Molec. gen. Genet. 162, 229-235 (1978) © by Springer-Verlag 1978
Genetic Defects in DNA Repair System and Enhancement of Intergenote Transformation Efficiency in Bacillus subtilis Marburg Kouji Matsumoto, Hideo Takahashi, Hiuga Saito, and Y6nosuke Ikeda Institute of Applied Microbiology,Tokyo University, Bunkyo-ku,Tokyo 113, Japan
Summary. Mechanisms of inefficiency in heterospecies transformation were studied with a transformation system consisting of Bacillus subtilis 168TI (trpC2 thy) as recipient and of D N A prepared from partially hybrid strains of B. subtilis which had incorporated trp + D N A of B.amyloIiquefaciens 203 (formerly, B.megaterium 203) in the chromosome (termed intergenote). The intergenote transformation was not so efficient as the corresponding homospecies transformation and the efficiency appeared to relate inversely with the length of heterologous portion in the intergenote. When a variety of ultraviolet light (UV) sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients for the inl;ergenote transformation, 2 out of 16 mutants exhibited significantly enhanced transformation efficiency of the trpC marker. Genetic studies by transformation showed that the trait relating to the enhancemen! of intergenote-transformation efficiency was always associated with the UV sensitivity, suggesting that these two traits are determined by a single gene. The efficiency of intergenote transformation was highly affected also by D N A concentration; the lower the concentration, the less the efficiency. When, however, the UV sensitive mutant was used as recipient, the effect of D N A concentration was largely diminished, suggesting the reduction of DNA-inactivating activity in the UV sensitive recipient. These results were discussed in relation to a possible excision-repair system selectively correcting the mismatched D N A in in the course of intergenote transformation.
et al., 1963; Ikeda et al., 1965). When D N A of heterologous portion which has been integrated previously in the chromosome of B. subtilis (termed intergenote, after Wilson and Young, 1972) is used to transform B.subtilis, the efficiency is much improved but still lower than that of the corresponding homospecies transformation. We assumed that mismatched D N A formed in the course of intergenote transformation might be corrected by yet unidentified mechanisms, similar to the excision-repair system postulated by Ephrussi-Taylor and Gray (1966) in Pneumococcus transformation. In Pneumococcus there are two types of point mutation: those transformed with low efficiency and those transformed with high efficiency. These authors formulated the hypothesis that there is a selective excision-repair system which acts on heteroduplexes involving donor strands of low efficiency markers. Studies have been undertaken on the assumption that the selective correction of the intergenote strands in heteroduplexes may be a cause of the inefficiency of intergenote transformation. In the present paper, we describe the effect of mutations in genes determining ultraviolet light (UV) sensitivity. The efficiency of intergenote transformation was enhanced when UV sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients.
Materials and Methods Bacterial Strains. Strains of Bacillus subtilis Marburg and B.amyloliquefaciens 203 (formerly, B.megaterium 203) used are listed in
Introduction Transformation of Bacillus subtilis by D N A of heterologous origin is usually very inefficient (Marmur Address offprint requests to: Dr. H. Saito
Table 1. Construction ofIntergenote. A strain 15 (trpB3) of B.subtilis Marburg was transformed to tryptophan independence by DNA of B.amyloliquefaciens 203 and several transformant colonies were isolated (Saito and Matsumoto, unpunished data). These isolates have integrated the heterologous portion around trp loci in the
0026-8925/78/162/0229/$01.40
K. Matsumoto et al. : D N A Repair and Intergenote Transformation in B. subtilis
230 Table 1. Bacterial strains Strain
Genotype
Origin or reference
B. subtilis
Marburg
SfrSmr 168TI 168TIM UVSI0TI UVSIOT UVSIOTIM UVS 10TA UVSI9TIM UVS80TI KE1 KE9 KE181 KE511 RSL 101
sul strA4 thy trpC2 thy trpC2 metl4 sul uvrAlO thy trpC2 uvrAlO thy sul uvrAlO thy trpC2 met14 sul uvrA 10 thy purB6 uvrA19 thy trpC2 metl4 sul recSO thy trpC2 uvrA1 thy trpC2 sul uvrA9 thy trpC2 sul uvrAlO thy trpC2 hisA1 metl4 sul strA4 uvrA 10 thy trp C2 argl 5 sul strA4 rfm strA leuA 111 purB6 leuA 8 metB5
