Bioscience, Biotechnology, and Biochemistry

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Sequence Analysis of Replication Origin of Plasmid pLS11 of Bacillus subtilis IFO 3022 Toshio Hara, Kazuhiko Nakajima, Hiroyuki Saito, Ayaaki Ishizaki, Seiya Ogata & Seinosuke Ueda To cite this article: Toshio Hara, Kazuhiko Nakajima, Hiroyuki Saito, Ayaaki Ishizaki, Seiya Ogata & Seinosuke Ueda (1992) Sequence Analysis of Replication Origin of Plasmid pLS11 of Bacillus subtilis IFO 3022, Bioscience, Biotechnology, and Biochemistry, 56:2, 223-227, DOI: 10.1271/ bbb.56.223 To link to this article: http://dx.doi.org/10.1271/bbb.56.223

Published online: 12 Jun 2014.

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Biosci. Biotech. Biochem., 56 (2), 223-227, 1992

Sequence Analysis of Replication Origin of Plasmid pLS 11 of Bacillus subtilis IFO 3022 Toshio HARA, Kazuhiko NAKAJIMA, * Hiroyuki SAITO, * Ayaaki ISHIZAKI, * Seiya OGATA, and Seinosuke UEDA** Microbial Genetics Division, Institute of Genetic Resources, and * Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan ** Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda, Kumamoto 860, Japan Received July 10, 1991

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The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS 11 of Bacillus suhti/is IFO 3022, which is responsible for l'-polyglutamate produCtion, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. suhti/is TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8%) identity), pBAA1 (92.8%), and pFTB14 (78.70/0) in Bacillus spp., pLP1 (42.1 % ) and pLABI000 (36.3%) in Lactohacillus spp., and pUBII0 (35.3%) and pC194 (37.40/0) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.

We have reported that a 5.7-kilobase pair (kb) plasmid designated pURl, which encodes the y-glutamyltranspeptidase gene responsible for y-polyglutamate production, is distributed widely in a number of Bacillus subtilis (natto) strains isolated from a fermented soybean food, natto. 1 ,2) B. subtilis and B. subtilis (natto) should be considered as one species, but these two bacilli are found to be classified separately on the basis of whether biotin is esential for p

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growth or not. 3,4) B. subtilis IFO 3022, which did not require biotin for the growth but produced a small amount of viscous substances, harbored a single plasmid pLSll (8.6 kb). 5) Recently, electron microscopy showed that a heteroduplex molecule between pURl and pLSll contains 1.11- and 1.24-kb double-stranded termini. 6 ) The 2.0-kb BstEll DNA fragment of pURl, including the l.11-kb double-stranded region, contains a 999-bp open reading frame, a promoter for the open reading frame,and a possible replication origin upstream of the promoter. 7) Analysis of the organization of various plasmids isolated from Gram-positive bacteria, such as pT18l,8) pC194,9) pE194,l°) pFTB14, 11) pLPl,12) pUBll0,13) pBAAl,14) and pLAB 1000,15) has shown that all of the information necessary for replication is located on fragments of about 1.5 kb. These fragments harbor a rep gene, encoding a protein essential for the initiation of replication (REP) and its corresponding target site. This communication reports that the 1.6-kb fragment of pLSII contains a 999-bp open reading frame, a promoter for the open reading frame, and a possible replication origin upstream of the promoter. Significant homology was observed between the amino acid sequence predicted from the 999-bp open reading frame and those of

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Fig. 1.

Derivation of P1asmids Used in This Study.

The chain of solid circles in the diagram indicates the DNA segment containing the Tmpr gene of B. subtilis TTK24. Heavy and thin lines represent the regions of pBR322 and pTLl2, respectively. Double lines represent the DNA fragment of pLSll. A, AatI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SphI.

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Fig. 2. Structure and Replication Activity of the Derived Fragments of the 1.6-kb ori Fragment. The open arrow in the restriction map indicates an open reading frame found in the sequence data (see Fig. 3). + and - indicate, respectively, ability and inability to replicate in the B. subtilis host. Derivatives were made by deletion with the indicated restriction enzyme or by Bal31 digestion (B) from the BglII site of pNH2.

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T.

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similar putative REP proteins encoded by the other wellknown gram-positive replicons.

Materials and Methods Bacterial strains and plasmids. Escherichia coli JMlOl and B. subtilis

et al.

