Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

Gene 399 (2007) 72 – 80 www.elsevier.com/locate/gene

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector Azusa Kuroki a,b , Naoto Ohtani a , Kenji Tsuge a , Masaru Tomita a,c , Mitsuhiro Itaya a,c,⁎ a

b

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan c Department of Environmental Information, Keio University, Fujisawa, Kanawaga, Japan Received 5 March 2007; received in revised form 25 April 2007; accepted 26 April 2007 Available online 6 May 2007 Received by R. Britton

Abstract The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA sizerelated inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges. © 2007 Elsevier B.V. All rights reserved. Keywords: BGM vector; Conjugational plasmid; Genetic transformation; Homologous recombination; Giant DNA cloning; pLS20

1. Introduction

Abbreviations: BGM vector, Bacillus subtilis genome vector; GpBR, BGM vector containing whole sequences of pBR322; BReT, Bacillus recombinational transfer method; CReT, conjugational plasmid-mediated DNA retrieval and transfer method; Tet, tetracycline; Ap, ampicillin; BS, blasticidin S; Cm, chloramphenicol; Em, erythromycin; Nm, neomycin; Spec, spectinomycin; cat, chloramphenicol resistance gene; spc, spectinomycin resistance gene; tet, tetracycline resistance gene; erm, erythromycin resistance gene; bsr, blasticidin S resistance gene; recA, gene encoding multifunctional protein involved in homologous recombination and DNA repair; leuB, gene encoding 3-isopropylmalate dehydrogenase; yjcI, gene encoding similar to cystathionine gammasynthase; proB, gene encoding glutamate 5-kinase; ∷, novel junction (fusion or insertion); CHEF, contour-clamped homogeneous electric field; kb, kilobase. ⁎ Corresponding author. Institute for Advanced Biosciences, Keio University, Nipponkoku, Tsuruoka, Yamagata 997-0017, Japan. Tel.: +81 235 29 0526; fax: +81 235 29 0529. E-mail address: [email protected] (M. Itaya). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.04.030

DNA cloning is the primary method used to understand genes and their functions. The most widely-used cloning system is based on the Escherichia coli host; it is versatile and covers a wide range of DNA fragment sizes (Sambrook et al., 1989). Plasmids and hosts able to clone large (N 100-kb) DNA fragments have been used in various genome analyses. They are the bacterial artificial chromosome (BAC)- (Shizuya et al., 1992) and the P1-derived artificial chromosome (PAC) system (Ioannou et al., 1994) propagated in E. coli for large DNA cloning, the transformation-associated recombination (TAR) system (Kouprina and Larionov, 2006) developed in yeast, and the Bacillus subtilis 168 genome-based Bacillus GenoMe (BGM) vector cloning system we established (Itaya, 1995). In the BGM cloning system, the B. subtilis genome is the cloning vehicle. Steps to integrate the target segment directly

A. Kuroki et al. / Gene 399 (2007) 72–80

into the B. subtilis genome take advantage of its inherent characteristic of competence development (Spizizen, 1958; Dubnau, 1999) and induced homologous recombination networks (Kidane and Graumann, 2005). We demonstrated the effectiveness of the BGM cloning system by cloning the entire (3500 kb) genome of Synechocystis PCC6803 (Itaya et al., 2005). The structural and genetic stability of the cloned DNA segments in the BGM vector (Kaneko et al., 2003; Itaya et al., 2005) may be attributable to single-copy replication as part of the B. subtilis genome. The cloned DNA in the BGM vector must be retrieved before delivery to a new host of interest and this additional step is different from other large DNA cloning systems in E. coli or yeast in which initial cloning results in the generation of plasmids. Therefore, we developed the Bacillus recombinational transfer (BReT) method; it is based on transformation-mediated gap repair (Tsuge and Itaya, 2001) to retrieve DNA cloned in BGM and yields plasmids. We have applied the BReT protocol (Fig. 1) to various DNA in the BGM vector (Tsuge and Itaya, 2001; Kaneko et al., 2003; Tomita et al., 2004a,b; Yonemura et al., 2007). It was found that BReT cannot be applied to strains deficient in competency development and genetic recombination, because it absolutely depends on the transformation nature of B. subtilis as used in BGM cloning. Moreover, in recombination-proficient cells, cloned fragments transferred in plasmids by BReT are subject to structural and genetic instability, presumably due to a possible recombination between the plasmid and the genome or