Mutation from Marburg Strain Originally given by F. Rothman Munakata (1970) Mutation of 168TI; Munakata (1970) Transformation of UVS10TI Munakata (1970) Munakata (1970) Munakata (1970) Mutation of 168TI; Munakata (1970) Transformation of 168TIM by D N A of GSY1027 a Transformation of 168TIM by D N A of her9 b Transformation of UVS10TIM by D N A of S7CH ° Transformation of UVS10TIM by D N A of S7CH c This laboratory This laboratory
B. amyloliquefaciens (formerly, B. megaterium) 203 203lys
lys
This laboratory
Intergenote strain Z5 H1 H5 T5
trp + (203, trp + (203, trp + (203, trp + (203,
Z5) H1) H5) T5)
This This This This
laboratory laboratory laboratory laboratory
Other strains listed in Table 2 are all derived by mutation of 168TI (Munakata 1970). Gene symbols: thy, trp, met, pur, his, arg, leu, and lys, are dependence, for growth, of thymine, tryptophan, methionine, adenine, histidine, arginine, leucine, and lysine, respectively. Symbols, sul, 1fro, and str, are resistance to sulfanyl amide, rifamycin or rifampicin, and streptomycin, respectively. Symbols, uvr and rec are ultraviolet light (UV) sensitivity and recombination deficiency, respectively. Phenotypes are shown by corresponding gene symbols with capitals replacing the first letters. Mit' (mitomycin C-insensitive) phenotype is determined by uvr + a b c
Given by H. Tanooka, National Cancer Center Research Institute, Tokyo Given by S. Okubo, Osaka University This laboratory
chromosome, which is termed as intergenote according to Wilson and Young (1972). The intergenote is designated, for example, trp + (203, Z5); the original and the hybrid strain numbers are shown in parenthesis. The longest hybrid portion, trp + (203, Z5), among various intergenote tested, appeared to cover at least several cistrons around trpC locus (Matsumoto and Saito, unpublished data).
DNA Preparation. D N A of intergenote strains was prepared by pH9-phenol method (Saito and Miura, 1963) with particular cautions, since the efficiency of intergenote transformation was quite variable according to differences in preparation. The preparation procedures included a step of RNase digestion between twice-repeated phenol treatment. The purified D N A sample was stored at 0 ° C and used within one month. When transformation was applied to make a new combination of markers, a crude D N A preparation dialyzed after the first phenol extraction was used for transformation.
Transformation Procedure. Recipient strains of B.subtilis Marburg were made competent by the method described by Bott and Wilson (1967) with minor modifications. At two and a half hours after cessation of logarithmic growth in Bott's competence medium, cells were collected and concentrated to 1/10 volume in the medium containing 15% glycerol to stock in liquid nitrogen. For use as recipient, the frozen cells were thawed at 37°C and diluted with 9 volumes of fresh Bott's competence medium. Maximal competence was usually attained between 60 and 120 min during incubation period at 37 ° C. The competent cells were incubated, unless otherwise stated, with 1 gg/ml of DNA for 30 rain and plated on selective media. Duplicate plates were usually used for scoring. The frequency of intergenote transformation (Trp +) was normalized by the transformation frequency of an unlinked homologous marker (mainly Thy+). This value, Trp+/Thy +, is termed as marker ratio.
Results Media. Spizizen's minimal medium was used as a basal medium for selection of prototrophic characters (Saito and Miura, 1963). Transformation of ultraviolet light (UV) sensitive to insensitive trait was selected and tested on nutrient agar containing 0.05 gg mitomycin C per ml. For selection of Str r trait, nutrient agar containing 500 gg streptomycin sulfate per ml was used.
Intergenote Transformation with V a r i o u s U V S e n s i t i v e R e c i p i e n t s Auxotrophic markers of B.subtilis 168TI (uvr + t h y t r p C 2 ) a n d a l s o o f v a r i o u s U V s e n s i t i v e m u -
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
231
Table 2. Effect of uvr mutations on the efficiency of intergenote transformation
Table 3. Intergenote transformation by DNA of strain Z5 with uvr + transformants of strain UVS10TIM as recipients
Donor
Recipient
Recipient Strain
(B.sub.)
