Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL

PLASMID

25,

113-120 (1991)

Characterization and Sequence Analysis of a Small Plasmid from Bacillus thuringiensis var. kurstaki strain HDI -DIPEL DAVID G. MCDOWELL AND NICHOLAS H. MANN’ Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom Received June 8, 1990; revised January 15, 1991 The complete nucleotide sequence of a small (2.055 kb) plasmid pHDZ from Bacillus thuringiensis var. kurstaki strain HD 1-DIPEL was obtained. The sequence encoded two open reading frames (ORFs) which corresponded to polypeptides of M, 26,447 and 9 122. Comparison of the sequence with those obtained for other plasmids from Gram-positive organisms suggested that pHD2 may belong to the extensive and highly interrelated family of plasmids exhibiting replication via a ssDNA intermediate; a putative nick site was proposed on the basis of sequence homology and one ORF exhibited distant homology with the site-specific topoisomemses encoded by the pT 18 1 family of staphylococcal plasmids, while the other ORF exhibited considerable similarity to a small polypeptide (RepA) encoded by plasmid pLS1. Constructs consisting of pHD2, pBR322, and the chloramphenicol resistance gene from pC 194 were capable of stable maintenance in B. thuringiensis var. israelensis, but were subject to apparently specific deletions in the heterologous host. The same constructs could not be established in Bacillus subtilis. 0 199 I Academx

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Bacillus thuringiensis is a Gram-positive bacterium represented by a wide variety of subspecies, which, during the course of sporulation, produce crystalline inclusions, the 6endotoxins, which are selectively toxic to a range of mainly lepidopteran and dipteran insect larvae (see Aronson et al., 1986; Whiteley and Schnepf, 1986; Rowe and Margaritas, 1987). Strains of B. thuringiensis commonly have complex plasmid profiles, with the number of plasmids ranging from 2 to 12 and the size ranging from 1.4 MDa to greater than 150 MDa (Lereclus et al., 1982; Carlton and Gonzalez, 1985). Interest in the plasmids of B. thuringiensis has focused predominantly on the larger plasmids which may have a role in 6-endotoxin production. However, three of the smaller plasmids of B. thuringiensis var. thuringiensis strain Hl . 1 have been partially characterized (Mahillon et al., 1988) and one ofthese, pG12, has been completely sequenced (Mahillon and Seurinck, 1988). ’ To whom correspondence

should be addressed.

Plasmids have been isolated from a wide range of Gram-positive bacteria and their characterization has led to the conclusion that the large majority of these plasmids are highly interrelated and replicate via a mechanism which involves a single-stranded DNA intermediate. The features common to this process are a plus origin of replication, a replication protein, and a minus origin (see Gruss and Ehrlich, 1989). It was the intention of this study to characterize one of the small plasmids of B. thuringiensis subspecies kurstaki HD l-DIPEL to establish whether it constituted a member of this extensive family of plasmids from other Gram-positive organisms. MATERIALS

AND METHODS

Bacterial strains, plasmids, and growth conditions. The bacterial strains used in this study were Escherichia co/i DHl (Hanahan, 1983) E. coli TG2 (see Sambrook et al., 1989), B. thuringiensis var. kurstaki strain HDI -DIPEL (obtained from Microbial Resources Ltd), B. thuringiensis subsp. israelen-

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sis strain IPS-78/11 (Ward and Ellar, 1983) and B. subtilis 168 (obtained from the National Collection of Industrial Bacteria). B. thuringiensis strains were grown in SR broth (Singer and Rogoff, 1968) and E. coli strains in LB broth. B. subtilis was grown for the preparation of competent cells as described by Bott and Wilson ( 1968). Solid media when required were prepared by the addition of 1.5% agar (Lab-M). X-Gal IPTG plates for use with strains carrying pUC8 or Ml3 vectors were prepared as described by Vieira and Messing (1982). E. coli cultures were grown at 37°C and all Bacillus cultures at 30°C. Antibiotics were used at the following concentrations: ampicillin, 100 pg ml-‘; chloramphenicol, 5 pg ml-‘; tetracycline, 10 pg ml-‘; kanamycin, 25 pg ml-‘. Plasmids pLG338 (Stoker et al., 1982), pUC8 (Vieira and Messing, 1982), pC194 (Horinouchi and Weisblum, 1982), and pBR322 (Bolivar et al., 1977) have already been described. Plasmid pHD2 is a 2.055-kb plasmid from B. thuringiensis var. kurstaki strain HDI-DIPEL and plasmids pDM 100, pDM200, pCC100, pCC200, and pCC300 were constructed during the course of this study. DNA purification. Plasmid DNA was isolated on a small scalefrom both E. coli and B. thuringiensis by the alkaline lysis method of Birnboim and Doly (1979) with the minor modification in the case of B. thuringiensis that the SDS2concentration was increased to 2% (w/v). Large-scale plasmid DNA preparation was carried out by the same process except that a cesium chloride/ethidium bromide density gradient step was included.