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between the plasmids. This requires provisional plasmid maintenance in the original BGM strain proficient for genetic recombination and delivery of the plasmid to recombinationdeficient strains of B. subtilis, i.e., a recA mutant. This process makes it necessary to isolate the BReT plasmid, however, problems arise in the preparation of intact DNA, especially if the DNA size is large (Kaneko et al., 2005). We used interspecies plasmid transfer by conjugation to exploit an alternative method to deliver giant DNA to a different B. subtilis strain that does not require plasmid isolation. pLS20, a plasmid from a B. subtilis (natto) strain first reported by Tanaka and Koshikawa (1977), was subsequently shown capable of effective conjugal transfer to B. subtilis (Koehler and Thorne, 1987). We recently reported the effective delivery of the 65-kb pLS20cat that carries a chloramphenicol-resistant gene (cat) at the unique SalI site of pLS20, to B. subtilis strains, even to a recA mutant (Itaya et al., 2006). Here we report the effective genetic transfer of DNA from the genome to a plasmid as an alternative to the BReT method, followed by conjugational transfer-mediated delivery to another host, including a recA mutant. Hereafter, this protocol is called conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector; plasmids suitable for CReT are called pLSGETS (Fig. 1). We demonstrate that CReT made it possible to retrieve various cloned Synechocystis genome segments in BGM and that the efficiency of conjugational transfer of the resultant plasmid was not affected by DNA size increases.

Fig. 1. Retrieval of DNA segments in GpBR by the CReT method. The genome and plasmids are drawn separately in all cells. The pBR322 sequence common in GpBR and the pLSGETS plasmid are indicated by arrows (2.1 kb: tet-half) and arrowheads (2.3 kb: amp-half). DNA cloned in GpBR is indicated by a grey bold line in cells (ii)–(ix) and the marker gene in cloned DNA is identified by solid diamonds. The retrieval process of CReT exploited in this study is shown in cells from (i) to (iv). (i) The donor of plasmid pLSGEST101spc or pLSGETS103tet. (ii) Transcipients from the BGM clone. (iii) Homologous pairing, indicated by × between pBR sequences. (iv) Transfer of the genomic segment to the plasmid. (iii) and (iv) occur at a certain frequency during the growth of (ii). The cell (v) can be selected by the increased copy number of marker genes in the plasmid derived from the DNA segment in GpBR. BReT absolutely requires a competent proficient host and (vi) requires linearized DNA in vitro and uptake. (vii) Homologous recombination, indicated by × between pBR sequences. (viii) Filling-in step to yield a circular replicable plasmid. Cells (ix) with a filled-in plasmid can be selected with a plasmid-associated marker (not indicated here).

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2. Materials and methods 2.1. Bacterial strains and culture media The E. coli strain DH5α (F− Φ80dlacZΔM15 Δ(lacZYA-argF) − + U169 deoR recA1 endA1 hsdR17(rK , mK ) phoA supE44 λ− thi-1 gyrA96 relA1) was routinely used as the host for molecular cloning. The B. subtilis strains employed in this study are listed in Table 1. Luria–Bertani broth (LB) was used to grow E. coli and B. subtilis at 37 °C. To obtain solid media, agar was added (1.5% w/v) to LB for E. coli or antibiotic medium 3 (Difco, Sparks, MD) for B. subtilis. Tetracycline (Tet, 10 μg/ml) and ampicillin (Ap, 50 μg/ml) were added for DH5α selection. Blasticidin S (BS, 250 μg/ml), chloramphenicol (Cm, 5 μg/ml), erythromycin (Em, 5 μg/ml), neomycin (Nm, 3 μg/ml), spectinomycin (Spec, 50 μg/ml), and Tet (10 μg/ml) were added for the selection of B. subtilis strains carrying the corresponding antibiotic resistance gene at a single copy in the genome. Strains with multiple antibiotic resistance genes were tested by the replica plating method. 2.2. DNA manipulation Plasmids were prepared by the alkali-SDS method (Birnboim and Doly, 1979). Large-scale plasmid preparation was by the alkali-SDS method, followed by ultracentrifugation in a CsClEthidium Bromide gradient (Sambrook et al., 1989). Chromo-