168TI
Number of transformants Genotype a x 10-3/ml
uvr +
Trp +
Thy +
380
260
Trp +
168TI
uvr +
KEI KE9 UVS10TI
uvrA 1 uvrA9 uvrAlO
UVS18TI UVS19TI UVS39TI UVS42TI UVS 100TI UVS102TI UVS191TI
uvrA18 uvrA19 uvrA39 uvrA42 uvrA 100 uvrAlO uvrA191
UVS46TI UVS105TI UVS108TI UVS 109TI UVSII4TI
uvrB46 uvrB10 uvrBl08 uvrBl09 uvrBI14
UVS 142TI uvr142 UVS80TI rec80 a
3400 330 110
0.11 0.05 0.07
40 240 31 24 9.8 2.0 8.0 2}. 460 18 9.0
380 450 350 85 53 160 72 100 240 4200 130 97
0.11 0.10 0.69 0.37 0.45 0.06 0.03 0.08 0.09 0.11 0.14 0.09
4.0 1.6 5.2 4.4 72 60 2.7
54 3I 41 93 210 230 14
0.07 0.05 0.13 0.05 0.34 0.26 0.19
1.4 0.71
Trp +
Trp +
Trp +
Thy +
Met +
Thy +
Met +
1.46
370 15 '7.5 43
Marker ratio
Thy +
SfrSmr (Intergenote) Z5:trp + (203, Z5)
Number of transformants x 10- 3/ml
28 0.05 6.5 0.11
Other genotypes are thy trpC2 for all recipients
tants ( u v r t h y trpC2) were t r a n s f o r m e d by D N A of the intergenote strain Z5 ( t h y + t r p + (203, Z5)) (Table 2). The efficiency of intergenote t r a n s f o r m a t i o n with u v r + recipient, expressed as ratio of ]-rp + (intergenote marker) to T h y + ( h o m o l o g o u s marker) was 0.05-0.11, which was significantly lower t h a n the corr e s p o n d i n g m a r k e r ratio of h o m o l o g o u s t r a n s f o r m a tion, 1.46. A m o n g U V sensitive m u t a n t s , m a n y were similar to u v r + recipients, whereas U V S 1 0 T I a n d U V S 1 1 4 T I exhibited e n h a n c e d efficiency of the intergenote transf o r m a t i o n . The t r a n s f o r m a t i o n experiments were usually repeated more t h a n twice a n d a part of the results are s h o w n as triplicate data in T a b l e 2. Alt h o u g h the t r a n s f o r m a t i o n efficiency with the same cross varied considerably, the e n h a n c e m e n t of intergen o t e - t r a n s f o r m a t i o n efficiency by the two u v r m u t a tions was obvious. The f l u c t u a t i o n was due m a i n l y to v a r i a t i o n in the c o m p e t e n c e developed in recipient cells.