Recombinant DNA procedures and DNA sequencing. All enzymes for recombinant DNA procedures, including restriction enzymes and T4 DNA ligase, were purchased from Amersham International plc and were used in accordance with the manufacturer’s instructions. Southern hybridization was

2 Abbreviations used: SDS, sodium dodecyl sulfate; ORF, open reading frame; SD, Shine-Ialgamo.

carried out as described by Maniatis et al. ( 1982). Sequencing reactions were carried out by the dideoxy method using an M 13 sequencing kit (Gibco BRL). The oligonucleotides used for primer extension sequencing reactions were synthesized by the P-cyanoethyl phosphoramidate method using an Applied Biosystems 380B machine. The oligonucleotides were not further purified. Sequence analysis. Sequence data were compiled and manipulated using the Microgenie (Beckman) sequence analysis program, as were nucleic acid alignments for putative nick sites. Sequencehomologies to published sequenceswere obtained utilizing the Seqnet computer facility at Daresbury. Protein alignments were carried out using the Gap program of the University of Wisconsin Genetics Computing Group Package(Devereux et al., 1984). Transformation. Transformation of E. coli was carried out by the CaCl, procedure of Mandel and Higa ( 1970). Transformation of B. thuringiensis var. israelensiswas carried out by the electroporation protocol of Bone and Ellar (1989) using a gene pulser electroporation system (Bio-Rad Laboratories Ltd). Transformation of B. subtilis was carried out by the method of Bott and Wilson (1968). Analysis of plasmid stability. B. thuringiensis subsp. israelensis transformants carrying plasmid pCC200 were grown in 50 ml SR broth in 250-ml shake flasks in the absence of antibiotic selection with subculturing for approximately 100 generations. Every 10 generations ( 1 subculture) appropriately diluted samples were plated out onto SR plates and individual colonies replica plated onto SR plates with and without chloramphenicol(5 pg ml-‘). RESULTS

AND DISCUSSION

The strain B. thuringiensis var. kurstaki HDl-DIPEL has been reported by Lereclus et al. (1982) and Kronstadt et al. (1983) to contain between 8 and 12 plasmids with sizes ranging from 1.5 to 120 MDa. Plasmid profiles obtained during this study were largely

CHARACTERIZATION

OF A SMALL B. thuringiensis PLASMID

similar to those previously reported. The smallest of the plasmids, which in our hands gave an apparent size of 2 kb, was purified by electrophoresis in a 0.5% agarosegel and subsequent electroelution. The 2-kb plasmid, designated pHD2, was refractory to digestion with a variety of endonucleases (including EcoRl, Pstl, BamHl, and &z/l), but appeared to contain a single site for HindIII. Consequently, an attempt was made to ligate the pHD2, linearized with HindIII, into the Hind111 site of pUC8 (phosphatase treated). Following transformation into E. coli TG2, white colonies were obtained on X-Gal IPTG plates, but screening of these colonies did not reveal any recombinants containing a 2-kb insert. Similar problems have been reported for the cloning of pGI1 from B. thuringiensis var. thuringiensis (Mahillon et al., 1988). It was thought that the pHD2 could not be cloned at high copy number and consequently, the low-copy-number vector pLG338 was employed. This vector has two Hind111 sites, one within the promoter of the tetracycline resistance gene and the other within the kanamycin resistance gene. pHD2 restricted with Hind111 was ligated with the HindIII-restricted pLG338 and transformed into E. coli DH 1. Kanamycin-resistant transformants were selected for; this ensured that the internal 2.6-kb fragment of pLG338 had been reinserted in the correct orientation. Colonies were screenedfor tetracycline sensitivity, and plasmid DNA was isolated from a number of tetracycline-sensitive colonies and restricted with HindIII. All such isolates gave the predicted restriction pattern, and one clone was used to prepare plasmid DNA on a large scale. The 2-kb insert was then subcloned into the Hind111 site of pBR322. Transformants containing the 2-kb Hind111 insert were obtained, suggesting that the problems encountered in attempts to clone it into pUC8 resulted either from the very high copy number of pUC8 or from the fact that plasmids carrying this insert have a lower efficiency of transformation. Southern blotting using pHD2 from B. thuringiensis as a probe confirmed the identity of the 2.0-kb Hind111

