Vol. 139, No. 3

JOURNAL OF BACTERIOLOGY, Sept. 1979, p. 775-782 002 1-9193/79/09-0775-08$02.00/0

recE4-Independent Recombination Between Homologous Deoxyribonucleic Acid Segments of Bacillus subtilis Plasmids TERUO TANAKA Laboratory of Microbial Chemistry, Mitsubishi-Kasei Institute of Life Sciences, Minamiooya 11, Machidashi, Tokyo, Japan Received for publication 25 June 1979

A plasmid (pLS104) carrying a tandem repetition of the leu region of the Bacillus subtilis chromosome arose spontaneously from pLS103, which carried a single copy of the leu region. Plasmid preparations from strains harboring pLS104 also contained the original plasmid, pLS103, and, in some preparations, plasmids carrying three or four repetitions of the leu region. These plasmids were shown to be generated by recombination between homologous deoxyribonucleic acid (DNA) segments in the tandemly repeated DNA regions on the plasmids, but not by recombinations between specific DNA sites. These phenomena were observed in a recE4 background, showing that recombination of the homologous DNA sequences does not require the recE gene product(s).

It has been reported that in Bacillus subtilis transformation, transformants are not obtained when the recipient cells carry the recE4 mutation (3). This phenomenon was attributed to the fact that the transforming DNA is not integrated into the host chromosomal DNA (3). This character is important for the construction of recombinant DNA molecules which contain sequences homologous to those of the host chromosomal DNA, since the presence of the recE4 mutation in the recipient ensures autonomous replication of the plasmids which would otherwise be integrated into the host chromosome. Thus, trp and leu genes of B. subtilis 168 have been cloned in the homologous strain carrying the recE4 mutation (6, 13). Keggins et al. (5) reported that the recE4 gene product is necessary for recombination between homologous DNA segments cloned on two compatible plasmids in the same cell. This result suggested that at least intermolecular recombination does not take place in the presence of the recE4 mutation. During the study of plasmids harboring the leucine gene region of B. subtilis in recE4 cells (13), a plasmid arose spontaneously which carried tandemly repeated leu gene regions. This plasmid exhibited recombination giving rise to various size classes of plasmids, depending on the extent of repetition of the leu gene region. Previously we have reported that a plasmid (pLS102) consisting of a B. subtilis (B. natto) plasmid and two EcoRI fragments of B. subtilis DNA carrying the leu gene region in tandem frequently recombined in a recE4 background to generate a smaller plasmid (pLS103) which had

a single EcoRI fragment carrying the leu region

(13). These observations in recE4 bacteria are in contrast to that of Keggins et al. described above, and they seemed to be useful for further characterization of the recE4 mutation. In this paper, evidence is presented that this recE4-independent recombination can take place between homologous DNA regions but that it does not involve any specific DNA sequences, such as those involved in the amplification of antibiotic resistance genes in certain R factors (4, 9, 10, 18). MATERIALS AND METHODS Materials. Restriction endonucleases and preparation of plasmids have been described previously (12). B. subtilis strains used are listed in Table 1. Molecular weight determination. Molecular weights of the plasmids were determined by either agarose gel electrophoresis or electron microscopy. For agarose gel analysis, the supercoiled forms of the following plasmids were used for molecular weight standards: ColEl (4.2 x 106 [2]), RSF1O10 (5.5 x 106 [14]), RSF2124 (7.3 x 106 [11]), pLS102 (10.7 x 106 [13]), RSF101O trp (16.3 x 10' [7]), and RSF2124 trp (18.1 x 106 [7]). For electron microscopy, RSF1010 was used as a molecular weight standard. Molecular weights of the DNA fragments obtained by digestion with restriction endonucleases were determined by co-electrophoresis with EcoRI or HindIlI fragments of bacteriophage A DNA (1, 15). Analysis of plasmids in agarose gels. Lysates (0.4 ml) were made from cells grown in 10 ml of AA medium (13), and the nucleic acids were precipitated by 2 volumes of ethanol. The precipitate was dissolved in 0.12 ml of TES buffer (13) containing 0.4% Sarkosyl and incubated for 20 min at 37°C after the addition of 5 j.g of RNase I (Sigma). Samples (30,l) were electro-

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TABLE 1. B. subtilis strains tuse(d Sfitraill

l)estriptioll

)escriptionl StraiD

RM125 MI112

Mu 15

arg-15 leuA8 rM- mu arg- 15 icuA8 thr-5 recE4 rm m aorg-15 leuiAS rccE4 r mM

Source (reference)

