Temperature-dependent Plasmid Integration into and Excision from the Chromosome of Bacillus stearothermophilus

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1 Journal of General Microbiology (1986), 132, Printed in Great Britain 1951 Temperature-dependent Plasmid Integration into and Excision from the Chromosome of Bacillus stearothermophilus By JUN-ICHI KOIZUMI, ZHANG MIN, TADAYUKI IMANAKA AND SHUICHI AIBA* Department of Fermentation Technology, Faculty of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565, Japan (Received 1 October 1985 ; revised 29 January 1986) A transformant of Bacillus stearothermophilus carrying a recombinant plasmid, plp 1 1 (9.5 MDa), on which the penicillinase gene (penp) and kanamycin resistance gene (kan) were located was subjected to mutagenesis, and a mutant plasmid (9.5 MDa; penp kan), designated ptra 1 17, was obtained. A transformant of B. stearothermophilus carrying ptra 1 17 could grow at 63 "C in medium containing kanamycin, whereas a transformant carrying plp 1 1 could not. Although ptrall7 was detected as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 "C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 "C. If the culture temperature was lowered to 48 "C from 63 "C, a new plasmid (1 0.7 MDa ; penp kan), designated ptrz 1 17, could be detected as ccc DNA; the size of this plasmid suggested that it was ptra117 plus a 1.2 MDa DNA fragment of the host chromosome, and this was confirmed by Southern hybridization. ptrz90 (7.9 MDa; kan) was constructed from ptrzll7 by the deletion of a 2.8 MDa DNA fragment that contained penp. Fresh transformants of B. stearothermophilus that carried either ptrzll7 or ptrz90 could grow at 65 "C. INTRODUCTION It has been reported that the plasmid ptb90, coding for resistance to kanamycin (Km) and tetracycline (Tc), can transform the thermophile Baciffus stearothermophilus and serve as a vehicle for the transfer of foreign DNAs into the micro-organism (Imanaka et al., 1982). The penicillinase (PCase) genes of Bacillus ficheniformis have been cloned into the EcoRI site of ptb90, yielding a recombinant plasmid, plpl1, the markers of which (Kmr Tcr PCase+) were expressed in the B. stearothermophilus transformant strain (Fujii et al., 1982). However, during cultivation of the transformant the copy number and stability of plpll decreased with the increase in culture temperature from 48 "C up to around 60 "C. Continued culture of the transformant was hardly possible at temperatures above 50 "C, primarily due to instability of the plasmid. The transformant could not grow at 63 "C in a medium containing Km, although this temperature was optimal for the growth of the B. stearothermophilus host (Aiba & Koizumi, 1984; Koizumi et al., 1985). These results prompted us to search for a more useful vector plasmid that could be used at higher temperatures. A new plasmid, ptra 1 17, was derived from plpl1 via mutagenesis. ptra 1 17 integrated into the host chromosome at 63 "C, but excision of the integrated plasmid from the chromosome yielded another plasmid, ptrzll7, that could be used at elevated temperatures. The purpose of this work was to study the temperature-dependent integration and excision of the mutant plasmid ptra Abbreviation: PCase, penicillinase SGM

2 1952 J.-I. KOIZUMI AND OTHERS METHODS Mdiu und muteriuls. L medium has been described previously (Aiba & Koizumi, 1984). It was supplemented with 0.25% (w/v) glucose for LG medium, or with Km for LKm medium. The concentration of Km used (pg ml-i) is indicated as a subscript (for example LKm,,,,, contains 100 pg Km ml-i). Solid media contained 2% agar. LPVA agar, used to detect penicillinase-producing colonies, was L medium supplemented with polyvinyl alcohol (0.75%) (Imanaka et a/., 1981h). Unless otherwise specified, all chemicals came from the sources given by Imanaka et ul. (I981 u, 1982). Bucteriu