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fragment cloned into pBR322 (data not shown) and the plasmid was designated pDM 100. A preliminary restriction map revealed the presence of a single Clal site within pHD2, consequently, a second construct, pDM200, was made in which pHD2 was cloned into pBR322 at the C/al site. Initial attempts to sequence the 2.0-kb fragment cloned in pDM 100 and pDM200 employed cloning of random fragments generated by sonication into M 13mpl8 and M 13mpl9. Although significant amounts of nucleotide sequence were generated in this fashion, it became clear that certain regions of the plasmid were not appearing among the recombinants at an appreciable frequency. Consequently, this approach was supplemented by directed cloning, utilizing the Tuql , HindIII, and Clu 1 sites present in the pHD2. Regions unsequenced by these two approaches were completed by primer extension sequencing. The complete nucleotide sequence of pHD2 is presented in Fig. 1. The A+T content of the plasmid was 65.4% and the sequence revealed two ORFs, potentially encoding polypeptides of M, (A) 26,447 and (B) 9 122. Possible - 10 and -35 regions exhibiting good homology with the Bacillus subtilis Esigma43 consensus promoter sequence (Graves and Rabinowitz, 1986) were found upstream from ORFs A and B. However, in the case of ORF A the spacing between the putative - 10 and - 35 sequencesis only 10 bases. Putative SD sequences were also found for both ORFs (Fig. 1). Of the many Gram-positive plasmids so far isolated and characterized, the vast majority are members of a family of highly related plasmids which replicate via a singlestranded intermediate. All theseplasmids possessa plus origin, a replication protein (Rep), and a minus origin. Many plus origins have been characterized and are found to be situated upstream from, or within, the gene encoding the Rep protein. These plus origins fall into three groups on the basis of sequence homology (Gruss and Ehrlich, 1989). Analysis of the pHD2 sequencefor possible regions of homology with these plus origins revealed

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10 20 30 40 50 60 70 80 90 100 110 120 TTCCTCGTGAAAGTGTAGTTGATCCAGAGAATGTTGAGTTTTTATTGGATAATGCTATTAGTTCTTATTTAGGGCAATTAGAAATTT

130 140 150 160 170 180 190 200 210 220 230 240 CAATGTGTGGGTAAATTGTAGAAATGTGGCGAAGACATTTTCGGACATTCTAATAGCCGAAAATCGTGTACAAAATGACATGTTTA Nick Site -35 -10 ORFA M 0 250 260 270 280 290 300 310 320 330 3:: 350 360 GTTTATTTGGATAGGCTAATGATTAAGTATAAAGATGTAACAGAGAAACAATTTAGTGATGTTTTAACTAAAATATCGTCAAAGCAG VYtDRtMIKYKDVTEK~FSDVtTKISSK~IFtPNTPIRSE 370 380 390 400 410 420 430 440 450 460 470 480 CATGGGACGTCTGTTAGAGATTATCATAGAGTTATACATATTGGATATGGTGAAGGTGCAGTTTATATAGGGTGGAAACATAATTC HGTSVRDYHRVIHIGYGEGAVYIGWKHNSEKEKDSYDHKV 490 500 510 520 530 540 550 560 570 580 590 600 GATTTTAACCCTTCTAAATTTGAAAATAACGAGTTGCAAAAAGATAGTTATGAAAAAGTGTTTGAAACCGTTTTTCATACGTTAAATG DFNPSKFENNELQKDSYEKVFETVFHTLNAVLKSNKRVVY 610 620 630 640 650 660 670 680 690 700 710 720 GGTATGG~ATTGCTTTTGATATAGAGCGTCATATGAGTGATATTGTGTCTTATAGTAAAACAGGAAAGCAACAGGATAGACATAAA GMDIAFDIEREMSDIVSYSKTGKQQDRHKGTVYYGNRNKD 730 740 750 760 770 780 790 800 810 820 830 840 GGATATTTGAAGATATATGATAAGAAAAAGGAGTTATATAATCATTTTAAAAGAATGATAGAAGAAGAGAATTTGACTCGTATTGAG GYtKIYDKKKELYNHFKRMIEEENlTRIEYSWRDSDGVYY 850 860 810 880 890 900 910 920 930 940 950 960 GACGAAATAAGGAAGAGTCCTCCGTTTAGTATTGATGAATCTTATACATTCTCGATTTTAATTTGAATAATGTTAAAGGGGCATTAA DEIRKSPPFSIDESYTFSILIU XiadIII 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 TGGATATGAAAGAGTTCCCTCGTAGAACTAAAGAGAGTATAAAAAAAGCCCTTGAAGAAATGGATCACTTGGCGGTGGACCCCAT

