- Email: [email protected]
Cloning vectors for Streptococcus thermophilus derived from a native plasmid
FEMS Microbiology Letters 216 (2002) 43^47
www.fems-microbiology.org
Cloning vectors for Streptococcus thermophilus derived from a native plasmid Ping Su
a;b;
, Karen Jury a , Gwen E. Allison a , Wing Yee Wong b , Woojin S. Kim b , Chun-Qiang Liu b , Tony Vancov b , Noel W. Dunn b b
Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia a DSM Food Specialties, P.O. Box 83, Moorebank, NSW 2170, Australia
Received 27 May 2002 ; received in revised form 2 August 2002; accepted 2 September 2002 First published online 21 September 2002
Abstract A 3.5-kb native plasmid (pND103) was identified in Streptococcus thermophilus ST2-1. Preliminary sequence analysis indicated that pND103 belongs to group I S. thermophilus plasmids. A region of approximately 2 kb appears to contain three components: a plus origin of replication (ori) typical of plasmids that replicate via rolling circle replication; a gene encoding a replication protein (rep); and a gene encoding a small heat shock protein (hsp). pND103 was then used to construct S. thermophilus/Escherichia coli hybrid cloning vectors by ligating different portions of pND103 to an origin-probe vector (pND330) composed of pUC19 and a Gram-positive erythromycin resistance gene. The shuttle vectors (pND913, pND914 and pND915) were successfully introduced back into plasmid-free S. thermophilus ST3-1 as well as to Lactococcus lactis LM0230 and E. coli JM109. Segregational and structural stability study indicated that these vectors can be maintained in these hosts. The results indicated that pND913, pND914 and pND915 are potential shuttle cloning vectors for S. thermophilus. ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptococcus thermophilus ; Cloning vector; Plasmid ; pND913
1. Introduction Streptococcus thermophilus, an important microorganism in dairy food fermentation, is widely used as a starter culture for the production of yoghurt and certain cheese varieties. This industrially important species is a member of a diverse group of facultative anaerobes commonly referred to as lactic acid bacteria (LAB), which are used in various combinations to yield speci¢c fermented foods. The application of gene technology to S. thermophilus would be greatly facilitated by the development of additional e¡ective cloning vectors. Hybrid vectors with shuttle capacity incorporating the replication function of native S. thermophilus plasmids were reported by Somkuti et al. [9]. Very few plasmids have been found in S. thermophilus
(reviewed in [5]). Plasmids that have been reported range in size from 2 to 25.5 kb and, only in very few cases, multiple plasmids have been found to coexist in one strain [5]. Based on DNA homology, Janzen et al. [3] proposed ¢ve di¡erent groups of plasmids in S. thermophilus : group I consists of small plasmids, ranging in size from 2.1 to 3.5 kb; group II, 4.2 kb ; group III, 5.2 kb; group IV, 6.8 kb; and group V, 7.4 kb. In this paper we report the genetic organization of a native plasmid, pND103, from S. thermophilus ST2-1 and its use to construct hybrid cloning vectors for S. thermophilus.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions
* Corresponding author. Fax: +61 (2) 93136700. E-mail address : [email protected] (P. Su).
Strains used include: ST2-1, a wild-type strain of S. thermophilus; ST3-1, a plasmid-free strain of S. thermophilus; LM0230, a plasmid-free derivative of Lactococcus lactis C2 [2] and Escherichia coli JM109 [12]. Plasmids
0378-1097 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 9 7 0 - 9
FEMSLE 10673 31-10-02
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P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
used were: pGEM-7Zf(+), an E. coli vector encoding ampicillin resistance (Promega, Madison, WI, USA) ; pND330, an origin-probe vector composed of pUC19 and a Gram-positive erythromycin resistance gene [4]; and pGKV210, an E. coli/LAB shuttle vector [11]. The identi¢cation of pND103 and construction of pND913, pND914 and pND915 are described in the text. L. lactis strains were incubated statically at 30‡C in M17 medium [10] supplemented with 0.5% (w/v) glucose (M17G). S. thermophilus strains were propagated at 37‡C anaerobically in M17 medium supplemented with 0.5% (w/v) lactose (M17L). E. coli cultures were grown at 37‡C with shaking in Luria broth (LB) [7]. When appropriate, antibiotics were added as follows : for E. coli, 100 Wg ml31 ampicillin (Amp) or 100 Wg ml31 carbenicillin (Carb), for L. lactis, 5 Wg ml31 erythromycin (Em), and for S. thermophilus, 5 Wg ml31 Em.