Ml112 (arg-15, leuB8, thr-5, recE4) were used for a cloning host. The plasmids pLS 11 2) and pATE U) were been described previously,2) and plasmid pTL12 carrying the dihydrofolate reductase gene was kindly provided by Tanaka and Kawano. 16 ) Media. LB broth and Panassay broth (Difco) for both B. subtilis and

Sphl 50 5'-GCATGCTGCGGAGATAACACGGGCTGCTGCGCGAATGATGCGGGCATGGGTGCGTTGATCGTGCCGAGACTGAAA 100 150 AGGCAAAATTTCAAAAAAAAATCTCCCCCCCTACGGAAGAATCCTTTTGATCTTTTGTTTTGGGTTTTAAAAAAAGCCG 200 GCTGTTTTTTCACCGTCTTTTTCGATTTTGGCGAAGCAAATCGGGTCTTTTCTTATCTTGATACTATATAGAAACACAT

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CATTTTTCAAAATTAGGTCAAAGCCTTGTGTATCAAGGGTTTGATGGTTC~GTAAAAACTCCTTCTG~ • 2 •

.

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350

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~AAGGTGTCGAATCAA~TAATAGAATGCTAGAGAACTAGCTC~~TTTTTTGTTGATTTATTCATCTGAAA 4

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400 450 ATG ATT ATA AGC ATC CTG AAG ATA AAA CCG CAA CAG GTA AAA AGC GGG ATT GGA AGG GGA Met lIe lIe Ser lIe Leu Lys lIe Lys Pro GIn GIn Val Lys Ser Gly lIe Gly Arg Gly 500 AAA AGA GAC GGA CGA ACC TCA TGG CGG ACT TAC GAA GCG TTA GAG AGT AAG ATT GGG GCA Lys Arg Asp Gly Arg Thr Ser Trp Arg Thr Tyr Glu Ala Leu Glu Ser Lys lIe Gly Ala 550 CCT TAC TAT GGC AAA AAG GCT GAA AAA CTA ATT AGT TGT GCA GAG TAT CTT TCG TTT AAG Pro Tyr Tyr Gly Lys Lys Ala Glu Lys Leu lIe Ser Cys Ala Glu Tyr Leu Ser Phe Lys

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600 AGA GAC CGG AGA CCG GGC AAG TTA AAA CTG TAT CAA GCC CAT TTT TGT AAA GTG AGG TTA Arg Asp Arg Arg Pro Gly Lys Leu Lys Leu Tyr GIn Ala His Phe Cys Lys Val Arg Leu 650 TGT CCG ATG TGT CGG TGG CGC AGG TCG TTA AAA ATT GCT TAT CAC AAT AAG TTG ATC GTA Cys Pro Met Cys Arg Trp Arg Arg Ser Leu Lys rle Ala Tyr His Asn Lys Leu rle Val 700 750 GAG GAA GCC AAT AGA CAG TAC TGC TGC GGA TGG ATT TTT CTC ACG CTG ACG ATT CGA AAT Glu Glu Ala Asn Arg GIn Tyr Cys Cys Gly Trp lIe Phe Leu Thr Leu Thr lIe Arg Asn 800 GTA AAG GGA GAA CGG CTG AAG CAC CAA ATT TCT GCG ATG ATG GAA GGC TTT AGG AAA CTG Val Lys Gly Glu Arg Leu Lys His GIn lIe Ser Ala Met Met Glu Gly Phe Arg Lys Leu 850 TTC CAG TAC AAA AAA GTA AAA ACT TCG GTT CTT GGA TTT TTC AGA GCT TTA GGA ATT GCC Phe GIn Tyr Lys Lys Val Lys Thr Ser Val Leu Gly Phe Phe Arg Ala Leu Gly lle Ala 900 AAA AAT CAT GAA GAA GAT ACA TAT CAT CCT CAT TTT CAT GTG TTG ATA CCA GTA AGG AAA Lys Asn His Glu Glu Asp Thr Tyr His Pro His Phe His Val Leu lIe Pro Val Arg LY5 950 AAT TAT TTT GGG AAA AAC TAT ATT AAG CAG GCG GAG TGG ACG AGC CTT TGG AAA AAG GCG Asn Tyr Phe Gly Lys Asn Tyr lIe Lys GIn Ala Glu Trp Thr Ser Leu Trp Lys Lys Ala 1050 1000 ATG AAA TTG GAT TAC ACT CCA ATT GTC GAT ATT CGT CGA GTG AAA GGT AAA GCT GTT CTT Met Lys Leu Asp Tyr Thr Pro rle Val Asp lIe Arg Arg Val Lys Gly Lys Ala Val Leu 1100 GAC GCT GAA CAG ATT GAA AAC GAT GTG CGG AAC GCA ATG ATG GAG CAA AAA GCT GTT CTC Asp Ala Glu GIn lIe Glu Asn Asp Val Arg Asn Ala Met Met Glu GIn Lys Ala Val Leu ll50 GAA ATC TCT AAA TAT CCG GTT AAG GAT ACG GAT GTT GTG CGC GGT AAT AAG GTG ACT GAA Glu lIe Ser Lys Tyr Pro Val Lys Asp Thr Asp Val Val Arg Gly Asn Lys Val Thr Glu 1200 GAC AAT CTG AAC ACG GTG CTT TAC TTG GAT GAT GCG TTG GCA GCT CGA AGG TTA ATG GGA Asp Asn Leu Asn Thr Val Leu Tyr Leu Asp Asp Ala Leu Ala Ala Arg Arg Leu Met Gly 1250 TAC GGT GGC ATG TTG AAG GAG ATA CAT AAA GAG CTG AAT CTT GGT GAT GCG GAG GAC GGC Tyr Gly Gly Met Leu Lys Glu lIe His Lys Glu Leu Asn Leu Gly Asp Ala Glu Asp Gly 1300 1350 GAT CTG GTC AAG ATT GAG GAA GAA GAT GAC GAG GTG CAA AAT GGT GCA TTT GAG GTT ATG Asp Leu Val Lys lIe Glu Glu Glu Asp Asp Glu Val GIn Asn Gly Ala Phe Glu Val Met 1400 Haelll GCT TAT TGG CAT CCT GGC ATT AAA AAT TAC ATA ATC AAA TAA AAAAACCTTTAGAAGGCCTGCTT Ala Ty~ Trp His Pro Gly lIe Lys Asn Tyr lIe lIe Lys 1450 TTTTAACTAACCCATTTGTATTGTGTTGAAATATGTTTTGTTCGTTTTTTATCTTTGTAGCTTTTTATGATTAATTGTC 1500 1550 TATGATAACCTAGGGAATCTTTACATTGTCTGTGTTGAATAGTTACATAAACTGCTTTGCTCTTAGGATAGTTTAAATA HindIII 1606 TACTGTTGTCATTGCTCCGAAAACAAAAGCTT-3'