somal DNA for B. subtilis transformation was prepared by a liquid isolation method (Saito and Miura, 1963). Type II restriction enzymes and DNA ligation kit were purchased from Toyobo (Tokyo, Japan) and Takara (Shiga, Japan) respectively. Primers were synthesized by Nihon Gene Research Laboratories Inc. (Miyagi, Japan). The preparation and transformation of competent E. coli and B. subtilis cells were as previously described (Anagnostopoulos and Spizizen, 1961; Mandel and Higa, 1970). 2.3. Preparation of intact B. subtilis chromosomal DNA and Southern hybridization Intact unsheared chromosomal DNA for contour-clamped homogeneous electric field (CHEF) gel electrophoresis was prepared in agarose gel plugs as described elsewhere (Itaya and Tanaka, 1991). Agarose gel (1.0% w/v) in TBE solution (50 mM Tris–Borate, pH 8.0, 1.0 mM EDTA) used in CHEF electrophoresis was run as described in the legend to Fig. 5. Agarose gel (1.0% w/v) in TAE solution (50 mM Tris–Acetate, pH 8.0, 1.0 mM EDTA) was used for conventional gel electrophoresis at room temperature. After electrophoresis, DNA bands were stained for 15 min with Ethidium Bromide solution and detected under UV light. For Southern hybridization (Southern, 1975) we used a nonradioactive labelling nucleotide, digoxigenin-11-dUTP. Probes were prepared with the PCR DIG probe synthesis kit (Roche, Mannheim, Germany). The specific primers were CAT-ORF-F2

Table 1 B. subtilis strains used in this study Strains

Genotypes

168trpC2 (= 1A1) RM125 BEST2137

trpC2

BEST4119 BEST6225 BEST7003 BEST7016 BEST7017 BEST7018 BEST7019 BEST8630 BEST40401 BEST21274 BEST21278 BEST21290 BEST21292 BEST21299 BEST21300 BEST21317 d BEST21305 BEST21320 BEST21322 BEST21335 BEST21309 a b c d

leu, arg, hsdRM trpC2 leuB::pBRTc proB::pBRBS yjcI::pBREm leuB::neo trpC2 yjcI::cat b leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo proB::pBRTc leu, arg, hsdRM proB::pBR(25.7 kb: spc) leu, arg, hsdRM proB::pBR(39.7 kb: spc) leu, arg, hsdRM proB::pBR(54.2 kb: spc) leu, arg, hsdRM proB::pBR(90.3 kb: spc) trpC2 recA362::tet leu, arg, hsdRM leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo proB::pBRTc leu, arg, hsdRM Δ(yvfC-yveP)::pr-neo proB::pBRTc trpC2 trpC2 trpC2 yjcI::cat leu, arg, hsdRM proB::pBR(25.7 kb: spc) leu, arg, hsdRM proB::pBR(39.7 kb: spc) leu, arg, hsdRM proB::pBR(54.2 kb: spc) leu, arg, hsdRM proB::pBR(90.3 kb: spc) trpC2 leuB::pBRTc proB::pBRBS yjcI::pBREm leuB::neo

Plasmid

pLS20cat pLS20cat pLSGETS101spc pLSGETS101spc pLSGETS103tet pLSGETS101spc pLSGETS103tet pLSGETS101spc pLSGETS2001 pLSGETS2002 pLSGETS2003 pLSGETS2004 pLSGETS101spc

Antibiotic markers

References and genetic crosses a

TetR, EmR, BSR, NmR

Itaya and Tanaka (1997b) Itaya (1993)

CmR NmR TetR, NmR SpecR, NmR SpecR, NmR SpecR, NmR SpecR, NmR TetR CmR NmR, CmR NmS, SpecR, CmR TetR, NmS, SpecR, CmR c hyper TetR, NmR, SpecS, CmR SpecR, CmR TetR, CmR, SpecR, CmR, EmS, c hyper SpecR, TetS, CmR c hyper SpecR, TetS, CmR c hyper SpecR, TetS, CmR c hyper SpecR, TetS, CmR TetR, EmR, BSR, NmR, SpecR, CmR

Itaya and Tanaka (1990) Itaya et al. (2003) Itaya et al. (2005) Itaya et al. (2003) Itaya et al. (2003) Itaya et al. (2003) Itaya et al. (2003) Itaya et al. (2003) Itaya et al. (2003) [TF] BEST6225 × BEST40401 [TF] pBEAZ211 × BEST21274 [CT] pLSGETS101spc× BEST7003 [CReT] BEST21290 [TF] pLSGETS101spc × 1A1 [TF] pLSGETS103tet × 1A1 [CT] BEST21299 × BEST4119 [CReT] BEST21300 × BEST7016 [CReT] BEST21300 × BEST7017 [CReT] BEST21300 × BEST7018 [CReT] BEST21300 × BEST7019 [CT] BEST21299 × BEST2137

[TF], transformation; [CT], conjugational transfer; [CReT], CReT method. Previously denoted as metD::cat (Itaya and Tanaka, 1990). Three copies of antibiotic resistance gene contained in plasmid in addition to the one in GpBR. Strain that spontaneously lost pGETS112(erm) which is used to label BEST4119 with EmR prior to conjugational transfer with BEST21299.