2.3. Nucleotide sequencing and analysis Both DNA strands were sequenced using an Applied Biosystems (Foster City, CA, USA) 377 DNA sequencer, following the manufacturer’s protocol. Sequencing of DNA from pND103 was initiated using two primers designed according to the sequence of plasmid pGEM7Zf(+). Based on the sequences obtained, 20-mer oligonucleotide primers were then synthesized and used to ‘walk’ along the DNA template. Recording and analysis of the nucleotide sequence were carried out using the AutoAssembler1 DNA sequence assembly software (Applied Biosystems) and the ANGIS Software System operated by the Australian Genomic Information Center, University of Sydney. Amino acid sequences were compared with all the sequences held in the database by the ‘BLASTP’ program. Nucleotide sequence was searched for 310 and 335 sequences using the ‘Findpatterns’ program.
2.2. DNA manipulation and cloning techniques Lactococcal plasmid DNA was isolated by the method of Anderson and McKay [1]. Plasmids from E. coli were isolated by the alkaline lysis method [7]. Restriction digestion and molecular cloning were as described by Sambrook et al. [7]. Restriction endonucleases and T4 DNA ligase were purchased from Boehringer-Mannheim (Germany) and used as recommended by the manufacturer. Recovery of DNA fragments from agarose gels was performed using a QIAEX gel extraction kit (Qiagen, Chatsworth, CA, USA). L. lactis was transformed by electroporation as described by Powell et al. [6]. S. thermophilus was transformed by electroporation [8] and for E. coli, the CaCl2 transformation method was used [7].
3. Results and discussion 3.1. Characterization of a native plasmid from S. thermophilus ST2-1 A series of industrial strains was screened for plasmids. The strain S. thermophilus ST2-1 was found to harbor only one small plasmid (3.5 kb), and this plasmid was designated pND103. Restriction analysis indicated that pND103 carries a unique EcoRV site. This was used to clone pND103 into the SmaI site of the E. coli sequencing vector pGEM-7Zf(+). Both orientation insertions were obtained and the resulting plasmids were named pND846
Fig. 1. A: Gene organization of the native plasmid pND103 from S. thermophilus ST2-1. The restriction sites were con¢rmed by sequence analysis. The 335 and 310 regions, the ribosome binding site and start codon of the rep gene are underlined. A size reference is shown on the top. B: The nucleotide sequence shown is that of the putative promoter region for the rep gene.
FEMSLE 10673 31-10-02
P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
and pND847. Initially pND846 was used as the sequencing template but this proved to be unstable. pND847 with pND103 cloned in the reverse orientation was stable. Sequence analysis of the open reading frames (ORFs) suggested that instability of pND846 may have been due to the pGEM vector’s LacZ promoter reading into pND103’s open reading frames. Preliminary sequence analysis revealed four potential ORFs in pND103 (Fig. 1) and indicated that pND103 belongs to group I of the S. thermophilus plasmids of which sequence information is available for pST1 (2.8 kb) and pER341 (2.8 kb) [3]. A region (ORF1, ORF2, ORF3 and the ori) of approximately 2 kb is present in all four plasmids and is highly homologous. ORF3 encodes a replication protein (rep) and ORF1 encodes a small heat shock protein (hsp). The presence of a heat
45
shock protein could be important in aiding S. thermophilus survival at elevated growth temperatures although the hsp gene in some cases is not necessary for replication in S. thermophilus [5]. Somkuti et al. have recently investigated the e¡ects of heat shock on the expression of the plasmid-encoded hsp gene. They found that the hsp was heat stress-regulated but did not show a positive impact to cell survival [8]. The degree of sequence identity in the region containing ori, rep, and hsp is approximately 90% among the four plasmids. The ori region and the promoter of rep are also very similar between pND103 and pST1. As for the pST1 plasmid, the ribosome binding site (RBS) is an unusual 50 bases in front of the start codon for the rep gene (Fig. 1). An explanation for this could be the presence of a hairpin structure in between the RBS and the start codon thus reducing the e¡ective distance. ORF2
Fig. 2. Cloning strategy and restriction maps of plasmids pND330, pND847 and pND913. pND913 contains the ori region from the native plasmid pND103.