Parents : 168TIM UVS10TIM
44 42
1300 110
2100 250
0.03 0.38
0.02 0.17
uvr + transformants :" W-10 W-16 W-19 W-21 W-23
16 16 11 14 6
330 320 440 460 230
590 550 640 600 380
0.05 0.05 0.03 0.03 0.03
0.03 0.03 0.02 0.02 0.02
a uvr + transformants were selected as mitomycin C-insensitive transformants (Mitr)
Genetic Relation of the uvrAlO
Mutation
to t h e E n h a n c e m e n t
of Intergenote- Transformation
Efficiency
The u v r A l O m u t a t i o n in the strain U V S 1 0 T I M was t r a n s f o r m e d to u v r + by D N A of 168TI at 0.05 gg/ml. U n d e r these conditions, t r a n s f o r m a t i o n of single genes is p r e d o m i n a n t a n d multiple t r a n s f o r m a t i o n is rare. The u v r + t r a n s f o r m a n t s were selected on plates c o n t a i n i n g m i t o m y c i n C a n d then the isolates were tested for U V sensitivity. I n t e r g e n o t e t r a n s f o r m a t i o n by D N A of strain Z5 (trp + (203, Z5)) was p e r f o r m e d with the five u v r + isolates ( t h y trpC2 m e t l 4 ) as recipients (Table 3). All exhibited the i n t e r g e n o t e - t r a n s f o r m a t i o n efficiency similar to that of the p a r e n t a l strain 168TI, indicating a close relation between u v r A l O a n d the e n h a n c e m e n t . Table 3 includes also t r a n s f o r m a t i o n frequencies of a n o t h e r h o m o l o g o u s marker, Met + . The m a r k e r ratio, T r p + / M e t +, also indicates the inefficiency of intergenote t r a n s f o r m a t i o n , as does the ratio, T r p + / Thy ÷ . Reciprocally to the cross described above, the strain 168TIM was t r a n s f o r m e d to U V sensitive by D N A of strain KE181 or KE511 ( s t r A 4 u v r A l O ) . Since direct selection of U V sensitive t r a n s f o r m a n t s was difficult, d o u b l e t r a n s f o r m a t i o n of s t r A a n d u v r A was carried out with D N A at a s a t u r a t i n g c o n c e n t r a tion (5 gg/ml). U n d e r these conditions, d o u b l e transf o r m a t i o n of two u n l i n k e d m a r k e r s occurs frequently. T h u s Str r t r a n s f o r m a n t s were p r i m a r i l y selected a n d a m o n g these isolates, U V sensitive t r a n s f o r m a n t s were detected as m i t o m y c i n sensitive (Mit s) clones.
232
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
Table 4. Intergenote transformation by DNA of strain Z5 with
Table 5. Efficiency of transformation by various intergenote DNA
uvrAlO transformants of strain 168 TIM as recipients Donor Recipient
Parents : 168TIM UVS10TIM
Recipient
b/a
Number of transformants x 10 3/ml
Marker ratio
168TIM
Trp +
Trp ÷
Ttp +
Thy +
Met +
Number of transformants x 10 ~/ml
Number of (a) transformants Trp + x 10 3/ml
(b) Trp +
Trp + Thy +
Thy + Trp + Thy +
Thy +
0.02 0.18
0.01 0.09
380
260
1.5
200
97
2.1
1.4
250 160 140 160
0.39 0.22 0.14 0.04
120 85 20 21
94 51 23 53
1.3 1.7 0.87 0.40
3.3 7.7 6.2 10
14 13
Thy +
570 74
Met +
1100 150
B. sub tiffs ." SfrSmr
uvrAlO transformants (I):" S-2 40 180 S-5 4.5 28 S-11 120 780 S-12 150 580 S-13 90 340 S-14 79 330
1400 1300 760 710
0.22 0.16 0.15 0.26 0.26 0.24
0.09 0.12 0.12 0.11
uvrA 10 transformants (II) :a S-3 60 230 S-4 180 560 S-22 96 430 S-23 53 140 S-39 120 320 S-40 130 560
660 930 840 380 870 980
0.26 0.32 0.22 0.38 0.38 0.23
0.09 0.19 0.11 0.14 0.14 0.13
a uvrA10 donors were strains KE181 for (I) and KE511 for (II). uvrA 10 transformants were obtained as double transformants, Mit t Strr; the latter was used for primary selection
Intergenote: T5 H5 H1 Z5
97 35 19 5.8
168 i
10 10
~10
10 s o