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(5′-ATGAACTTTAATAAAATTGATTTAG) and CAT-STOP-R (5′-TAAAGCCAGTCATTAGGCC). pHASH102 (Ohashi et al., 2003) was the template DNA for the cat gene. Specific bands were visualized with CDP-star (Sigma-Aldrich, St. Louis, MO, USA) and exposure to X-ray film. 2.4. Conjugational transfer assay The conjugation process involves mixing of the donor and recipient on a solid surface (Koehler and Thorne, 1987) or in liquid culture (Itaya et al., 2006); however, we adopted the liquid culture-mix protocol as the standard assay. Briefly, following the inoculation of stationary culture into LB at 0.5%, the 2-h cultures of both donor (0.15 ml) and recipient cells (0.15 ml) were mixed and incubated for 15 min at 37 °C without shaking. After spreading 0.1 ml of the aliquots onto antibiotic medium 3 plates containing the appropriate antibiotics, the plates were incubated at 37 °C to select transcipients.

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2.6. Construction of pLSGETS plasmids The CReT plasmid pLSGETS101spc, comprised of the B. subtilis conjugational plasmid pLS20 and the pBR322 sequence, was constructed by following 3 steps from (i) to (iii) as illustrated in Fig. 2a: In step (i), the HindIII fragment of pLS20cat carrying the cat gene was cloned into the unique HindIII site of pGETS109 (Tomita et al., 2004b); this yielded the Cm-resistant plasmid pBEAZ203 cloned in RM125. In step (ii), pBEAZ208 carries a Spec resistance gene (spc) between tet-half and amp-half of the pBR322 sequence (see also Fig. 1). It was linearized with PvuII and ligated with the blunt-ended BglII site of pBEAZ203 to produce pBEAZ211. Finally in step (iii), pBEAZ211 was linearized with PmaCI and used to transform BEST21274. As a result of homologous recombination between linear pBEAZ211 and the residential pLS20cat, BEST21278 carrying pLSGETS101spc was selected by Spec and Cm. The ability of conjugational transfer of pLSGETS101spc was measured using our standard assay (Itaya et al., 2006).

2.5. Preparation of concentration-gradient plates 3. Results Antibiotic medium 3 (25 ml) containing antibiotics and agar was poured into a rectangular plastic container (6.5 × 8.5 × 1.0 cm, see Fig. 4) tilted to obtain a slanted surface. After solidification, the container was untilted and 25 ml of antibiotic-free agar medium was poured to fill the container and render a flat surface. The plates were then left at room temperature for at least 15 h prior to use. The concentration gradient on the surface remained effective for a few days.

3.1. Verification of the structure of the CReT plasmid and its ability for conjugational transfer The BGM vector has a pBR322 sequence as a cloning locus; it is called GpBR (genomic pBR) (Itaya et al., 2005; Kaneko et al., 2005). The 2 GpBR halves always remain at both ends of the cloned DNA (see Figs. 1 and 3). Retrieval of the cloned

Fig. 2. pLS20cat derivative and measuring of their copy number. a) Preparation of pLSGETS101spc. For construction details see Section 2.6. The relevant restriction enzyme sites are B: BglII, H: HindIII, S: SalI, and Pm: PmaCI. pBEAZ211 was linearized with PmaCI prior to the transformation of BEST21274. Insertion was via homologous recombination (indicated by ×). BEST21278 was selected by the formation of pLAGETS101spc due to the acquisition of the spc gene (solid diamond) from pBEAZ208. pBEAZ208 derived from pCISP304B which lacks a BamHI site of the Tet resistance gene (tet) of pCISP303B (Itaya et al., 2000), b) Lane 1 includes approximately 10 ng of a HindIII digest of total DNA of BEST21317 prepared in agarose plugs. DNA diluted 1/2 and 1/4 are shown. The labelled cat probe identifies 2 signals. The intensity ratio of pLSGETS101spc (3.421 kb) to the genome (2.577 kb from yjcI∷cat) was approximately 1:3. Intensity comparison between the diluted lanes yielded results consistent with the copy number.

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Fig. 3. pLSGETS plasmids constructed with the CReT method. The bold circle (bottom) represents the B. subtilis 168 genome; oriC, terC, and relevant genetic marker loci are shown. GpBR in Fig. 1 was installed at the proB locus in all BGM clones with BEST names (column at the left of the figure). tet (solid rectangle) or Synechocystis genome DNA segments (grey line; sizes to the left) are transferred to the pLSGETS plasmids shown at the top of the figure. spc is indicated by solid diamonds (Itaya et al., 2003). The SalI sites (represented by S) are consistent with the SalI restriction enzyme analysis presented in Fig. 5.