Fig. 3.

Nucleotide Sequence of the 1.6-kb SphI-HindIII DNA Fragment.

Nucleotide residues are numbered in the 5'-to-3' direction, beginning with the 5'-end residue originated from the SphI site. The deduced amino acid sequence is given below the nucleotide sequence. Putative promoter elements ( - 35, - 10 and the ribosome binding site) are underlined.

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reading frames (ORFs), we found only one large frame (Fig. 2) designated rep, which consisted of 999 bp and encoded a protein molecule with 333 amino acids with a Mr of37,138. Several conserved regulatory sequences similar to the E. coli and Bacillus consensus promoter sequences 22 ) were DNA manipulations. Plasmid DNAs from B. szibtiliswere prepared and purified as described previously.2) Restrictiqn endonucleases, T4DNA observed 5' upstream to the rep ORF. ligase, and bacterial alkaline phosphatase were purchased from Takara Plasmid pNH2 was digested with Bg/U and then treated Shuzo Co., Ltd., and used as recommended .by the manufacture. 20 min with exonuclease Ba131 under the conditions in for Degradation of DNA with exonuclease Bal31 (Takara Shuzo Co., Ltd.) which about 50 bp per min were removed from each end of was done by the procedure of Legerski et al. IS ) the DNA molecule. After ligation by T4 ligase, the DNA Methods for transformation and assenssment of ori function. E. co/twas was transformed into E. coli and subsequently introduced transformed by the method of Morrison, 19) and B. subtilis was transformed into B. subtilis by protoplast transformation. Tmpr by using protoplasted cells. 20 ) transformants, which contain 1.4-kb fragment 5 in Fig. 2, were obtained at high efficiencies with a 168-bp deletion DNA sequencing. DNA fragments were subcloned into plasmids pUCl8 and pUCI9, and DNA sequencing was done by the dideoxy chain plasmid generated with Ba131 (fragment 5 in Fig. 2), while termination method 21 ) with sequenase (United States Biochemical no transformants were obtained with a similar 255-bp Corporation, Ohio, U.S.A.). Nucleotide and amino acid sequences were deletion plasmid (fragment 6 in Fig. 2). It suggests that the analyzed by the Hitachi DNASIS system. putative replication origin of pLS 11 is between position 168 and 255. To discover the sequences essential for replication, a homology search was done to find whether there are Results and Discussion sequences within this region conserved in the registered Delimitation of the replication origin To facilitate the identification of the replication region Gram-positive replicons. A 34-bp sequence was conserved of pLS 11, we used the trimethoprim-resistant (Tmpr) di- in four plasmids, pLSll, pUHl, pBAAl, and pFTB14 in hydro folate reductase gene of B. subtilis 168. A schematic Bacillus spp. (Fig. 4), and the 14-bp sequence within this presentation of the constructed plasmids is given in Fig. 1. region was conserved in all eight plasmids in Gram-positive The source of the dihydrofolate reductase-coding gene was. bacteria including Lactobacillus spp. and Staphylococcus a Tmpr strain, TTK24, of B. subtilis 168 17 ) and has been spp. This conserved 14-bp sequence is found in the 55-bp cloned in the pBR322 plasmid of Escherichia coli. DNAs region of pC194 shown by Gros et al. 23 ) to have origin from pTL 12, carrying the Tmpr dihydrofolate reductase activity. Within this 14-bp sequence, in addition, the gene, which was constructed by Tanaka and Kawano, 16) sequence CTTGAT A is the sequence at which nicking of and pBR322 were both treated with EcoRI and HindUI, the plus-strand occurs in