I n t e r g e n o t e t r a n s f o r m a t i o n o f the U V sensitive isolates as r e c i p i e n t by D N A o f s t r a i n Z5 is s h o w n in T a b l e 4. I n t h e s e cases also, t h e e n h a n c e m e n t o f interg e n o t e - t r a n s f o r m a t i o n f r e q u e n c y was f o u n d to be c l o s e l y a s s o c i a t e d w i t h t h e U V sensitivity. E x a m i n a t i o n o f uvr + r e v e r t a n t s f r o m t h e s t r a i n U V S 1 0 , if t h e r e v e r s i o n o c c u r r e d , w o u l d o f f e r a g o o d e v i d e n c e for the g e n e t i c i d e n t i t y o f t h e t w o traits. W h e n , h o w e v e r , m o r e t h a n 1011 cells o f U V S 1 0 T I M were plated on the medium containing mitomycin C, n o r e v e r t a n t c o l o n y a p p e a r e d e v e n a f t e r i n c u b a t i o n for s e v e r a l days. T h e f a i l u r e to f i n d r e v e r s i o n o f t h e U V sensitivity o f U V S 1 0 T I M , leaves a q u e s t i o n w h e t h e r the t r a i t is a c t u a l l y d e t e r m i n e d by a single g e n e o r not. T h e t r a n s f o r m a t i o n f r e q u e n c i e s o f u v r A l O to uvr + a n d o f the r e c i p r o c a l c r o s s w e r e likely to be t h o s e o f single events, b u t f u r t h e r e x a m i n e d as f o l l o w s . T h e s t r a i n U V S 1 0 T I M was t r a n s f o r m e d w i t h D N A o f R S L at the s a t u r a t i n g c o n c e n t r a t i o n o f D N A (5 g g / m l ) . P r i m a r i l y M e t + t r a n s f o r m a n t s were selected and then they were tested for the unselected m a r k e r s : T h y +, T r p + a n d M i t r ( m i t o m y c i n C insensitivity). T h e f r e q u e n c i e s o f d o u b l e t r a n s f o r mants among Met + isolates: Thy+Met+/Met+, Trp+Met+/Met + and MitrMet+/Met + were 4.3%, 5 . 2 % a n d 2 . 3 % , r e s p e c t i v e l y . S i n c e T h y ÷, M e t ÷ a n d
UVS10TIM
_
$19
I
I
$80
I
i
01
gy0 x.-
i---
10"
0.01
'
0.1
'
0.01 0.1 1 10 H1 DNA / u g / m [
1
10
Fig. 1. Transformation frequency of homologous marker and intergenote marker. Transformation frequency of homologous marker (Thy+: - - o - - ) and intergenote marker (Trp+: o--) plotted as a function of DNA concentration. Donor was strain HI: tip + (203, H1). Recipients were 168TIM (uvr + rec+), uvsl0TIM (uvrAlO), UVS19TIM (uvrA19), and UVSSOTI (recSO), indicated in the figures as 168, S10, S19, and $80, respectively
T r p + t r a n s f o r m a t i o n has b e e n k n o w n to be single events, t h e s i m i l a r i t y o f f r e q u e n c i e s suggests t h a t every trait is d e t e r m i n e d by a single gene.
T r a n s f o r m a t i o n by D N A C o m p r i s i n g o f Various L e n g t h o f H e t e r o l o g o u s R e g i o n Intergenote DNA prepared from different clones of p r i m a r y t r a n s f o r m a n t s f r o m the h e t e r o s p e c i e s t r a n s -
K. Matsumoto et al. : D N A Repair and lntergenote Transformation in B. subtilis
0.5 0.4 ...~0.3 ~0.2 v O
,.0.1 x..
2~
//
0.05 I
0.01
I
I
I
0.1 1 10 Z5 DNA ,,ug/m[
Fig. 2. Dependence of the marker ratio on D N A concentration and on the recipient strain. D o n o r was strain Z5: trp + (203, Z5). Recipients were 168TIM (uvr+): - - o and UVS10TIM (uvrA10): --0--
formation was examined for transformation efficiency (Table 5). The individual intergenote D N A exhibited unique efficiency with 168TIM as recipient. The difference in frequencies appeared to reflect the length of heterologous portion in the intergenotes. Transformation experiments with closely linked markers suggested that the lower the frequency the longer the heterologous portion (Saito and Matsumoto, unpublished results). With UVS10TIM as recipient the transformation efficiecies of intergenote D N A were enhanced and the magnitude of enhancement (column b/a in Table 5) appeared to be related to the length of the heterologous portion in the intergenote.