DNA in GpBR involves homologous recombination between pBR322 sequences of GpBR halves and the pLSGETS plasmid. To this end, as illustrated in Fig. 2a, the conjugational plasmid pLS20cat was converted to pLSGETS101spc containing the counter-pBR322 sequence to carry out the CReT process. The structure of pLSGETS101spc was verified by digestion with various enzymes; the result of SalI digestion is shown in Fig. 5a. We confirmed the ability for conjugational transfer to several strains, including competency-deficient strains (data not shown). Appropriate selection was required to monitor the retrieval of DNA from the genome (single-copy) to pLSGETS (multicopy). Earlier we noticed that the pLS20cat strain exhibited more resistance to Cm than the strain carrying a single cat gene in the genome (unpublished observations). This provided an experimental basis to perform DNA retrieval with our protocol using increased antibiotic concentrations. Multi-copy numbers are vital for effective selection based on differences in the copy number of the antibiotic resistance gene. 3.2. Copy number of pLSGETS Neither Tanaka and Koshikawa (1977) who first isolated and characterized pLS20, nor Koehler and Thorne (1987) reported the number of pLS20 per cell. To make use of copy numberdependent selection, we experimentally determined the precise number of pLSGETS101spc in B. subtilis (Fig. 2b). We estimated the copy number of pLSGETS by comparing the cat gene number in the genome and in plasmids using Southern hybridization,

positing that one copy of genomic cat per cell exists in the stationary stage (Itaya and Tanaka, 1991, 1997a) and every single cell carries the cat gene-containing plasmid (Itaya et al., 2006). A strain containing the cat gene in both the genome and the plasmid, BEST21317, was prepared as described in Table 1. The preparation of DNA from BEST21317 in agarose gel plugs assures the recovery of total cellular DNA irrespective of pLSGETS101spc and the genome. For Southern hybridization we used the cat gene as a probe. The amount of DNA of cat gene was quantitated by measuring luminescence intensity at various amounts of input DNA (Fig. 2b). The average copy number of pLSGETS101spc was found to be 3 per cell. 3.3. Retrieval of the GpBR segment to the pLSGETS plasmid We investigated the ability of CReT plasmid to retrieve the cloned DNA in GpBR. If the parental strain has a marker gene in GpBR, its copy number should be 4 per cell after the DNA fragment is retrieved by the plasmid, i.e., 1 copy in GpBR and 3 copies in the pLSGETS plasmid. Therefore, CReT cells can be selected on account of the increased copy number of marker genes in GpBR. To verify the retrievability of the CReT plasmid, we used tet gene in GpBR. BEST7003 is one of the standard BGM vector strains (Itaya et al., 2003, 2005; Kaneko et al., 2003, 2005) that feature a tet in GpBR. pLSGETS101spc was delivered to BEST7003 by conjugational transfer, yielding BEST21290. We expected the tet to replace the spc of pLSGETS101spc (Fig. 3). Such gene replacement should occur spontaneously during cell

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growth by the mechanism illustrated in Fig. 1. Aliquots (0.1 ml) of BEST21290 cultures grown in LB containing Cm were spread on Tet concentration-gradient plates (Tet: 0–150 μg/ml). As shown in Fig. 4a, after 44-h incubation at 30 °C, colonies were formed in areas where the Tet concentration exceeded 50 μg/ml; in these areas parental BEST7003 failed to give rise to colonies. Of 50 colonies tested, 44 exhibited Spec sensitivity; the 6 Spec-resistant colonies were not further analyzed. Randomly examined 4 Specsensitive clones manifested structurally identical plasmids; hereafter these are called pLSGETS103tet. The structure of pLSGETS103tet was verified by digestion with various restriction enzymes; the result of SalI digestion is shown in Fig. 5a. The pLSGETS103tet copy number, measured by using a similar Southern hybridization method and a tet probe, was estimated as 3 (data not shown). Spec sensitivity was indicative of the absence of pLSGETS101spc in the BEST21292 strain attributable to rapid segregation during selection at high Tet concentrations (50 μg/ml). The conversion frequency from pLSGETS101spc to pLSGETS103tet (approximately 10− 6) was roughly comparable with B. subtilis cellular events brought about by homologous recombination (Toda et al., 1996). pLSGETS103tet exhibited unaltered conjugational transfer efficiency compared with pLSGETS101spc (data not shown). pLSGETS103tet can also be used as a CReT plasmid. 3.4. Transfer of giant DNA in the GpBR locus of BGM clones: the first CReT process We investigated the DNA size transferable to the pLSGETS plasmid using DNA segments originating from the Synechocystis PCC6803 genome integrated into GpBR (Itaya et al., 2003). As the 4 Synechocystis segments, 25.7 kb in BEST7016, 39.7 kb in BEST7017, 54.2 kb in BEST7018, and 90.3 kb in BEST7019, possessed spc in common (Fig. 3); it was used as an effective selection marker for cloning in GpBR (Itaya et al.,