FEMSLE 10673 31-10-02
46
P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
shows no signi¢cant homology to any sequence at the amino acid level. ORF4 has a potential RBS but shows no signi¢cant homology to any known sequences at the amino acid level. Two very long stretches of ‘AT’-rich regions between ORF3 and ORF4 are present. 3.2. Construction of cloning vectors based on pND103 Plasmid pND103 was developed into E. coli/S. thermophilus shuttle vectors by ligating di¡erent portions of pND103 to pND330. Sequence analysis indicated that the ori region lies on the longer (2.1-kb) PvuII^EcoRV fragment (Fig. 1). To clone the ori region only pND847 (Fig. 2) was double-digested with PvuII and BamHI [BamHI is in the multicloning site, originally from pGEM7Zf(+)], then ligated to pND330 digested with SmaI and BamHI. The ligation mixture was precipitated with ethanol and electroporated into LM0230. Transformants were obtained on the M17G+0.5 M sucrose+Em selection plates. One of the clones was puri¢ed and the plasmid harbored in the strain designated pND913. The plasmid pro¢le of pND913 was checked with restriction enzyme analysis and it was con¢rmed that the ori region from pND103 had been cloned into pND330. The successful direct selection of the S. thermophilus ori region in Lactococcus indicates that the S. thermophilus origin of replication is functional in a lactococcal background. Using a similar procedure as above the whole pND103 was also cloned into pND330. pND847 was cut with XbaI and BamHI, then ligated into pND330 cut with the same two enzymes. This gave plasmid pND914. pND915 was generated in the same way but using BamHI and KpnI. Plasmid extraction and restriction analysis con¢rmed that pND914 and pND915 contain the complete pND103 in di¡erent orientations (data not shown). The plasmid pER341 has been used to construct hybrid vectors for S. thermophilus [8]. pER341 di¡ers from pND103 in size and in orientation of the rep and hsp genes. 3.3. Segregational and structural stability in E. coli To transform pND913, pND914 and pND915 into E. coli, competent cells of E. coli JM109 were prepared as the host strain. Selective plates LB+Carb were used. All of the three plasmids were transformed at similar frequencies (Table 1). Segregational stability was examined after approximately 30 generations without selective pressure. No more than 5% of plasmid loss was observed for shuttle vectors pND913, pND914 and pND915 over the 30 generations. As a result, all the constructs were shown to be reasonably stable in E. coli JM109. Structural stability of pND913, pND914 and pND915 harbored in JM109 was also studied. Transformants were grown in LB broth in the presence of Amp pressure for 30 generations, transformants (¢ve colonies of each strain) were puri¢ed and checked for plasmid pro¢le and con-
Table 1 Transformation frequencies of pND913, pND914 and pND915 Plasmid
pND913 pND914 pND915 pGKV210
Transformants (per Wg31 of DNA) Into E. coli JM109
Into S. thermophilus ST3-1
7U104 5U104 6U104 6U104
8U103 3U103 2U103 7U103
¢rmed by restriction analysis. It was observed that the structures of the three shuttle vectors were stably maintained for 30 generations. No obvious alteration in the size of any of the plasmids was observed. 3.4. Transformation and stability in S. thermophilus pND913 was extracted from LM0230 and used to transform S. thermophilus ST3-1. The plasmid content of ¢ve transformants was checked and the presence of pND913 con¢rmed. To ensure that the transformants were ST3-1 and not contaminants, pulsed ¢eld gel electrophoresis was conducted on one transformant. The transformant and ST3-1 gave exactly the same pattern when digested with SmaI (data not shown). Transformation experiments aimed at introducing pND914, pND915, pGKV210 into ST3-1 were also successful (Table 1). Stability, both segregational and structural, of the pND103-derived plasmids (pND913/914/915) and pGKV210 in the ST3-1 background was evaluated to determine how the stability of pND103-based vectors compares to the well characterised pGKV210, and to determine the most stable variant of pND103 that would be used in future heterologous gene technology experiments. No plasmid rearrangement was observed with pND913, pND914 and pND915 and all plasmids had similar stability in ST3-1 to that observed in E. coli. pND913 may be more suitable for cloning work because of its smaller size.