Effect of DNA Concentration on the Efficiency of Intergenote Transformation Transformation of both intergenote and homologous markers was examined at various D N A concentra-
233
tions with 168TI and three UV sensitive mutants as recipient (Fig. 1). The transformation frequency increased with D N A concentration but exhibited saturation at almost definite concentration level, except for the case of UVS80TI recipient. The strain UVS80TI requires a much higher concentration of D N A for saturation than other recipients. This phenomenon appears to be a common feature of recombination deficient mutants ofB.subtilis (Hoch et al., 1967), and suggests the enhanced inactivation of transforming D N A as a result of defects in Rec functions. Figure 2 illustrates the marker ratio of intergenote marker (Trp +) to homologous marker (Thy +) plotted as a function of D N A concentration. Difference between the recipients, 168TIM and UVS10TIM, is obvious. With 168TIM as recipient, the marker ratio is markedly reduced with decrease of D N A concentration, whereas UVS10TIM exhibits fairly constant marker ratio at every concentration tested. These results suggest the presence of a DNA-inactivating function which acts especially on the intergenote transformation; the uvrAlO mutation may diminish this function.
Effect of uvrAlO Mutation on Heterospecies Transjormation In contrast to the intergenote transformation, when D N A of B.amyloIiquefaciens 203 was used to transform UVS10TIM, transformation frequency to Trp ÷ was not enhanced by uvrA10 at all (Table 6). Another marker purB6 was also examined, since this marker was known to be transformed by the heterologous D N A with appreciably higher frequency comparable to the intergenote transformation of trp marker (Saito et al., 1973). Although we expected the enhancement of Pur + transformation frequency with uvrAlO recipient, the results were again negative (Table 6) suggest-
Table 6. Effect of uvrAlO on the heterospecies transformation by D N A of B. amyloliquejaciens 203 Donor
Number of transformants/0.1 mI Trp +
Thy *
Put +
Recipient a
SfrSmr (B. subtilis) 203lys (B. amyloliquefaciens 203) ~
Trp +
Thy +
Pur +
Recipient a
168TI M
I01
UVS 10TI M
UVS 10TA
(uvr +trpC2 thy)
(uvr +purB6)
(uvrA 10 trp C2 thy)
(uvrA 10 purB6)
35,000 6
210,000 8,100
56,000 4
210,000 2,600
Relevant genotypes are shown in parentheses
26,000 1
30,000 1
234
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
ing some difference between heterospecies transformation and intergenote transformation.
Discussion Intergenote strains can be constructed by integration of the heterospecies D N A segment into B.subtilis chromosome. When such intergenote D N A was used to transform B.subtilis, recombinant D N A having a mismatched region between double stranded portions would be formed. If the donor D N A strand of the mismatched region were preferentially corrected, it would result in the inefficiency of intergenote transformation as observed in this study. In transformation of Pneumococcus, a selective excision repair system was postulated to act on low efficiency markers (Ephrussi-Taylor and Gray, 1966). Guild and Shoemaker (1974) reported that competition by nonisogenic but homologous D N A leads to a rise in the number of transformants of the low efficiency marker, suggesting the similarity in recognition of nonisogenic D N A and of low-efficiency marker DNA. A mechanism which asymmetrically corrects a mismatched region was found in B.subtilis by transfection of heteroduplex SPP1 D N A constructed in vitro with wild and mutant phage (Spatz and Trautner, 1970). The similar correction mechanism was posturated in transformation system of B.subtilis to account for the formation of pure clones of transformants on nonselective media, because, if heteroduplex D N A persisted, not pure but mixed clones would be formed (Bresler et al., 1971), The data presented in this paper suggest the presence of a correction mechanism for intergenote transformation. A m o n g sixteen UV sensitive mutants tested, two exhibited enhanced efficiencies of intergenote transformation. The mutations, uvrAlO and uvrB114, relating to the enhancement, locate at two unlinked loci concerning with the host-cell reactivation (Munakata, 1977). Thus it is likely that the correction of the mismatched region may be carried out by a function related to excision repair of UV damage in DNA. However, the genetic relationship is yet uncertain since other uvr mutations including extensively deleted mutations, uvrA191 and uvrBl09, have no effect on the enhancement. In Escherichia coli, Nevers and Spatz (1975) reported that the correction of heteroduplex D N A of phage lambda was deficient in uvrD and uvrE mutants. The frequency of heterospecies transformation was not enhanced by the uvrA10 mutation. Since the efficiency of heterospecies transformation is generally more than two orders lower than that of intergenote transformation, an additional mechanism may be
concerned with the former inefficiency. Although host-controlled restriction had been suspected to operate in the heterospecies transformation, Trautner et al. (1974) reported that a restriction-modification system of B.subtilis R, which was active on both phages and transducing DNA, was inert for transforming DNA. This result seems to be reasonable, since transforming D N A integrates as a single strand into the host chromosome already modified. Thus the inefficiency of heterospecies transformation may be due mainly to the low efficiency in pairing of D N A strands. In heterospecies transformation of Pneumococcus by Streptococcus D N A , the transformation frequency rised with D N A concentration increasing up to a higher concentration level than the saturation level of homospecies transformation (Chen and Ravin, 1966). The shift of saturation concentration was explained as a result of internal inactivation of heterospecies DNA. Effect of D N A concentration on the marker ratio of intergenote to homologous markers, as described in the present paper, suggests the selective inactivation ofintergenote DNA, resulting in the inefficiency as observed. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Japan.