und p1usmid.s. The bacterial strain used was B. stc.crrothermc~phi1u.s CU21 (Smr)(Imanaka et a/., 1982). The plasmids used are listed in Table 1. The restriction endonuclease cleavage maps of the plasmids and the procedure for their construction are shown in Fig. I. Plasmids that are not included in Table 1 but appear in Fig. 1 have been used in previous work (Imanaka et a/., 1981 u, h, 1984; Fujii ef ul., 1982), and they are shown here only to facilitate understanding of the derivation of the various plasmids. Prepurution of' plusmid und chromosomul DNA. Plasmid DNA was prepared by the alkaline lysis method described by Maniatis et ul. ( 1982), and purified by CsCl/ethidium bromide equilibrium density-gradient centrifugation (Maniatis rt ul., 1982) when necessary. Chromosomal DNA was prepared from a sarkosyl lysate of cells as described previously (Koizumi PI ul., 1985), and centrifuged. The DNA fragment used as a probe in Southern hybridization was recovered from low-melting-temperature agarose gel (Maniatis et al., 1982). TrunsJormution. Transformation of B. stearothermophilus protoplasts with plasmid DNA was done as described by Imanaka rt ul. (1982). Transformants were selected on regeneration agar containing Km (25 pg ml-i) and stored on LKmSo,, agar at 4 "C. Transformants of B. steurothermophilusphi1u.s were grown in shaken culture containing 20 ml LKm,,,,, medium. For temperature shift experiments, a 10 to 20 h culture of transformants was inoculated (inoculum size: 1%) into medium at the same temperature and at the temperature required. MutugeniJsis und scrwning procedures for mutunt plusmids. B. steurothrrmclphilus carrying plp 1 1 was grown in LKm,,,,, at 48 "C to mid-exponential phase and was harvested by centrifugation. The cells were suspended in L broth containing 10 pg NTG ml-i and 10 mm-citric acid, and were incubated at 48 "C for 15 min. Fresh L broth (20ml) was added and the cells were harvested by centrifugation, washed twice with cold L broth and resuspended in 50 ml of the same broth. The survival ratio after the mutagenic treatment was about For the initial selection of mutants, I50 pi re-suspended bacteria was spread on LKmSoo agar, and incubated at 60 "C overnight. The total number of cells inoculated on the plates was about 3 x 10'. All colonies that appeared on the first screening plates were transferred to LPVA agar to test for penicillinase-producing (PCase+) colonies. Colonies that did not produce penicillinase were discarded, because a chromosomal mutation might enable the cells to pass the first screening. Inocula from individual PCase+ colonies were checked for growth in LKm,,, broth at 63 C (the third screening); the transformant harbouring plpl1 could not grow in these conditions (Aiba & Koizumi, 1984). Whenever the inoculum could grow, it was assumed that the original colonies, from which the inoculum had been taken, carried mutant plasmids. B. stearothermophilus was transformed with plasmids extracted from these original colonies, which had been cultured in LKm,,, broth at 48 "C, and the screening procedures described above were repeated. Aguroso gel eii~ctr(~phorc.sis. Electrophoresis was done in a horizontal slab gel containing 0.8 % agarose as described by Imanaka et ul. (1981 a). Low-melting-temperature agarose gel containing 1.0% agarose was used for the recovery of DNA fragments; the recovery procedure has been described previously (Imanaka et a/., 1985). Nick trun.htion und Southern hyhritlizution. Nick translated DNA of the probe fragment used in Southern hybridization was prepared by using 32P-labelled deoxycytidine 5'-triphosphate (Amersham) as described by Maniatis et ul. (1982). DNAase I was from Takara Shuzo Co. and E. coli DNA polymerase I from Boehringer. DNA fragments separated by agarose gel electrophoresis were transferred to a nitrocellulose membrane filter (Schleicher & Schull) by