1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 TTAAGAACTATACTCGTTTATGATATTAGCGTGTGCTTCTCTGTGTGTAAGAGGGTGTCAATATGCTGCTCTCTTTTTGTTTTTGTTA

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 ACATTGTTCTGTGTATCTTATGTTGATATTGTGTTTAAACTGATGTTATATTTATGTAGTACGATATACAAGAGGTGATTAGATGAGT -35 -10 SD ORFB MVRVNTRISK 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 AAAGTTAAATGATTGGTTGGACGAGTATAGCAAAGAAAGTGGTGTACCGAAAAGCACTTTAGTTCATTTAGCTTTAGAGAATTATG KtNDWtDEYSKESGVPKSTLVHtAtENYVN~KVMtE~MPK 1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560 GATGCAACAAATGTTGAGTATGATGTTTGAAAATGTAACCAAGGGAATATGTTTGAGTTGAAGTAACGGTTATGTTTCGAAAATGT MQQHLSMMFENVTQQQLNQKGNHFELKU 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 GAGATGCACITCCATAAACTAAATGCGTAGTTGGTGTGGCTGAAGTTTGCCCGCCACCTACTCATTTAGAATATCCGTGCATGGG ClaI 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 ATGAGTTGTAATTATTATTCACTTGGGTGAACGTTCTATGACTGAAAGGAAACCCATGGCACTTAACTGTGTAAAT~GCACTTATTT

1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 GACTTMGGGAGCGACATGAATGAAGTGAAGTGCAGACAGAAGGTAAGGTGACGGAGTGGACTCTCAAAGGACACGACGACGC

1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 AGCGTACGGATCGGCGAGGTAACGGAGGTGTAGGAGCAGATTGATAGAAAGTGAGGGTAACAATTTGAAACTGACAGAAAGAC 2050 TAAAGTTGTTTGGAG FIG. 1. Complete nucleotide sequence of plasmid pHD2. Open reading frames (ORF) A and B and possible Shine-Dalgarno sequences(SD) are indicated. Putative - 10 and -35 sequencesfor the two ORFs are underlined, as is the potential nick site upstream from ORF A. The positions of the Hind111and Clal sites are indicated.

a sequenceupstream from ORF A which had considerable homology, particularly over the immediate region of the nick site, with the

plus origins of the Staphylococcus aureus plasmids pT 181, pC22 1, and pS 194 (Projan and Novick, 1988) (Fig. 2). Plasmids which

CHARACTERIZATION pC223 448 pC221 1234 pT181 116 PHD2 126 pS194 3406 pUEill2 1305

OF A SMALL B. thuringiensis

PLASMID

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CTTAGARAATCACGATTCAGACAATTTTTCTAMACCGGCTTCT'MTAGCCGGTTAGACGCACATTTCGTGTGCACTTCT 359 -------MTMGGATTTAGA~TTT~CT~CCGGTCT'MTAGCCGGTTMGTGGTMTTTTTTTACCACCCCTCAACCAGA 1317 TTTGGAAAATCACGATTTAGACAATTTTTCT~CCGTCT'MTAGCCGGTTGGACGCACATACTGTGTGCATATCTGllT 27 TGTGGGTIIRRTTGTA~T~~GMGACATTTC~CATTCT'MTAGCCGMMTC~GTACAAlUTGACATGTTTAATAAAA 215 MGTTTGGAAAAGGlWUCTCTTTTCTARRACC~ATACTCT'MTAGCCGGTTAMCCGA~TATTATGTACACCCCCGAA 3495 MGCATAGGMGAG~TTCAAATTTTGTTAARACCGG'MTAGCCGGTTMGPGGTCTTT~TCTCMCCCM 1394 ~_~~~_-~~~__/_l_lII II I I_;_; I; 1I I; 1;; I ;___;____111_1/__1___1__111___11__11_ I I III, I________I__I____,((,