References [1] Anderson, D.G. and McKay, L.L. (1983) Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46, 549^552. [2] Efstathiou, J.D. and McKay, L.L. (1977) Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis. J. Bacteriol. 130, 257^265. [3] Janzen, T., Kleinschmidt, J., Heve, H. and Geis, A. (1992) Sequencing and characterization of pST1, a cryptic plasmid from Streptococcus thermophilus. FEMS Microbiol. Lett. 95, 175^180. [4] Liu, C.Q., Khunajakr, N., Lian, G.C., Deng, Y.M., Charoenchai, P. and Dunn, N.W. (1997) Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis. Plasmid 38, 79^90. [5] Mercenier, A. (1990) Molecular genetics of Streptococcus thermophilus. FEMS Microbiol. Rev. 87, 61^77. [6] Powell, I.B., Achen, M.G., Hillier, A.J. and Davidson, B.E. (1988) A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Appl. Environ. Microbiol. 54, 655^660.
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P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47 [7] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [8] Somkuti, G.A., Solaiman, D.K.Y. and Steinberg, D.H. (1998) Structural and functional properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus. Plasmid 40, 61^72. [9] Somkuti, G.A. and Steinberg, D.H. (1999) Promoter activity of the pER341-borne STPhsp in heterologous gene expression in Escherichia coli and Streptococcus thermophilus. FEMS Microbiol. Lett. 95, 175^ 180.
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[10] Terzaghi, B.E. and Sandine, W.E. (1975) Improved medium for lactic streptococci and their bacteriophages. Appl. Environ. Microbiol. 29, 807^813. [11] van der Vossen, J.M., Kok, B.M.J. and Venema, G. (1985) Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis. Appl. Environ. Microbiol. 50, 540^542. [12] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119.
FEMSLE 10673 31-10-02
www.fems-microbiology.org
Cloning vectors for Streptococcus thermophilus derived from a native plasmid Ping Su
a;b;
, Karen Jury a , Gwen E. Allison a , Wing Yee Wong b , Woojin S. Kim b , Chun-Qiang Liu b , Tony Vancov b , Noel W. Dunn b b
Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia a DSM Food Specialties, P.O. Box 83, Moorebank, NSW 2170, Australia
Received 27 May 2002 ; received in revised form 2 August 2002; accepted 2 September 2002 First published online 21 September 2002
Abstract A 3.5-kb native plasmid (pND103) was identified in Streptococcus thermophilus ST2-1. Preliminary sequence analysis indicated that pND103 belongs to group I S. thermophilus plasmids. A region of approximately 2 kb appears to contain three components: a plus origin of replication (ori) typical of plasmids that replicate via rolling circle replication; a gene encoding a replication protein (rep); and a gene encoding a small heat shock protein (hsp). pND103 was then used to construct S. thermophilus/Escherichia coli hybrid cloning vectors by ligating different portions of pND103 to an origin-probe vector (pND330) composed of pUC19 and a Gram-positive erythromycin resistance gene. The shuttle vectors (pND913, pND914 and pND915) were successfully introduced back into plasmid-free S. thermophilus ST3-1 as well as to Lactococcus lactis LM0230 and E. coli JM109. Segregational and structural stability study indicated that these vectors can be maintained in these hosts. The results indicated that pND913, pND914 and pND915 are potential shuttle cloning vectors for S. thermophilus. ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptococcus thermophilus ; Cloning vector; Plasmid ; pND913
1. Introduction Streptococcus thermophilus, an important microorganism in dairy food fermentation, is widely used as a starter culture for the production of yoghurt and certain cheese varieties. This industrially important species is a member of a diverse group of facultative anaerobes commonly referred to as lactic acid bacteria (LAB), which are used in various combinations to yield speci¢c fermented foods. The application of gene technology to S. thermophilus would be greatly facilitated by the development of additional e¡ective cloning vectors. Hybrid vectors with shuttle capacity incorporating the replication function of native S. thermophilus plasmids were reported by Somkuti et al. [9]. Very few plasmids have been found in S. thermophilus
(reviewed in [5]). Plasmids that have been reported range in size from 2 to 25.5 kb and, only in very few cases, multiple plasmids have been found to coexist in one strain [5]. Based on DNA homology, Janzen et al. [3] proposed ¢ve di¡erent groups of plasmids in S. thermophilus : group I consists of small plasmids, ranging in size from 2.1 to 3.5 kb; group II, 4.2 kb ; group III, 5.2 kb; group IV, 6.8 kb; and group V, 7.4 kb. In this paper we report the genetic organization of a native plasmid, pND103, from S. thermophilus ST2-1 and its use to construct hybrid cloning vectors for S. thermophilus.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions
* Corresponding author. Fax: +61 (2) 93136700. E-mail address : [email protected] (P. Su).