Southern blotting (Maniatis et ul., 1982). The filter was preincubated at 65 "C for 2 to 4 h in an aqueous solution containing 6 x SSC (I x SSC: 120 mm- NaCI, 15 mm-sodium citrate, ph 74), 5 x Denhardt's solution (Maniatis et al., 1982), 0.5% SDS and l0opg denatured calf thymus DNA m1-i. Hybridization was done in the above aqueous solution, supplemented with denatured "'P-labelled probe DNA (0.6 to 6 x 10' c.p.m. pg-' ) and 0-01 M-EDTA, for 16 hat 65 "C. The filter was then washed for 3 h with variousconcentrationsof SSC and SDS as described by Maniatisetal. (1982). Finally, the nitrocellulose filter was dried in air and exposed at - 70 "C against Fuji X-ray film (RX). RESULTS AND DISCUSSION Behaviour of' mutant plasmids The 45 strains that passed the third screening for mutant plasmids were subjected to a repetition of the screening procedures (see Methods); only one of the strains passed. This candidate strain was cultured at 48 "C and plasmid DNA was extracted. The plasmid

3 Plasmid integration and excision I953., Fig. 1. A genealogical map of the ptr series of plasmids, ptra117, ptrz117, ptrz90 and ptrz919. Cleavage sites of EcoRI (O), Hind111 (A), BglII (B), PstI (P) and XbaI (X) are indicated. *, Digestion with EcoRI followed by ligation with T4 DNA ligase;., the 2.8 MDa EcoRI fragment that containspenppeni- (for pttel l)orpenppeni+ (for ptte21) (Imanaka etal., 1981 b); the 2.9 MDa EcoRI fragment; B, the 1.2 MDa DNA fragment; 0, the 1.0 MDa EcoRI fragment; H, the EcoRI fragment that carries the Tc' determinant. Plasmid ptb90 plpl1 ptral I7 ptrz 1 17 ptrz90 ptrz9 19 pttb42 ptb9 13 Table 1. Plasmids used Relevant characteristics* kan Tc' kan penp peni Tc' kan penp peni Tc' Itc kan penp peni Tc' Etc kan Tc' Etc kan Etc kan penp peni+ Tc' kan Reference Imanaka et al. (1982) Fujii et al. (1982) This work This work This work This work Imanaka et al. (1981 6) Imanaka et al. (1984) * kan, Structural gene of kanamycin nucleotidyltransferase, responsible for resistance to kanamycin (Km') (Matsumura et al., 1984) ; penp, structural gene of penicillinase; penl, inactivated regulator gene of penicillinase; peni+, intact regulator gene of penicillinase (ptte21 in Fig. 1); Tc', resistance to tetracycline; Itc, integrative at elevated temperature into chromosome ; Etc, excised from thermophile chromosome. (designated ptra 1 17) had the same size and cleavage map as plp 1 1 (Fig. 1 ; Fig. 4, lanes A and C). B. stearothermophilus carrying either plpl1 or ptb90 could not grow in LKm,,, broth at 63 "C, but carrying ptra 1 17 it could (Table 2). The elevated upper growth-temperature of the transformant (63 "C) was apparently due to the plasmid, because no specific treatments were

4 1954 J.-I. KOIZUMI AND OTHERS Fig. 2. Agarose gel electrophoresis of plasmid DNA. Lane A, mutant plasmid ptra117 from a transformant of B. srearothermophilus cultured at 48 "C in LKm,,o medium; lane B, the same culture at 63 "C (no plasmid could be detected); lane C, a new plasmid, ptrzll7, which emerged when the culture temperature was shifted from 63 to 48 "C. Table 2. Growth of' B. stearotherrnophilus transformants in LKmloo medium at various temperatures The host strain B. stearothermophilus CU21 could grow at 67 "C in L broth without Km. Data for ptrz919 were omitted. +, Growth (an increase of OD660 after several hours or overnight culture); -, no growth (no increase of ODbbo after overnight culture). Plasmid 48 "C 55 "C 59 "C 63 "C 65 "C 67 "C plpl1 ptb90 + ptra I 17 -I- ptrzl17 ptrz imposed on the host strain other than the transformation procedure. Accordingly, ptrall7 was defined as a mutant plasmid of plpl1. The mutant plasmid ptra 117 could not be detected by agarose gel electrophoresis after alkaline extraction of DNA from the transformant of B. stearotherrnophilus cultured at 63 "C. However, plasmid DNA could be detected in an extract from the