FIG. 2. Comparison of replications origins. The sequencesof the replication origins of the pT 181 family of plasmids are aligned with a homologous region of pHD2. Nucleotides in the pHD2 sequencewhich are conserved in one or more pT 181 plasmid family sequencesare indicated by a vertical bar. Nonconserved nucleotides are indicated by a horizontal bar. The position ofthe nick site is indicated by a short bar in the sequence.

have homologous plus origins tend to exhibit homology for their Rep proteins. A database comparison of the amino acid sequence of the polypeptide encoded by ORF A revealed regions of homology with the Rep proteins of pT 181, pS 194, pC22 1, and pC223, an alignment of ORF A with RepC of pTl8 1 is shown in Fig. 3. Although the overall homology between ORF A and these highly conserved Rep proteins is low, there are more highly conserved regions. It is proposed on the basis of these homologies that pHD2 is related, albeit distantly, to this family of S. aureu~ plasmids in terms of its replication mechanisms and that ORF A encodes a Rep protein with site-specific topoisomerase activity for the nick site. In the case of pG12, the

other B. thuringiensis plasmid for which the complete nucleotide sequence is available (Mahillon and Seurinck, 1988), there is an ORF which exhibits sequencehomology with Rep proteins and a casette (Josson et al., 1990) which exhibits homology with the mob regions of pT 181 and pE 194. Comparison of the sequences of the two B. thuringiensis plasmids pGI2 and pHD2 did not reveal significant regions of homology, which is in keeping with the report (Mahillon et al., 1988) that B. thuringiensis strain DIPEL contains plasmids that are related to pGI1 and pG13, but not to pG12. ORF B of pHD2 was found to exhibit considerable homology (Fig. 4) with a small polypeptide, RepA (45 amino acids) encoded by

l-ORFA

62 MQWLDRLMIKYKDVT-----EKQFSDVLTKISSKQIFLPNTPIRSEHGTSVRDYHRVIHIGYGEGA I.. I... .:.:..:I ::: e:.;.;: ; : I...::. *I..:. :: MYKNNHANHSNHLENHDLDNFSKTGYSNSRLDAHTVCISDPKLSFDRMTIVGNLNRDNAQALSKFMSVE---PQIRLWDILQTKFKA 1-RepC 90

63 156 WIGWKHNSEKEKDSYDMKVDFNPSKFENNELQKDSYEKVFKGT~Y .I...,,, ,. III.. 4 . 4lII.I I :: * , , :. . . .,.,,,.I .:I;. . I..: . I,,.... . . . ..I. *I***,!, .,...:;: .: ..:, /.,. WIEYDKVKADSWDRRNMRIEFNPNKLTRDEM-----------IWLKQNIISYMEDDGFTRLDLAFDFEEDLSDYYAMSDKAVKKTIFYG~GK 91 179

157 223 GNRNI(DGYLKIYDKKKELYF~IEEENLTRIEYSWREIRKSPPFSIDESYTFSILI----------------------. ..I.., I., ,.,..:: I., ::. : .I.( 1 I:.: ..*.,,*,,.~ 1: :::. :I. . . I . ..I / .I., G~SNRFIRIYNKKQERKDNADAEVMSEHLWRVEIELKDCFSDLHILQPDWXTIQRTADRAIVFMLLSDEEEWGKLHRNSRTKYKNLI 180 279 ----------------------------------SPVDLTDLMKSTLKANEKQLQKQIDFWQHEFKFWK 280 314 FIG. 3. Comparison of replication proteins. The amino acid sequence predicted by ORF A of pHD2 is aligned by the Gap program with the RepC protein of pT 181. Identities are indicated by a vertical bar and conservative and semiconservative substitutions by double dots and single dots, respectively.

118

MCDOWELL AND MANN ORFB RepA

1 M-VRVNTRISKKLNDWLDEYSKESGVPKSTLVHLALENYVNQKVMLEQMP .,,,/I I, : I:. .:, I f : .I!.:: .,,(,I ... . I:.. : I..: 1 MKKRLTITLSESVLENLEKMAREMGLSKSAMISVALENYKKGQEK-----

49 45

FIG. 4. Comparison of putative replication control proteins. The first 49 amino acid residuesof ORF B of pHD2 are aligned by the Gap program with the complete amino acid sequence of the RepA protein of pLS1. Identities are indicated by a vertical bar and conservative and semiconservative substitutions by double dots and single dots, respectively.

