Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5378–5382, May 1997 Microbiology Experimental surgery to create subgenomes of Bacillus subtilis 168 (recombinationyneomycin resistanceymain genomeycovalently closed circular DNAyreplication origin) MITSUHIRO ITAYA* AND TERUO TANAKA† *Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan; and †School of Marine Science and Technology, Tokai University, Shimizu, Shizuoka 424, Japan Communicated by John Roth, University of Utah, Salt Lake City, UT, February 25, 1997 (received for review March 1, 1996) ABSTRACT The 4,188-kb circular genome of Bacillus to term the autonomously replicating entity a subgenome of subtilis 168 was artificially dissected into two stable circular bacteria. chromosomes in vivo, one being the 3,878-kb main genome and the other the 310-kb subgenome that was recovered as co- MATERIALS AND METHODS valently closed circular DNA in CsCl-ethidium bromide ul- tracentrifugation. The minimal requirements to physically Bacterial Strains and Plasmids. B. subtilis 168 trpC2 was separate the 310-kb DNA segment out of the genome were two obtained from the Bacillus Genetic Stock Center (Columbus, interrepeat homologous sequences and an origin of DNA OH). All the BEST strains in this report are derived from this replication between them. The subgenome originated from the by integration of antibiotic resistance markers. The SfiI-NotI 1,255–1,551-kb region of the B. subtilis genome was essential physical map (14) of the B. subtilis 168 genome and the I-CeuI for the cell to survive because the subgenome was not lost from map, equivalent to the ribosomal RNA gene operon (rrn) map the cell. The finding that the B. subtilis genome has a potential (17) are described in Fig. 1. Preparation and transformation of to be divided and the resulting two replicons stably main- E. coli JA221 (F2 hsdR hsdM1 trp leu lacY recA1, ref. 14) tained may shed light on origins and formation mechanisms competent cells were done by the method of Mandel and Higa of giant plasmids or second chromosomes present in many (19). Protocols for preparation and transformation of compe- bacteria. Similar excision or its reversal process, i.e., integra- tent B. subtilis cells were described (14). Luria–Bertani broth tion of large sized covalently closed circular DNA pieces into (20) was used for growth of E. coli, and Luria–Bertani and the main genome, implies significant roles of subgenomes in antibiotic medium 3 (Difco) for B. subtilis. Nutritional require- the exchange of genetic information and size variation of ments for B. subtilis were tested using Spizizen plates with bacterial genomes in bacterial evolution. appropriate amino acids (21). Bacteria were grown at 378Cin all media. pNEXT33 and pNEXT41 (the NotI-linking clones carrying The bacterial chromosome structure has been thought to be NotI site at 1,255 and 3,791 kb, respectively) were described stably maintained under constant environments (1), although previously (14, 22). In this study we used two truncated some plasticity tolerating DNA rearrangements such as inver- neomycin resistance gene alleles; one carries a deletion at the sion, deletion, and duplication has been reported (2–4). There C terminus (ne), while the other carries a deletion at N are several bacteria that have a second chromosome (chro- terminus (eo). Construction of pBEST518 that carries the [ne] mosome II) (5–7) or megabase-sized plasmids (7–11), although cassette and pBEST524B that carries the [eo] cassette was the origins and dynamics of their formation mechanisms are described previously (23). not clear, and the discrimination of plasmids from second The [ne] and [eo] gene cassettes used in this study were chromosomes has not been well defined. Meanwhile there are prepared from pBEST518 and pBEST524B. Briefly, the 2.4-kb numerous examples that DNA pieces shuttle between an [ne] cassette composed of a (ne) segment (1.1 kb) and the autonomously replicating state and an integrated form in the spectinomycin resistance gene (spc, 1.3 kb), the 1.4-kb [eo] host genome. Well known examples are, the genome of cassette composed of a (eo) segment (0.79 kb) and the Escherichia coli phage lambda that exists in two states, one the blasticidin S resistance gene (bsr, 0.6 kb) were excised with lysogenic state, and the other the state showing lytic cycle (12) NotI from pBEST518 and pBEST524B, respectively. The [ne] and the F-factor that shuttles between the Hfr and episome cassette and the [eo] cassette were inserted into the NotI sites forms (13). of pNEXT33 and pNEXT41, respectively, resulting in B. subtilis 168, an endospore forming gram-positive soil pNEXT33FA and pNEXT41GB. bacterium, has a single 4,188-kb circular genome whose struc- The oriN sequence is the replication origin of a 70-kb ture is considered to be stably maintained (1, 14). This low-copy number plasmid pLS32 obtained from a B. natto bacterium carries no indigenous plasmid