+) pEI94(+) pTISI(+) Plasmids ori-DIG ori-DIG ori-DIG

p2977 - - -

p2983A - - -p2983B - - -

p2990 - - -p2991 - - -p2992 + - -

pt38 + - -pbS - - -

pLBL4 + - -pLH I ,pLH2,pLH3 + - -

pCI94 + - -

pE194 - + -pTI81 - - + pHV33 + - -pAI-6 - - -

Ml3-phage - - + pV517 + - -

Ill

triction fragments, with exception of these, generated with Sspl and Dral (4 frag­ments), Alul (11 fragments), Apol (6 frag­ments), which were too numerous and too small in size. The Hindlll single site was chosen as reference point for the alignment of fragments. There are no sites for: Aval, Avail, Asnl, BamHI, Bgll, Bg!II, Clal, Cfrl, EcoRI, Kpnl, Neil, Nrul, Nsil, Mbol, Mspl, Pstl, Pvul, Sail, SauJA, Smal, Xbal, Xhol. Localization of dso of the plasmid pt38 The availability of the DIG-labeled pC 194-dso sequence enabled us to determine the restriction fragment, carrying the dso of re­plication of pt38 by hybridization methods (Fig. 4).

After double digestions of pt38 plasmid DNA with appropriate endonucleases, the cleaved DNA fragments were separated in 1 % agarose gel and transferred to nylon membrane in alkaline conditions. The cleaved and separated fragments were hy­bridized with 5 'DIG-pC 194dso oligonu­cleotide probe. The detection results reve-

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M 2 3 4 5 6 2 3 4 5 6

Fig.4. The analysis of location of dso (plus-origin of replication) of pt38 by restriction digest; separating of fragments in agarose gel and hybridization with DIG-labeled pC194-dso. A. Separating of restriction fragments of pt38 in 1% agarose gel. B. Hybridization with DIG-labeled pC194-dso. No of lane and probe: I. pt38/EcoRV, 2. pt38/Hindlll, 3. pt38/Haelii, 4. pt38/Hindlii!EcoRV, 5. pt38/Haelii!EcoRV, 6. pt38/HaeiiUHindiii.

aled that the dso of pt38 is positioned on the 1.9 kb restriction fragment HindliV EcoRV.

There are several major conclusions from this work. This is the first comprehensive survey of the plasmid content of strepto­coccal Bulgarian endemic yogurt strains. Several, varying in size plasmids were found, in contrast to previous works, claiming paucity of plasmids in S. thermo­phi/us.

It is important to note that as a starter culture S. thermophilus require elevated processing temperature. Many features of these bacteria, including the synthesis of heat-shock proteins and the enzymes of restriction/modification system have plas­mid determination (25). Interestingly, most industrial strains in many countries are plasmid free (one often-cited explanation is that plasmids may exert a negative effect

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during the selection of industrial strains), while the best Bulgarian yogurt starters were found to contain plasmids. This may have significant theoretical and practical implications. Additionally, the new plas­mids were separated into three groups on the base of DNA homology, which con­firmed the diversity of the streptococcal plasmids and the fact, that there is no ho­mology between them and the plasmids from lactobacilli (26, 29).

The presented results expand the know­ledge on the mode of replication of small plasmids of Gram-positive microorganisms. To the moment the pC 194 group of RCR plasmids included several plasmids, con­taining hsp (the gene, coding heat-shock protein) or genes for the restric­tion/modification system. Our study sup­plemented the pC 194 family with two new plasmids (pt38 and p2992) for which the

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single stranded DNA intermediates were experimentally demonstrated.

The S. thermoplzilus plasmid pt38 may be a potential source of streptococcal (Gram­positive) dso in future cloning vector con­struction experiments. It is highly appropri­ate for this because of its relatively small size, apparently stable maintenance and the three unique restriction sites. The known localization of the dso makes possible to create deletion variants of pt38 and to clone the plus-origin fragment in a high copy Gram-negative plasmid in order to develop a shuttle vector system for Gram-positive and Gram-negative hosts.

Acknowledgments We thank the ELBY Center for Research, Development and Production of Milk Products, for kindly supplying of the Streptococcus tlzermophilus industrial starters used in this work.

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at 0

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ch 2

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