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2003). Upon delivery of pLSGETS103tet to the 4 recipient strains by conjugal transfer, transcipients were selected with normal working concentrations of Tet, the plasmid-associated marker, and by Spec, a common marker for the 4 recipients. Transcipients grown at 37 °C in LB medium in the presence of Cm were spread on Spec concentration-gradient plates (Spec: 0–500 μg/ml). As a result, colonies of all the transcipients were formed in areas where the Spec concentration exceeded even 125 μg/ml, while parental strains failed to give rise to colonies in the areas (a result of BEST7016 is representatively shown in Fig. 4b). We expected the spc to replace the tet of pLSGETS103 (Fig. 3) if the CReT method was successful. Loss of the original tet marker, which was determined by examining Tet resistance in at least 25 colonies of 4 kinds of transcipients derived from BEST7016, -7017, -7018, and -7019, was 52%, 52%, 16%, and 5%, respectively. The retrieval efficiency, which is determined by the loss of original tet marker in transcipients in CReT method, was dramatically reduced as the size of the target Synechocystis DNA became larger. Two randomly selected plasmids from each of the 4 Tet-sensitive transcipients exhibited the identical expected structure and the plasmids were named pLSGETS2001, -2002, -2003, and -2004, respectively. Digestion with various restriction enzymes confirmed their fine structure; SalI data are shown in Fig. 5b. The conjugational transfer of the 4 kinds of plasmid to the B. subtilis recA mutant strain BEST8630 (recA::tet) resulted in a constant number of transcipients selected under standard assay conditions. It should be emphasized that we observed no apparent DNA size-dependent reduction. Highly purified plasmids from the recA transcipients were identical to the originals. As shown in Fig. 5b, there were no structural alterations during the conjugational transfer process regardless of the insert. These recA mutant transcipients proved to be effective donors in the conjugational transfer to other B. subtilis strains regardless of the Synechocystis DNA insert (unpublished

Fig. 4. Selection at increased antibiotic concentrations. The antibiotic concentration increases linearly from left (0 μg/ml) to right (Tet: 150 μg/ml, Spec: 500 μg/ml). Stationary cultures (0.1 ml) of a) BEST21290 and b) BEST21305 were plated and incubated for 44 h at 30 °C. Colonies formed in the high antibiotic concentration range. For details on the preparation of the concentration-gradient plates see Section 2.5.

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Fig. 5. SalI fragments of conjugational plasmids. The pLSGETS structure was analyzed by CHEF. a) pLSGETS101spc and pLSGETS103tet were from BEST21284 and BEST21300, respectively. The running conditions were 3 V cm− 1, 3-s pulse time, and 9-h running time. The SalI fragment numbers and sizes, including the fragments indicated by arrowheads, were consistent with the changes schematically presented in Fig. 4a. b) pLSGETS2001, -2002, -2003, and -2004 in lanes I were isolated from strains corresponding to (v) in Fig. 1. Lanes A include pLSGETS plasmids isolated from recA transcipients. The running conditions were 3 V cm− 1, 6-s pulse time, and 14-h running time. All plasmids were prepared by the ultracentrifuge method. Lambda/HindIII marker fragments were run in lane M; sizes are indicated on the left.