although there are a strain (24). Plasmid pBET131 (14.3 kb) is composed of an number of foreign plasmids capable of replication in this host oriN-carrying 7-kb BamHI segment from pLS32, 5.4-kb (15, 16). In this study we show evidence that a 310-kb genomic pUH101 (a pBR322 derivative plasmid carrying the chloram- DNA segment of this bacterium can be excised and replicate phenicol resistance gene), and a 1.9-kb fragment carrying the as an independent replicon from the genome. The established tetracycline resistance gene (T.T., Y. Ohshiro, and M. Ogura, covalently closed circular form of DNA was found in the cell unpublished work). To integrate pBET131 in the BEST5018 and its presence was essential for the cell to grow. We propose genome, a pBR322 sequence was first created at 1,375 kb of the BEST5018 genome. The pBR322 sequence marked with eryth- The publication costs of this article were defrayed in part by page charge romycin resistance gene of BEST2124 (25) was transferred to payment. This article must therefore be hereby marked ‘‘advertisement’’ in the 1,375-kb site of BEST5018 by transformation, resulted in R R R accordance with 18 U.S.C. §1734 solely to indicate this fact. BEST4170 (Spc ,BS , pBR322::Em at 1,375 kb). BEST4173 (Fig. 1) was obtained from BEST4170 by replacement of the Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y945378-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviation: cccDNA, covalently closed circular DNA. 5378 Downloaded by guest on September 29, 2021 Microbiology: Itaya and Tanaka Proc. Natl. Acad. Sci. USA 94 (1997) 5379 FIG.1. SfiI-NotI-I-CeuI map of the B. subtilis 168 genome. The physical map of the 4,188-kb B. subtilis genome for NotI (inner circle), SfiI (outer circle), and I-CeuI map (equivalent of the rrn map) is shown. The sizes of the I-CeuI fragments .100 kb are shown by arcs with two arrowheads. The loci of oriC and terC have been determined (14, 18). Strain BEST4173 has the [ne] cassette at 1,255 kb, the [eo] cassette at 1,551 kb, and the oriN (closed circle) at 1,375 kb of the physical map as described in the materials and methods section. The spectinomycin resistance gene (spc) included in the [ne] cassette and the blasticidin S resistance gene (bsr) in the [eo] cassette are shown. The 300-kb DNA segment (1,255–1,551 kb) is shown by double arcs. erythromycin marker with the cat-oriN-tet array of pBET131. somal homologous recombination between two truncated BEST4173, selected by chloramphenicol and tetracycline, neomycin resistance gene alleles (indicated as [ne] and [eo] in showed erythromycin sensitive. Fig. 1) integrated at the ends of the DNA region to be Plasmid transformants of E. coli were selected by tetracy- rearranged. Integration of the [ne] cassette at 1,255 kb was cline, 15 mgyml; chloramphenicol, 5 mgyml; spectinomycin, 100 done by transformation of linearized pNEXT33FA, selecting mgyml; and ampicillin, 100 mgyml. Transformants of B. subtilis for spectinomycin resistance transformant BEST2161. Inte- were selected by chloramphenicol, 5 mgyml; blasticidin S, 500 gration of the [eo] cassette at 1,551 kb of the BEST2161 mgyml; tetracycline, 15 mgyml, and spectinomycin, 50 mgyml. genome was similarly done using linearized pNEXT41GB. In Vitro DNA Manipulations. Endonucleases and T4 DNA BEST5018 was obtained as blasticidin S resistant transformant ligase were obtained from Toyobo (Osaka), except for SfiI and of BEST2161. Presence and orientation of the [ne] and [eo] I-CeuI from New England Biolabs, NotI from Takara Shuzo cassettes in the BEST5018 genome was verified by Southern (Kyoto), and I-SceI from Boehringer Mannheim. hybridization using both plasmids as probes (data not shown). Preparation of B. subtilis Chromosomal DNA and Southern Strain BEST5018 did not produce a spontaneous neomycin- Hybridization. Intact unsheared chromosomal DNA for con- resistant recombinants. tour-clamped homogeneous electric field gel electrophoretic To test if about 300-kb genomic segment (1,255–1,551 kb) analysis was prepared in agarose plugs as described previously can be established as an autonomous replicon, strain (1, 14, 18). Running conditions are described in the Fig. 3. BEST4173 was constructed from BEST5018 as described in Chromosomal DNA for analysis by conventional gel electro- Materials and Methods. Presence of the [ne], [eo], and oriN of phoresis was prepared by a liquid isolation method (26). BEST4173 shown in Fig. 1 was verified by Southern hybrid- Agarose gel (1.0% wtyvol) in TBE solution (50 mM ization experiments using pNEXT33FA, pNEXT41GB, and Triszborate, pH 8.0y1.0 mM EDTA) was used for conventional pBET131 as probes (data not shown). It was expected that a gel electrophoresis at room temperature. After electrophoresis recombinational event occurring between the common 590-bp the DNA was stained in ethidium bromide solution (600 homologous region of the integrated [ne] and [eo] sequence ngyml) for 15 min and photographed.