transformant when cultured at 48 "C (Fig. 2, lanes B and A). At 63 "C, all of the transformants retained the original phenotype (Kmr PCase+) despite the apparent disappearance of ptra117, on which kan andpenp genes were encoded. When the transformants cultured in LKmloo broth at 63 "C were transferred to LG medium at 63 "C, there was no loss of Kmr, even after several generations in the absence of Km as selective pressure. A B. stearothermophilus strain carrying ptra117 was grown at 63 "C in LKmIoo broth and was used to inoculate fresh LKm,,, medium incubated at 48 "C. Electrophoresis of plasmid DNA prepared from the culture at 48 "C showed a new and larger plasmid (designated ptrzll7) (Fig. 2, lane C). The appearance of an extrachromosomal element as ccc DNA was triggered by a shift-down of culture temperature from 63 to 48 "C, and was also observed after a shift from 63 to 54 "C (data not shown). When the temperature was shifted alternately up and down between 48 and 63 C in a subsequent inoculation of B. stearothermophilus carrying ptrzll7 into LKm,,, medium, no new plasmids other than ptrzll7 could be detected. Plasmid ptrzll7 could be detected as ccc DNA throughout this cycle of alternating temperature change. However, if B. stearothermophilus lacking ptrzll7 was transformed with ptrzll7, the plasmid disappeared when the culture was first shifted up to 63 "C; shift-down of the culture to 48 "C was followed by the reappearance of the unchanged ptrzll7. Restriction endonuclease analysis of ptrzll7

5 Plasmid integration and excision 1955 showed that a 1.2 MDa DNA fragment of unknown origin and with single Hind111 site had inserted into the 2.9 MDa EcoRI fragment of ptrall7, yielding a new 4.1 MDa EcoRI fragment; no other changes in cleavage sites were found between ptrall7 and ptrzll7 (Fig. 1). ptrz90 and ptrz919 (Fig. 1) were derivatives of ptrzll7 that lacked the 2.8 MDa EcoRI fragment containing penicillinase genes penp and penl, and were obtained by digestion of ptrzll7 with EcoRI, ligation with T4 ligase and transformation of B. stearothermophilus with the ligation mixture, selecting for Kmr PCase- transformants. Table 2 compares the growth at various temperatures of B. stearothermophilus harbouring the mutant plasmids derived in this study. When transformed with ptrzll7, the strain could grow at higher temperatures than when it harboured either plpl1 or ptrall7 (Table 2). At 59 "C the transformant harbouring the original plasmid, plpl1, required more than 10 h before the OD660 of the culture broth reached 0.6, while transformants harbouring ptrall7 or ptrzll7 required less than 5 h. The generation time of the transformant harbouring plpl1 was about 3-4 h, while those of transformants harbouring ptrall7 or ptrzll7 were about 0.6 h. A difference in growth rate at 59 "C was also observed between transformants carrying ptb90 and ptrz90; the generation time for the former was about 1.6 h, and for the latter about 0-6 h. Southern hybridization The above results suggested that the mutant plasmid ptrall7 was integrated into the chromosome at 63 "C and excised from it at 48 "C, yielding another mutant plasmid, ptrzll7. This possibility was tested by Southern hybridization. ptrall7 and ptrzll7 were extracted from the transformants that had been cultured in LKm,,, medium at 48 "C. The plasmids were digested with PstI, (Fig. 3, lanes A and B), transferred to a nitrocellulose membrane by Southern blotting and hybridized to 2P-labelled pttb42 DNA (Fig. 3, lanes a and b). Plasmid pttb42 (Fig. 1) was used as a probe since it lacks the 1.0 MDa EcoRI fragment of ptrall7 and ptrzll7, which is known to hybridize to the host chromosome (Imanaka et al., 1984). The use of pttb42 was justified by the absence of hybridization to the plasmid-free host chromosome (Fig. 3, lanes e and f). As expected, pttb42 (probe) hybridized with both ptrall7 and ptrzll7 (Fig. 3, lanes a and b), because all three plasmids had homologous DNA fragments (Fig. 1). Chromosomal DNA was extracted from transformants carrying ptra 1 17 in LKm, oo broth at 63 "C, and from the plasmid-free host cells in L broth at 63 "C, and was digested with XbaI and PstI. After electrophoresis and Southern hybridization, a thick band of hybridization was observed with DNA from transformants carrying ptra117 (Fig. 3, lanes c and d; band indicated with open triangle). It was inferred that the thick band was due to ptrzll7 existing as non-ccc DNA, because the band had an almost identical mobility in electrophoresis to that of ptrz117 digested with PstI (Fig. 3, lane b). A slight difference in mobility between the thick band in lane c and that in lane d (or in lane b) might be due to the difference in migration pattern of the chromosomal DNA digest as background (Fig. 3, lanes C and D). The probe also hybridized to other, smaller fragments of DNA from transformants carrying ptrall7 (Fig. 3, lanes c and d; bands indicated with filled triangles). The mobilities of these additional bands differed from that of ptrall7 linearized with PstI (Fig. 3, lane a), which suggested that ptrall7 had integrated into the host chromosome at 63 "C. If ptra117 had integrated into the chromosome by flanking homology involving the 1.0 MDa EcoRI fragment of ptrall7 that had a single XbaI site and homology with the chromosome (Fig. 3b), then digestion of the product with XbaI should give a DNA fragment that hybridized to the probe and was larger than ptra117. Since the observed bands were smaller than ptrall7, the integration may have been homology-independent (Fig. 3 b). The origin of the 1.2 MDa DNA fragment that was contained within the 4.1 MDa EcoRI fragment of ptrzll7 (Fig. 4b) was examined by Southern hybridization. ptrall7 and plpl1 were double digested with EcoRI and BgIII and the chromosome of plasmid-free B. stearothermophihs was digested with PstI. The fragments were electrophoresed on an agarose gel (Fig. 4, lanes A, B and C), transferred to a nitrocellulose membrane filter by Southern blotting

6 1956 J.-I. KOIZUMI AND OTHERS (b) Homology-independent integration x i / I Integration HK B W 1 Digestion with Xbal x x C hrcmosome A Flanking homology integration Fig. 3. (a) Agarose gel electrophoresis and Southern hybridization. Plasmid and chromosomal DNA were digested with restriction endonucleases and electrophoresed on an agarose gel. Lane A, ptrall7 digested with PstI ; lane B, ptrzll7 digested with PstI ; lane C, XbaI digest of the chromosome from B. srearothermophilus carrying ptrall7 and cultured at 63 C; lane D, PstI digest of the same chromosome as used in lane C; lane E, XbaI digest of the chromosome from plasmid-free B. stearothermophilus; lane F, PstI digest of the same chromosome as used in lane E; lane L, I cz857 S7 digested with HindIII. The blotted gel was hybridized (lanes a to f) to 32P-labelled pttb42 DNA (Fig. 1). (Lanes a to f correspond to lanes A to F.) (6) Schematic diagram of ptral17. 0, The 1.0 MDa DNA fragment; H, the rest of the plasmid, which ought to hybridize to the probe, pttb42 (the use of this symbol here is different from that in Fig. 1). X, Cleavage site of XbaI; P, cleavage site of PstI. Schemes for homology-dependent and homology-independent integration of ptra 1 17 into the chromosome are shown. With homologyindependent integration, two XbaI fragments should hybridize to pttb42, whereas with integration through flanking homology, only one XbaI fragment should hybridize. and hybridized to the 32P-labelled 4.1 MDa EcoRI fragment of ptrzll7 (Fig. 4, lanes a, b and c) and to 32P-labelled plasmid ptb913 (Fig. 4, lanes a, b and c ). The 4.1 MDa probe fragment was prepared from an EcoRI digest of ptrz919 separated on a low-melting-temperature agarose gel, while ptb913 was prepared by in vivo deletion (Fig. 1).