References Bott, K.F., Wilson, G.A. : Development of competence in Bacillus subtilis transformation system. J. Bact. 94, 562-570 (1967) Bresler, S.E., Kreneva, R.A., Kushev, V.V.: Molecular heterozygores in Bacillus subtilis and their correction. Molec. gen Genet. 113, 204-213 (1971) Chert, K.C., Ravin, A.W. : Heterospecifictransformation in Pneumococcus and Streptococcus. I. Relative efficiency and specificity of DNA helping effect. J. molec. Biol. 22, 109-121 (1966) Ephrussi-Taylor, H., Gray, T.C.: Genetic studies of recombining DNA in pneumococcaltransformation. J. gen. Physiol. 49, Part 2, 211-231 (1966) Guild, W.R., Shoemaker, N.B.: Intracellular competition for a mismatch recognition system and marker-specific rescue of transforming DNA from inactivation by ultraviolet irradiation. Molec. gen. Genet. 128, 291-300 (1974) Hoch, J., Barat, M., Anagnostopoulos, C.: Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J. Bact. 93, 1925-1937 (1967) Ikeda, Y., Saito, H., Miura, K., Takagi, J., Aoki, H. : DNA base composition, susceptibility to bacteriophages and interspecific transformation as a criteria for classification in the genus Bacillus. J. gen. appl. Microbiol. 11, 181-190 (1965) Marmur, J., Seaman, E., Levine, J.: Interspecific transformation in Bacillus. J. Bact. 85, 461-467 (1963) Munakata, N. : Repair of ultraviolet-induced damage in transforming DNA of Bacillus subtilis. Jap. J. Genet. 45, 1-9 (1970) Munakata, N.: Mapping of the genes controlling excision repair of pyrimidin photoproducts in Bacillus subtilis. Molec. gen. Genet. 156, 49-54 (1977)
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis Nevers, P., Spatz, H.Ch. : Escherichia coli mutants uvrD and uw'E deficient in gene conversion of 2-heteroduplexes. Molec. gen. Genet. 139, 233 243 (1975) Saito, H., Miura, K.: Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. biophys. Acta (Amst.) 72, 619 629 (1963) Saito, H., Takahashi, H., Ikeda, Y. : Gene conservation in Bacillus subtilis. In: Genetics of industrial microorganisms, Vol. 1 (Van~k, Z., Ho~t'filek, Z., Cudliu, J., eds.), pp. 89-94. Prague: Academia 1973 Spatz, H.Ch., Trautner, A.: One way to do experiments on gene conversion? Transfection with heteroduplex SPP1 DNA. Molec. gen. Genet. 109, 84 106 (1970)
235
Trautner, T.A., Pawlek, B., Bron, S., Anagnostopoulos, C.: Restriction and modificaton in Bacillus subtilis. Molec. gen. Genet. 131, 181 191 (1974) Wilson, G.A., Young, F.E. : Intergenote transformation of Bacillus subtilis genospecies. J. Bact. 111, 705 716 (1972)
Communicated
by T.Yura
Received December 20, 1977
Genetic Defects in DNA Repair System and Enhancement of Intergenote Transformation Efficiency in Bacillus subtilis Marburg Kouji Matsumoto, Hideo Takahashi, Hiuga Saito, and Y6nosuke Ikeda Institute of Applied Microbiology,Tokyo University, Bunkyo-ku,Tokyo 113, Japan
Summary. Mechanisms of inefficiency in heterospecies transformation were studied with a transformation system consisting of Bacillus subtilis 168TI (trpC2 thy) as recipient and of D N A prepared from partially hybrid strains of B. subtilis which had incorporated trp + D N A of B.amyloIiquefaciens 203 (formerly, B.megaterium 203) in the chromosome (termed intergenote). The intergenote transformation was not so efficient as the corresponding homospecies transformation and the efficiency appeared to relate inversely with the length of heterologous portion in the intergenote. When a variety of ultraviolet light (UV) sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients for the inl;ergenote transformation, 