Uozumi (16) This laboratory" (13) This laboratory

(13) Spizizen (17) lcuB6 tr)C2 BRt44 Zahler (17) leuB 110 trpC2 C tT269 Zahler (17) leuCl13 trp C2 CU272 Zahler (17) leuC7 trpC2 C1U741 T'he presence of recE4 was confirmed by susceptibility to mitomycin C. Back mutation to rec+ was less than 2 x 10 9. MI112 and M1115 were not transformed to Leu+ by using DNA from B. subtilis 168 thy trpC.2 under the condition where 10' Leu+ transformaints of RM125 were obtained. in 0.7°; agarose gels (14). Construction of pLS108, pLS110, and pLS111. pLS108 was constructed by digestion of pLS107 with EcoRI* (8) and by ligation with T4 DNA ligase. For pLSl 10 and pLS1 11, a BarmNI fragment (see Fig. 6) of pLS102 (13) was isolated from agarose gels as previously described (14) and added to the BamNI digest of pLS108 or pLS204, and the mixture was treated with T4 DNA ligase (14). Construction of pLS110 or pLSI1 was carried out in B. subtilis M1112 or MI115 cells, respectively, and the cells harboring those plasmids were found by screening large colonies. This is

phoresed

the leu region in tandem [3]) produced large colonies when transformed into M1112 and that preparations of pLS102 were always accompanied by pLS103 (13). These results suggested that pLS 104 carried homologous DNA sequences in duplicate in its molecule and that it was converted to pLS103 by in vivo recombination. To test whether this was the case, physical mapping of pLS104 was first carried out. The molecular weight of pLS104 was estimated by agarose gel electrophoresis (9.1 x 106) and electron microscopy [(9.2 ± 0.2) x 106]. pLS104 was cleaved by BamNI, SmaI, and HpaI twice, with the cleavage sites of each restriction enzyme being separated by 2.6 x 106 daltons, and the relative positions of the cleavage sites of the three enzymes were found to be identical (data not shown) with those on pLS103 (see Fig. 3a). The cleavage products of each restriction enzyme had molecular weights of 2.6 x 106 and 6.5 x 10', the sum of which was in agreement with the molecular of weight pLS104. pLS103 and pLS104 were digested with HindlIl and separated by agarose gel electrophoresis as shown in Fig. 2. The digestion patterns of pLS103 and pLS104 were identical except that an extra band (A') was present in pLS104 above band A (Fig. 2b) and that fluorescences of bands D and I were strong for their molecular weight. Quantitation by densitometric tracing of the negative showed that approximately two molar equivalents of

based on the fact that the repetition of the leu genes on plasmids gives rise to large colonies, as shown in pLS102-carrying cells (13), and on the assumption that pLSI 10 and pLSII I should carry the intact leuA and leuiC genes respectively, in duplicate, since pLS 108 and pLS204 contain the leuA and leuC gene functions, respectively (data not shown).

RESULTS Characterization of pLS104, which carries the leu region in duplicate. During routine preparation of pLS103 from M1112, a plasmid (pLS104) larger than pLS103 (6.5 x 106 daltons [13]) was found to be present in one preparation of pLS 103 (Fig. la). Transformation of M1112 with this plasmid preparation produced two size classes of colonies. From the smaller colonies pLS103 was recovered, whereas the larger colonies carried pLS104 and, in addition, a small amount of pLS103 (Fig. lb). Purified pLS104 when transformed into M1112 produced large colonies from which both pLS104 and pLS 103 were again recovered (the same pattern as that in Fig. lb), showing that pLS104 must have recombined in a recE4 strain to generate pLS103. These observations were similar to the previous finding that pLS102 (a plasmid which carries two EcoRI fragments containing

FI(G 1. Agarose gel electrophoresis of pLS103 and pLSJ04. A plasmid (pLSI04) was found in a plasmid preparation obtained fronm MI112 cells carrying pLS103. (a) Plasmid preparation consisting of pLSJ03 and a small amount of pLS104; (b) plasmid pr-epar-ation from a larger colony obtained after transformation of M1WI112 with the plasmid preparation shown in (a).