7 Plasmid integration and excision 1957 Fig. 4. Examination of the origin of the 1.2 MDa fragment in ptrzll7. (a) Agarose gel electrophoresis and Southern hybridization. EcoRI and BglII double digests of ptrall7 (lane A) and plpl1 (lane C), a PstI digest of chromosomal DNA from B. stearothermophilus (lane B) and a &indiii digest of L c1857 S7 (lane L) were electrophoresed on an agarose gel. The double digests of ptrall7 and plpl1 yielded eight DNA fragments. Six of the fragments are numbered in order of size in (a) and (b). The two smallest fragments were hard to detect. The blotted gel was hybridized to a 4.1 MDa EcoRI fragment from ptrz919 (lanes a, b and c) and to ptb913 (lanes a, b and c ). (b) Diagrammatic interpretation of the results. M, EcoRI-BgiII fragments 2 and 6 of ptrall7 and plpl1, which hybridized with the 4.1 MDa fragment of ptrz919 and with ptb913; B, the 1.2 MDa fragment; 4, strong hybridization ;--A, weak hybridization. The PstI digest of the chromosome of plasmid-free host strain hybridized at five locations to the 4.1 MDa fragment (Fig. 4, lane b), which was further evidence that the 1.2 MDa DNA fragment of ptrzll7 had originated from the host chromosome. This evidence was supported by the absence of hybridization with probe ptb913, which was used as control and which lacked the 1.2 MDa DNA fragment (Fig. 4, lane b ). This is the first report of temperature-dependent integration of a plasmid into, and excision of a plasmid from, the host chromosome. Hence, the mutant plasmids ptrall7, ptrzll7 and ptrz90 obtained in this study are of significance; ptrall7 is integrated into the chromosome

8 1958 J.-I. KOIZUMI AND OTHERS of B. stearothermophilus on temperature shift-up, whereas ptrzll7, the product of the excision, is maintained stably in the host strain at 65 C. Further molecular and genetic studies are needed on the location and mode of integration of ptra117, and on the importance of the chromosomal DNA fragment carried by ptrzll7. REFERENCES AIBA, S. & KOIZUMI, J.-I. (1984). Effects of temperature on plasmid stability and penicillinase productivity in a transformant of Bacillus stearothermophilus. Biotechnology and Bioengineering 26, FUJII, M., IMANAKA, T. & AIBA, S. (1982). Molecular cloning and expression of penicillinase genes from Bacillus lichentformis in the thermophile Bacillus stearothermophilus. Journal of General Microbiology 128, IMANAKA, T., FUJII, M. & AIBA, S. (1981a). Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids. Journal of Bacteriology 146, IMANAKA, T., TANAKA, T., TSUNEKAWA, H. &AIBA, S. (1981 b). Cloning of the genes for penicillinase, penp and penl, of Bacillus lichenijormis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus lichenifbrmis. Journal of Bacteriology 147, IMANAKA, T., FUJII, M., ARAMORI, I. & AIBA, S. (1982). Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. Journal of Bacteriology 149, IMANAKA, T., ANO, T., FUJII, M. & AIBA, S. (1984). Two replication determinants of an antibioticresistance plasmid, ptbl9, from a thermophilic bacillus. Journal of ceneral Microbiology 130, IMANAKA, T., HIMENO, T. & AIBA, S. (1985). Effect of in oitro DNA rearrangement in the NHz-terminal region of the penicillinase gene from Bacillus lichenijormis on the mode of expression in Bacillus subtilis. Journal of General Microbiology 131, KOIZUMI, J.-I., MONDEN, Y. & AIBA, S. (1985). Effects of temperature and dilution rate on the copy number of recombinant plasmid in continuous culture of Bacillus stearothermophilus (plp 11 ). Biotechnology and Bioengineering 27, MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning : a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. MATSUMURA, M., KATAKURA, Y., IMANAKA, T. &AIBA, S. (1984). Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid publ10. Journal of Bacteriology 160,