Strains used include: ST2-1, a wild-type strain of S. thermophilus; ST3-1, a plasmid-free strain of S. thermophilus; LM0230, a plasmid-free derivative of Lactococcus lactis C2 [2] and Escherichia coli JM109 [12]. Plasmids
0378-1097 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 9 7 0 - 9
FEMSLE 10673 31-10-02
44
P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
used were: pGEM-7Zf(+), an E. coli vector encoding ampicillin resistance (Promega, Madison, WI, USA) ; pND330, an origin-probe vector composed of pUC19 and a Gram-positive erythromycin resistance gene [4]; and pGKV210, an E. coli/LAB shuttle vector [11]. The identi¢cation of pND103 and construction of pND913, pND914 and pND915 are described in the text. L. lactis strains were incubated statically at 30‡C in M17 medium [10] supplemented with 0.5% (w/v) glucose (M17G). S. thermophilus strains were propagated at 37‡C anaerobically in M17 medium supplemented with 0.5% (w/v) lactose (M17L). E. coli cultures were grown at 37‡C with shaking in Luria broth (LB) [7]. When appropriate, antibiotics were added as follows : for E. coli, 100 Wg ml31 ampicillin (Amp) or 100 Wg ml31 carbenicillin (Carb), for L. lactis, 5 Wg ml31 erythromycin (Em), and for S. thermophilus, 5 Wg ml31 Em.
2.3. Nucleotide sequencing and analysis Both DNA strands were sequenced using an Applied Biosystems (Foster City, CA, USA) 377 DNA sequencer, following the manufacturer’s protocol. Sequencing of DNA from pND103 was initiated using two primers designed according to the sequence of plasmid pGEM7Zf(+). Based on the sequences obtained, 20-mer oligonucleotide primers were then synthesized and used to ‘walk’ along the DNA template. Recording and analysis of the nucleotide sequence were carried out using the AutoAssembler1 DNA sequence assembly software (Applied Biosystems) and the ANGIS Software System operated by the Australian Genomic Information Center, University of Sydney. Amino acid sequences were compared with all the sequences held in the database by the ‘BLASTP’ program. Nucleotide sequence was searched for 310 and 335 sequences using the ‘Findpatterns’ program.
2.2. DNA manipulation and cloning techniques Lactococcal plasmid DNA was isolated by the method of Anderson and McKay [1]. Plasmids from E. coli were isolated by the alkaline lysis method [7]. Restriction digestion and molecular cloning were as described by Sambrook et al. [7]. Restriction endonucleases and T4 DNA ligase were purchased from Boehringer-Mannheim (Germany) and used as recommended by the manufacturer. Recovery of DNA fragments from agarose gels was performed using a QIAEX gel extraction kit (Qiagen, Chatsworth, CA, USA). L. lactis was transformed by electroporation as described by Powell et al. [6]. S. thermophilus was transformed by electroporation [8] and for E. coli, the CaCl2 transformation method was used [7].