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again the fluorescences of the cleavage products were strong for their molecular weights (data not shown). These results suggested that in pLS104, the D and I fragments were present in duplicate. When pLS104 was cleaved with both HindIII and HpaI, bands A' and C disappeared and three new DNA bands (1.26 x 106, 0.63 X 106, and 0.47 x 106 daltons) were generated. The smallest DNA fragment was present in two molar equivalents as estimated by densitometric analysis, and the 0.63 x 106- and 0.47 x 106dalton fragments were identical to two new DNA fragments which were found to be generated by HpaI cleavage of fragment C (data not shown). From these results, it was concluded that fragment A' was composed of 1.26 x 106- and 0.47 x 106-dalton fragments. Also, these results suggested that part of fragment C was present in fragment A'. pLS104 was next cleaved by double digestion with EcoRI and BamNI, and the resulting three DNA fragments (2.6 x 106, 2.3 x 106, and 1.9 x 106 daltons) were isolated from agarose gels (two molar equivalents of DNA were present in the region corresponding to the molecular weight of 2.3 x 106). When the three fragments were further digested with HindIII, it was found that the 2.6 x 106-dalton segment contained fragments D and A'; the 2.3 x 106_ dalton segment contained fragments D, C, G, and A; and the 1.9 x 106-dalton segment conFIG. 2. HindIII cleavage patterns of pLS103 (a) tained fragments B, F, and J. From these results and pLS104 (b). Data in parentheses indicate the size a physical map of pLS104 was constructed (Fig. of each fragment (in megadaltons). The figure is a 3b). composite photo of the weak and strong portions of Biological tests were performed on the DNA the gels. fragments. Both of the two BamNI fragments of

DNA fragments per plasmid were present in these bands. Fragments D and I were cleaved once by SmaI and BamNI, respectively, and

EI

a

b

FIG. 3. Restriction maps ofpLS103 (a) and pLS104 (b). The molecular weights ofpLS103 and pLS104 are 6.5 x 10i and 9.1 x 16P, respectively. HindIII cleavage sites are shown in the inner circles. The arrow in the map of pLS104 shows a repeating DNA segment (2.6 x 10" daltons). HindIII-A' is composed of parts of the HindIII B and C fragments (designated by AB and AC, respectively). The fused point (designated by the dotted line on the inner circle in [b]) is to the left of the HpaI site in fragment A' but is not located exactly. Molecular weights of the HindII fragments are giuen in parentheses in Fig. 2.

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pLS104 isolated from agarose gels had the ability of transforming leuA8, leuB6, leuBZ10, leuC7, and leuC113 markers (Table 2), indicating that the DNA regions corresponding to these markers were repeated in pLS104. Furthermore, the following results were obtained by transformation (data not shown): leuA8+, leuB6+, leuC7+, and leuC113+ were localized in the region between the BamNI and the Hpal sites (the region including segment D); using isolated HindlIl fragments, leuB110+ and leuC113+ were localized on fragments B and C, respectively, and both of these markers were found to be present in fragment A', indicating that fragment A' was made by fusion of fragments B and C. From these physical and biological tests, it was concluded that the DNA region (2.6 x 106 daltons) encompassing the BamNI, SmiaI, and HpaI sites and also including the leu markers described above was repeated tandemly in pLS104 and that the repeating segment extended from the fused point in fragment A' to either fragment B or fragment C (Fig. 3b). Generation of plasmids which carry three or four leu gene regions. During purification of an MI112 cell carrying pLS104, 2 out of 40 colonies were found to carry a larger plasmid (pLS 105) in addition to pLS 104. When these cells were spread on AA plates (13) and the lysates of 18 colonies were electrophoresed in agarose gels, it was found that 10 colonies harbored pLS104, 7 colonies harbored both pLS105 and pLS104, and 1 colony harbored an even

J. B3AC TFRIOL

larger plasmid (pLS106) in addition to pLS104 and pLS105. Representatives of each group are shown in Fig. 4. The molecular weights of pLS105 and pLS1O6 were approximately 11.7 x 10' and 14.3 x 10'3, respectively, as deternined by agarose gel electrophoresis. The molecular weights obtained by electron microscopy were (12.1 ± 0.3) x 10' and (14.7 ± 0.2) x 106 for pLS105 and pLSl06, respectively. The difference in molecular weight between pLS104 and pLS105 was 2.6 x 10, which corresponds to the difference between the molecular weights of pLS103 and pLS104. Digestion of pLS105 with BamnNI, iSmaI, HpaI, and HindIII gave a pattern identical with that of pLS104 (data not shown). Purified pLS1(5 from agarose gels gave rise to large colonies of M1112 from which either pLS105 accompanied by pLS104 and pLS103 or pLS 104 accompanied by pLS 103 were recovered, showing that pLS105 was converted to pLS104 and pLS103 by in vivo recombination. Compilation of these results led to the conclusion that pLS 105 was generated by the addition to pLS 104 of the 2.6 x 10'-dalton fragment described above. By a similar p)rocedure, a preparation consisting of pLS106, pLS105, pLS104, and pLS103 was digested with the restriction enzymes, and pLS106 was found to be a plasmid in which four 2.6 x 10-dalton fragments are repeated tandemly. The relationship between the plasmids described in this paper and the earlier paper (13) is shown in Fig. 5. Cells carrying pLS105 were susceptible to mitomycin C and could not be transformed to Arg'