data). Our results facilitate the retrieval of DNA segments from GpBR and their delivery to other BGM strains. The delivery of manipulated DNA to a recA mutant of B. subtilis assures longterm DNA preservation (Kaneko et al., 2005). 3.5. Selection by other antibiotics For more general applications, we also examined the effectiveness of other antibiotic resistance genes in CReT. We used the B. subtilis strain BEST2137 because it possesses 3 GpBRs with different antibiotic resistance gene markers, i.e. tet at leuB::pBRTc, erythromycin (erm) at yjcI::pBREm (formerly metD::pBREm), and blasticidin S (bsr) at proB::pBRBS (Itaya, 1993). pLSGET101spc was delivered to BEST2137 by conjugational transfer from BEST21299. The resultant transcipient BEST21309 was grown up in LB medium containing only Cm to maintain the plasmid. Equal aliquots (0.1 ml) of the stationary culture of BEST21309 were spread on concentrationgradient plates, antibiotic tested, and incubated at 30 °C for 44 h. The selection of Tet-resistant strains, out of leuB::pBRTc, was as effective as for BEST7003 and was used as the internal standard. Surprisingly, selection by Em was poor because BEST2137 exhibited unexpected resistance at all concentrations, up to 400 μg/ ml, used in this study. Because similar Em resistance was observed for other strains (BEST21007) possessing pBREm (data not shown), the inherent significantly high resistance of B. subtilis to Em may hamper selection with Em when the CReT method is used. With respect to BS, parental BEST2137 unexpectedly generated a number of colonies at BS concentrations of up to 1 mg/ml as high as BEST21309 (data not shown). The expected plasmid carrier could be discerned from spontaneous hyper BS-resistant clones by the loss of the original spc marker on replica plates. This low efficiency, 12% (3 out of 25) compared with 88% (44 out of 50) for Tet resistance, led us to cease using Em and BS in the CReT protocol.

4. Discussion The BGM cloning method has been applied for the stable cloning of particularly large DNA exceeding the limits of PCRmediated DNA cloning technology. Efficient, simple protocols are required to permit diversification of applications to the postcloning process in the BGM vector system. As our BReT method is totally dependent on the genetic competence inherently developed by B. subtilis, the application of BReT to strains with reduced competence was not successful and hampered the retrieval process (unpublished observations). Moreover, in the B. subtilis recA mutant in which most genetic recombination activity is lost, structural alterations are expected to be minimal, as is the case in the widely accepted E. coli recA cloning host (Chen et al., 2005). Based on findings in our earlier study on the shuttling of DNA by conjugational transfer to the recA mutant from another strain and vice versa, we explored a novel CReT method. At first glance, the pLS20-based CReT plasmids pLSGETS101spc and pLSGETS103tet, which are as large as 70 kb, seem too large to elaborate by the standards of current molecular cloning technology. While the pBR322 sequence on pLSGETS101spc can be expected to play a similar role upon application of the BReT method, it was not suitable for use with the BReT protocol because the efficiency of competence-mediated transformation was low (unpublished observation). This may be attributable to the large size, approximately 72.7 kb of pLSGETS101spc compared to the practically used BReT plasmids pGETS103 (7.2 kb, (Tsuge and Itaya, 2001)), pGETS109 (12.5 kb, (Tomita et al., 2004b)), and pGETS113 (12.0 kb, (Tomita et al., 2004b)). On the other hand, conjugation-mediated transformation proved to be practical and convenient for the retrieval of recombinant DNA out of the BGM vector. The DNA cloned in the GpBR retrieval process occurs spontaneously during normal incubation. An appropriate selection marker gene was required for

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the monitoring of the retrieval of DNA from the genome to the CReT plasmid. In this study we investigated several antibiotic resistance genes as potential selection markers and verified that tet and spc were effective while erm and bsr were not. To render the CReT method more powerful, other useful selection marker genes must be identified. The technical procedures involved in the CReT protocol are as follows: (i) transfer pLSGETS to the BGM clones by conjugation and select the transcipients by dual markers — donor and recipient, (ii) prepare stationary cultures of the transcipients in LB containing Cm and spread the cultures on gradient plates for antibiotics selection, and (iii) select colonies in the high concentration area of the gradient plate and screen for the loss of the original pLSGETS marker, (iv) transfer retrieved plasmids from transcipients to other strains by conjugation. Given the successful DNA shuttling among B. subtilis hosts including recA mutants and competency-deficient strains, several relevant advantages should be addressed. (a) The conjugational transfer process is technically simple. Only 15 min are required for the mixing of separately prepared donor and recipient cultures (Itaya et al., 2006). (b) Transcipients are selected on plates under appropriate selection conditions, most likely a combination of antibiotics. (c) The conjugational transfer efficiency for pLS20, estimated as 4 × 10− 4/donor/15 min at 37 °C (Itaya et al., 2006) was high enough to yield the required transcipients. (d) There was no obvious decrease in the efficiency of shuttling between B. subtilis strains despite large size differences; the smallest plasmid was 72.7 kb (pLSGETS101spc) and the largest was 160.5 kb (pLSGETS2004). This feature may allow the delivery of far larger DNA segments compared to natural competence-mediated transformation where efficiency decreased as the DNA size increased (Tsuge and Itaya, 2001; Itaya et al., 2003; Kaneko et al., 2005). (e) As CReT does not require the DNA isolation that is an absolute requirement for BReT, possible DNA damage due to physical shearing during normal in vitro isolation after retrieval can be avoided, particularly when the DNA size exceeds 100 kb (Kaneko et al., 2005). Although retrieval proceeds spontaneously during cell growth in widely-used media such as LB, there was an obvious size-dependent reduction in efficiency. This is consistent with the observation that the integration efficiency in GpBR is dramatically reduced and exhibits an inverse correlation with the DNA size to be integrated (Itaya et al., 2003). The reduction may account for the less probability of double homologous recombination due to the increase of physical distance in the genome. Hence, the 4.2-kb pBR322 sequence, i.e., a flanking sequence, plays a key role in the step of homologous recombination (Tsuge and Itaya, 2001; Kaneko et al., 2003; Tomita et al., 2004b,a; Kaneko et al., 2005). As its length may represent a bottleneck in the application to far larger DNA such as the entire Synechocystis genome cloned also in GpBR (Itaya et al., 2005), more suitable large sequences must be exploited. While pLS20 exhibited high genetic stability (Sakaya et al., 2006), there was strong incompatibility as demonstrated by an experiment in which 2 pLS20 derivatives with different antibiotic markers did not coexist without selection pressure (unpublished observations). Only Cm was added to LB medium