3. Results and discussion 3.1. Characterization of a native plasmid from S. thermophilus ST2-1 A series of industrial strains was screened for plasmids. The strain S. thermophilus ST2-1 was found to harbor only one small plasmid (3.5 kb), and this plasmid was designated pND103. Restriction analysis indicated that pND103 carries a unique EcoRV site. This was used to clone pND103 into the SmaI site of the E. coli sequencing vector pGEM-7Zf(+). Both orientation insertions were obtained and the resulting plasmids were named pND846
Fig. 1. A: Gene organization of the native plasmid pND103 from S. thermophilus ST2-1. The restriction sites were con¢rmed by sequence analysis. The 335 and 310 regions, the ribosome binding site and start codon of the rep gene are underlined. A size reference is shown on the top. B: The nucleotide sequence shown is that of the putative promoter region for the rep gene.
FEMSLE 10673 31-10-02
P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
and pND847. Initially pND846 was used as the sequencing template but this proved to be unstable. pND847 with pND103 cloned in the reverse orientation was stable. Sequence analysis of the open reading frames (ORFs) suggested that instability of pND846 may have been due to the pGEM vector’s LacZ promoter reading into pND103’s open reading frames. Preliminary sequence analysis revealed four potential ORFs in pND103 (Fig. 1) and indicated that pND103 belongs to group I of the S. thermophilus plasmids of which sequence information is available for pST1 (2.8 kb) and pER341 (2.8 kb) [3]. A region (ORF1, ORF2, ORF3 and the ori) of approximately 2 kb is present in all four plasmids and is highly homologous. ORF3 encodes a replication protein (rep) and ORF1 encodes a small heat shock protein (hsp). The presence of a heat
45
shock protein could be important in aiding S. thermophilus survival at elevated growth temperatures although the hsp gene in some cases is not necessary for replication in S. thermophilus [5]. Somkuti et al. have recently investigated the e¡ects of heat shock on the expression of the plasmid-encoded hsp gene. They found that the hsp was heat stress-regulated but did not show a positive impact to cell survival [8]. The degree of sequence identity in the region containing ori, rep, and hsp is approximately 90% among the four plasmids. The ori region and the promoter of rep are also very similar between pND103 and pST1. As for the pST1 plasmid, the ribosome binding site (RBS) is an unusual 50 bases in front of the start codon for the rep gene (Fig. 1). An explanation for this could be the presence of a hairpin structure in between the RBS and the start codon thus reducing the e¡ective distance. ORF2
Fig. 2. Cloning strategy and restriction maps of plasmids pND330, pND847 and pND913. pND913 contains the ori region from the native plasmid pND103.
FEMSLE 10673 31-10-02
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P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47
shows no signi¢cant homology to any sequence at the amino acid level. ORF4 has a potential RBS but shows no signi¢cant homology to any known sequences at the amino acid level. Two very long stretches of ‘AT’-rich regions between ORF3 and ORF4 are present. 3.2. Construction of cloning vectors based on pND103 Plasmid pND103 was developed into E. coli/S. thermophilus shuttle vectors by ligating di¡erent portions of pND103 to pND330. Sequence analysis indicated that the ori region lies on the longer (2.1-kb) PvuII^EcoRV fragment (Fig. 1). To clone the ori region only pND847 (Fig. 2) was double-digested with PvuII and BamHI [BamHI is in the multicloning site, originally from pGEM7Zf(+)], then ligated to pND330 digested with SmaI and BamHI. The ligation mixture was precipitated with ethanol and electroporated into LM0230. Transformants were obtained on the M17G+0.5 M sucrose+Em selection plates. One of the clones was puri¢ed and the plasmid harbored in the strain designated pND913. The plasmid pro¢le of pND913 was checked with restriction enzyme analysis and it was con¢rmed that the ori region from pND103 had been cloned into pND330. The successful direct selection of the S. thermophilus ori region in Lactococcus indicates that the S. thermophilus origin of replication is functional in a lactococcal background. Using a similar procedure as above the whole pND103 was also cloned into pND330. pND847 was cut with XbaI and BamHI, then ligated into pND330 cut with the same two enzymes. This gave plasmid pND914. pND915 was generated in the same way but using BamHI and KpnI. Plasmid extraction and restriction analysis con¢rmed that pND914 and pND915 contain the complete pND103 in di¡erent orientations (data not shown). The plasmid pER341 has been used to construct hybrid vectors for S. thermophilus [8]. pER341 di¡ers from pND103 in size and in orientation of the rep and hsp genes. 