TABLE 2. Transformation of leueine miarkers of B. subtilis rec' strains uwith the BanzNI fragments of pLS104 isolated from agarose gels"

" pLSI04 was cleaved with BamNI and electrophoresed in 0.77%r agarose gels. Gel slices (2 mm long) were cut out from two DNA regions (I and III) and from the middle (II) of each of the two bands. Transformation with these preparations was carried out as described previously (13). Electrophoresis is from right to left.

Fic. 4. Agarose gel electrophoresis of lysates pr-epored firom pLSl 04-c arryinig cells. Cells carrying pLSJ04 were spread on an AA plate, and lysates were maIie fromrz 18 colonies grown in the A4 medium (1.3). Thrl ee tYpical patterns mIre shown.

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pLS 1 05 (11 .7) FIG. 5. Relationship between plasmids generated by recombination in B. subtilis recE4 cells. The repeating segments carrying the leu region are depicted by the internal arcs. The arrows indicate the direction of recombination. The molecular weight (x10-t) of each plasmid is shown in parentheses.

Thr+ (data not shown), showing that the characteristics of the recE4 mutation (3) were preserved. The mutation rate of MI112 cells to mitomycin C resistance was less than 2 x 10-9, whereas cells carrying pLS105 appeared at a frequency of 2/40 among pLS104-carrying cells as described above. These observations demonstrate that generation of plasmids carrying various numbers of repetitions of the leu region is not due to reversion of recE4 to rec+. Recombination takes place between homologous DNA sequences, but not at specific sites on the plasmids. Generation of the various plasmids depicted in Fig. 5 could be explained by one of two mechanisms. One is general recombination which occurs at homologous DNA regions on the plasmids, and the other is site-speciflc recombination involving special DNA sequences which flank the region to be amplified. The latter phenomenon has been reported for the amplification of drug resistance genes on plasmids in gram-negative bac-

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teria (4, 9, 10) and Streptococcus faecalis (18). If the site-specific recombination takes place in pLS104, it should occur in the HindIll B and C fragments, since the ends of the repeating DNA segment were in these fragments (Fig. 3b). On the other hand, although pLS102 is also converted to pLS103 by in vivo recombination (Fig. 5) (13), any other plasmids which might have lost one of the repeating units have not been found in pLS102 preparations by using agarose gel electrophoresis (13) or electron microscopy. This excludes the possibility that the repeating segment is flanked at both ends by special DNA sequences which might cause sitespecific recombination and thus amplify the leu gene region. However, there still could have been a special DNA sequence at one end involved in recombination. To test this possibility, plasmids (pLS110 and pLS111) which carried only one end of the repeating segment in duplicate were constructed (Fig. 6). pLS108 (Fig. 6) was constructed by EcoRI* digestion of pLS107, ligation by T4-ligase, and subsequent transformation into MI112. Most (75%) of the HindlIl C fragment, which carried the HpaI site, was lost in pLS108 (Fig. 7a). Since the repeating segment contained the Hpal site, as described above (also shown in Fig. 3b), one end of the segment should have been lost in pLS108. In pLS204 (Fig. 6), there was no HindIlI B fragment (Fig. 7b), indicating that the other end of the repeating segment was lost in this plasmid. The addition of the BamNI fragment of pLS102 to the BamNI sites of pLS108 and pLS204 led to the construction of pLS110 and pLS111 (Fig. 6), in which the fragments B and C were repeated twice, respectively, in a tandem repetition. When plasmids were prepared from cells harboring these plasmids, it was found that pLS110 and pLS111 were accompanied by smaller plasmids (Fig. 8) which were indistinguishable from pLS108 and pLS204, respectively, as shown by digestion with restriction enzymes (data not shown). These results indicate that recombination took place between homologous DNA sequences in each plasmid and exclude the possibility that recombination involved specific DNA sequences which might have been present at both ends of the repeating unit. DISCUSSION Cloning of DNA segments homologous to the chromosomal DNA in B. subtilis has been carried out by use of recE4 mutant cells (6, 13). Recombination between homologous DNA sequences was not detected irrespective of whether they are on separate plasmids in the same cell (6) or on the chromosomal DNA and on a plas-

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