the initiation of replication of the mixed and ligated by T4 ligase, then pATEl was coliphage ¢XI74, 24) and this conserved sequence was found constructed. Plasmid pLS 11 was digested with SphI and in a hairpin region of ¢XI74. HindU I , and then the ends were filled in with the Klenow fragment to generate blunt ends. The DNA fragments were Amino acid sequence homology of pLSll REP protein with different Gram-positive replication proteins mixed and ligated to the AatI site of pATEl by T4 ligase, The amino acid sequence of the REP protein coding and then added to B. subtilis MIlI2 protoplasts. Several Tmpr colonies were obtained on AA agar plates containing region of pLS 11 was compared with a number of amino Tmp (1 mg/ml) and one of them was used for further study. acid sequence of proteins registered in GenBank by the A plasmid, pNH2, carried in such a Tmpr colony had a homology search system GENAS. 25 ) Homologies between molecular size of 8.2 kb (Fig. I). The physical map of pNH2 the predicted amino acid sequence of the REP protein of pLS 11 and those of the REP protein of p UH 1, pBAA 1, using various restriction enzymes is shown in Fig. 1. To define the boundaries of a functional unit of pLSll pFTBI4, pLPI, pLABIOOO, pUBllO, and pCI94 are replication, the 1.6-kb SphI-HindUI fragment ofpLSll was illustrated in Fig. 5. The REP protein encoded on pLSl1 digested with selected restriction endonucleases to obtain a showed a substantial degree of homology to three REP set of overlapping DNA fragments. The digests filled in proteins in Bacillus plasmids: 92.8% identity with with Klenow fragment were ligated with pATEl, introduced into E. coli by transformation, and selected for ampicillin pLSll S'-CAAATCGGGTCTT TTCTTATCTTGATA CTATATAGA-3' pUHl gAAATCGGGTCTT TTCTTATCTTGATA CTATATAGA resistance. The plasmid DNA preparations containing each pGAAl CAAATCGGGTCTT TTCTTATCTTGATA CTATATAGA generated fragment were tested for replication in B. subtilis. pFTB14 aAAATCGGGTCTT TTCTTATCTTGATA CTATATAGc pLPl tTtTc TTCTTATCTTGATA CTATta The results are summarized in Fig. 2. The constructed pLABIOOO GTtTc TTCTTATCTTGATA CTActa pUBIIO GTtcT TTCTTATCTTGATA Catata plasmid with the IA-kb SphI-HaeUI fragment (fragment 2 pC194 tTtcT TTCTTATCTTGATA aTAagg in Fig. 2) could replicate in the B. subtilis host, but the recombinant plasmid preparations containing the small 4>X174 GTgcT cccccAacTT~TA tTAata fragments (fragment 3 and 4 in Fig. 2) could not replicate.

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E. coli, Spizizen minimal medium for B. subtilis, and M9 minimal medium for E. coli were the same as those described previously. 7) The cells carrying trimethoprim-resistant (Tmpr) plasmids were grown in AA medium 17) containing 1 mg/ml of Tmp.

Nucleotide sequence of the replication region The nucleotides of the 1.6-kb SphI-HindUI fragment were sequenced by the method ofSanger. 21 ) Though the strategy is not shown, the nucleotides of both stands were sequenced using numerous restriction fragments to give enough overlapping regions (Fig. 3). Looking for possible open

Fig. 4. Homology at the Origin for Plus-strand Synthesis among Plasmids pLSll, pUHl,7) pBAAI,14) pFTBI4,11) pLPI,12) pLABIOOO,1S) pUBllO,13) pC194,9) and phage ¢X174.24) Sequences from the regions corresponding to the pLSll origin for replication are shown, and the same base of given replicons with that of pLS 11 is capitalized. The 14-bp sequence conserved in all eight plasmids in gram-positive replicons are boxed. The sequence CTTGAT A, which is the site at which nicking of the plus strand occurs in phage