3.3. Segregational and structural stability in E. coli To transform pND913, pND914 and pND915 into E. coli, competent cells of E. coli JM109 were prepared as the host strain. Selective plates LB+Carb were used. All of the three plasmids were transformed at similar frequencies (Table 1). Segregational stability was examined after approximately 30 generations without selective pressure. No more than 5% of plasmid loss was observed for shuttle vectors pND913, pND914 and pND915 over the 30 generations. As a result, all the constructs were shown to be reasonably stable in E. coli JM109. Structural stability of pND913, pND914 and pND915 harbored in JM109 was also studied. Transformants were grown in LB broth in the presence of Amp pressure for 30 generations, transformants (¢ve colonies of each strain) were puri¢ed and checked for plasmid pro¢le and con-
Table 1 Transformation frequencies of pND913, pND914 and pND915 Plasmid
pND913 pND914 pND915 pGKV210
Transformants (per Wg31 of DNA) Into E. coli JM109
Into S. thermophilus ST3-1
7U104 5U104 6U104 6U104
8U103 3U103 2U103 7U103
¢rmed by restriction analysis. It was observed that the structures of the three shuttle vectors were stably maintained for 30 generations. No obvious alteration in the size of any of the plasmids was observed. 3.4. Transformation and stability in S. thermophilus pND913 was extracted from LM0230 and used to transform S. thermophilus ST3-1. The plasmid content of ¢ve transformants was checked and the presence of pND913 con¢rmed. To ensure that the transformants were ST3-1 and not contaminants, pulsed ¢eld gel electrophoresis was conducted on one transformant. The transformant and ST3-1 gave exactly the same pattern when digested with SmaI (data not shown). Transformation experiments aimed at introducing pND914, pND915, pGKV210 into ST3-1 were also successful (Table 1). Stability, both segregational and structural, of the pND103-derived plasmids (pND913/914/915) and pGKV210 in the ST3-1 background was evaluated to determine how the stability of pND103-based vectors compares to the well characterised pGKV210, and to determine the most stable variant of pND103 that would be used in future heterologous gene technology experiments. No plasmid rearrangement was observed with pND913, pND914 and pND915 and all plasmids had similar stability in ST3-1 to that observed in E. coli. pND913 may be more suitable for cloning work because of its smaller size.
References [1] Anderson, D.G. and McKay, L.L. (1983) Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46, 549^552. [2] Efstathiou, J.D. and McKay, L.L. (1977) Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis. J. Bacteriol. 130, 257^265. [3] Janzen, T., Kleinschmidt, J., Heve, H. and Geis, A. (1992) Sequencing and characterization of pST1, a cryptic plasmid from Streptococcus thermophilus. FEMS Microbiol. Lett. 95, 175^180. [4] Liu, C.Q., Khunajakr, N., Lian, G.C., Deng, Y.M., Charoenchai, P. and Dunn, N.W. (1997) Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis. Plasmid 38, 79^90. [5] Mercenier, A. (1990) Molecular genetics of Streptococcus thermophilus. FEMS Microbiol. Rev. 87, 61^77. [6] Powell, I.B., Achen, M.G., Hillier, A.J. and Davidson, B.E. (1988) A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Appl. Environ. Microbiol. 54, 655^660.
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P. Su et al. / FEMS Microbiology Letters 216 (2002) 43^47 [7] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [8] Somkuti, G.A., Solaiman, D.K.Y. and Steinberg, D.H. (1998) Structural and functional properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus. Plasmid 40, 61^72. [9] Somkuti, G.A. and Steinberg, D.H. (1999) Promoter activity of the pER341-borne STPhsp in heterologous gene expression in Escherichia coli and Streptococcus thermophilus. FEMS Microbiol. Lett. 95, 175^ 180.
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[10] Terzaghi, B.E. and Sandine, W.E. (1975) Improved medium for lactic streptococci and their bacteriophages. Appl. Environ. Microbiol. 29, 807^813. [11] van der Vossen, J.M., Kok, B.M.J. and Venema, G. (1985) Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis. Appl. Environ. Microbiol. 50, 540^542. [12] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119.
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