plasmid pLS 1 (Lacks et al., 1986) which was constructed (Stassi et al., 1981) from a Streptococcusagalactiae plasmid pMV 158. RepA is translated from a polycistronic mRNA together with the replication protein RepB and it is suggestedthat RepA may be involved in the control of replication (Puyet et al., 1988). In this context it is worth noting that a related peptide may be encoded by pTl8 1, the preemptor sequenceof which contains a 9-bp repeat of the RepC SD sequence and could serve asthe ribosome binding site for a 46-codon ORF (Projan and Novick, 1988). The amino sequencepredicted by this ORF, however, exhibited only slight homology with those of ORF B of pHD2 and RepA of pLS 1 (data not shown). A characteristic of plasmids of the ssDNA family is that when they are introduced into other hosts, the minus origin of replication is not functional. The plasmid is capable of propagation, though, through nonspecific priming, albeit it at a reduced frequency, leading to the accumulation of ssDNA and causing pronounced segregational instability (Boe et al., 1989; de1Solar et al., 1987; Gruss et $., 1987). In order to provide additional evidence as to whether pHD2 replicates via a single-stranded mechanism, we decided to introduce it into two hosts, B. subtilis and B. thuringiensis var. israelensis(Bti) and to examine its structural and segregational stability. Since pBR322 is not capable of replication in either of these hosts, replication of pDM 100 and pDM200 would have to rely on the Gram-positive plasmid origin; however, in order to be able to identify transformants it would be necessaryto introduce a selectable marker. The chloramphenicol acetyltransferase (CAT) gene of pC194 is bounded by Mspl and Tuql sites. pC 194 was digested

with Mspl and Taq1and ligated with Cla 1-restricted pBR322. Transformation into E. coli DH 1 yielded ampicillin- and chloramphenicol-resistant colonies. A plasmid from one such colony characterized in more detail was shown to have an insert at the Clal site of pBR322, but still retained a single Clal site. From restriction mapping it became clear that the CAT gene in this construct had been generated by partial Taql restriction and still retained an internal Tuql site and that the Taql site involved in the ligation had recreated a Clal site. This plasmid was designated pCC 100 and was used to construct two further plasmids in which pHD2 restricted with Clul or Hind111was cloned into the single Clal site (pCC200) or the single Hind111 site (pCC300) of pCC 100, respectively. Transformants of B. thuringiensis var. israelensis expressing chloramphenicol resistance were obtained at a high frequency using both pCC200 and pCC300. However, no transformants were obtained with B. subtilis, although control transformations with pC 194yielded chloramphenicol-resistant colonies, suggestingthat the plus origin was not functional in B. subtilis. To test the segregational stability of the Bti transformants, one clone was propagated for approximately 100 generations under nonselective conditions in SR broth, during which time no chloramphenicol-sensitive clones were detected. Plasmid DNA was prepared from 12 independent Bti/pCC200 transformants and used to transform E. coli DH 1; chloramphenicolresistant transformants were obtained at high frequency, but no ampicillin-resistant transformants were obtained, suggestingthat deletions may have occurred during replication of B. thuringiensis var. israelensis. When such E. coli transformants were screened,

CHARACTERIZATION

OF A SMALL B. thuringiensis PLASMID

they were all found to be of the same size and had lost the EcoRl site and one Clul site, confirming that they were deleted forms of the original plasmid pCC200. Restriction mapping of the deleted plasmids indicated that in all casesthe deletion covered approximately the same region, running from a site within the pBR322 part of the plasmid through the boundary with the pHD2 component and extending to a site prior to the Hind111site of pHD2. Becauseof the orientation of pHD2 in pCC200, this deletion would not affect ORFA. In conclusion, the 2.055kb plasmid, pHD2, of B. thuringiensis var. kurstaki strain HDl-DIPEL encodes two ORFs, one of which (A) encodes a 26.5kDa polypeptide which may act as a site-specific topoisomerase, involved in plasmid replication, by virtue of regions of homology with topoisomerasesof the pT 181 group of plasmids. The second ORF (B) encodes a 9.1-kDa polypeptide which, over its amino-terminal half, exhibits considerable homology with the RepA polypeptide of pLS1, which is thought to be involved in the control of plasmid replication. pHD2 also contains a putative nick site, related to those of the pT 181 plasmid family, at which the site-specific topoisomerase may operate. On the basis of these homologies, it is suggestedthat pHD2 may be a member of the large family of plasmids from Gram-positive bacteria which replicate via a ssDNA intermediate. ACKNOWLEDGMENTS D.G.M. acknowledges receipt of a SERC-CASE postgraduate studentship. We thank Dr. A. Morby and Dr. M. A. McCrae for their help with the computer analysis of nucleic acid and protein sequences.

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