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for the maintenance of pLSGETS before exposure to increased antibiotic concentrations. As we did not examine the kinetics underlying the replacement of 2 original copies by 1 emerged plasmid molecule, we may have underestimated the frequency of the appearance of the correct plasmid carrier. Most DNA cloned in GpBR to date carries cat or erm as a selection marker for cloning for historical rather than technical reasons (Itaya, 1995; Itaya et al., 2000; Itaya et al., 2003; Itaya et al., 2005). In our study, selection by Em was ineffective. The cat gene, which originated from pLS20cat, persisted in all pLSGETS plasmids. To yield a wider range of applications of the CReT method, efforts are underway in our laboratory to replace the cat gene of pLSGETS. Acknowledgements We thank to Dr. S. Kaneko for useful discussion. This study was partially supported by research grant (No. 16380066) from the Ministry of Education, Culture, Sports, Science and Technology. References Anagnostopoulos, C., Spizizen, J., 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol 81, 741–746. Birnboim, H.C., Doly, J., 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523. Chen, I., Christie, P.J., Dubnau, D., 2005. The ins and outs of DNA transfer in bacteria. Science 310, 1456–1460. Dubnau, D., 1999. DNA uptake in bacteria. Annu. Rev. Microbiol. 53, 217–244. Ioannou, P.A., et al., 1994. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6, 84–89. Itaya, M., 1993. Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure. Mol. Gen. Genet. 241, 287–297. Itaya, M., 1995. Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome. Mol. Gen. Genet. 248, 9–16. Itaya, M., Tanaka, T., 1990. Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168. Mol. Gen. Genet. 223, 268–272. Itaya, M., Tanaka, T., 1991. Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method. J. Mol. Biol. 220, 631–648. Itaya, M., Tanaka, T., 1997a. Experimental surgery to create subgenomes of Bacillus subtilis 168. Proc. Natl. Acad. Sci. USA 94, 5378–5382. Itaya, M., Tanaka, T., 1997b. Predicted and unsuspected alterations of the genome structure of genetically defined Bacillus subtilis 168 strains. Biosci. Biotechnol. Biochem. 61, 56–64. Itaya, M., Nagata, T., Shiroishi, T., Fujita, K., Tsuge, K., 2000. Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector. J Biochem (Tokyo) 128, 869–875. Itaya, M., Fujita, K., Ikeuchi, M., Koizumi, M., Tsuge, K., 2003. Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector. J Biochem (Tokyo) 134, 513–519. Itaya, M., Tsuge, K., Koizumi, M., Fujita, K., 2005. Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc. Natl. Acad. Sci. USA 102, 15971–15976. Itaya, M., Sakaya, N., Matsunaga, S., Fujita, K., Kaneko, S., 2006. Conjugational transfer kinetics of pLS20 between Bacillus subtilis in liquid medium. Biosci. Biotechnol. Biochem. 70, 740–742. Kaneko, S., Tsuge, K., Takeuchi, T., Itaya, M., 2003. Conversion of sub-megasized DNA to desired structures using a novel Bacillus subtilis genome vector. Nucleic Acids Res. 31, e112.

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