2 out of 16 mutants exhibited significantly enhanced transformation efficiency of the trpC marker. Genetic studies by transformation showed that the trait relating to the enhancemen! of intergenote-transformation efficiency was always associated with the UV sensitivity, suggesting that these two traits are determined by a single gene. The efficiency of intergenote transformation was highly affected also by D N A concentration; the lower the concentration, the less the efficiency. When, however, the UV sensitive mutant was used as recipient, the effect of D N A concentration was largely diminished, suggesting the reduction of DNA-inactivating activity in the UV sensitive recipient. These results were discussed in relation to a possible excision-repair system selectively correcting the mismatched D N A in in the course of intergenote transformation.
et al., 1963; Ikeda et al., 1965). When D N A of heterologous portion which has been integrated previously in the chromosome of B. subtilis (termed intergenote, after Wilson and Young, 1972) is used to transform B.subtilis, the efficiency is much improved but still lower than that of the corresponding homospecies transformation. We assumed that mismatched D N A formed in the course of intergenote transformation might be corrected by yet unidentified mechanisms, similar to the excision-repair system postulated by Ephrussi-Taylor and Gray (1966) in Pneumococcus transformation. In Pneumococcus there are two types of point mutation: those transformed with low efficiency and those transformed with high efficiency. These authors formulated the hypothesis that there is a selective excision-repair system which acts on heteroduplexes involving donor strands of low efficiency markers. Studies have been undertaken on the assumption that the selective correction of the intergenote strands in heteroduplexes may be a cause of the inefficiency of intergenote transformation. In the present paper, we describe the effect of mutations in genes determining ultraviolet light (UV) sensitivity. The efficiency of intergenote transformation was enhanced when UV sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients.
Materials and Methods Bacterial Strains. Strains of Bacillus subtilis Marburg and B.amyloliquefaciens 203 (formerly, B.megaterium 203) used are listed in
Introduction Transformation of Bacillus subtilis by D N A of heterologous origin is usually very inefficient (Marmur Address offprint requests to: Dr. H. Saito
Table 1. Construction ofIntergenote. A strain 15 (trpB3) of B.subtilis Marburg was transformed to tryptophan independence by DNA of B.amyloliquefaciens 203 and several transformant colonies were isolated (Saito and Matsumoto, unpunished data). These isolates have integrated the heterologous portion around trp loci in the
0026-8925/78/162/0229/$01.40
K. Matsumoto et al. : D N A Repair and Intergenote Transformation in B. subtilis
230 Table 1. Bacterial strains Strain
Genotype
Origin or reference
B. subtilis
Marburg
SfrSmr 168TI 168TIM UVSI0TI UVSIOT UVSIOTIM UVS 10TA UVSI9TIM UVS80TI KE1 KE9 KE181 KE511 RSL 101
sul strA4 thy trpC2 thy trpC2 metl4 sul uvrAlO thy trpC2 uvrAlO thy sul uvrAlO thy trpC2 met14 sul uvrA 10 thy purB6 uvrA19 thy trpC2 metl4 sul recSO thy trpC2 uvrA1 thy trpC2 sul uvrA9 thy trpC2 sul uvrAlO thy trpC2 hisA1 metl4 sul strA4 uvrA 10 thy trp C2 argl 5 sul strA4 rfm strA leuA 111 purB6 leuA 8 metB5
Mutation from Marburg Strain Originally given by F. Rothman Munakata (1970) Mutation of 168TI; Munakata (1970) Transformation of UVS10TI Munakata (1970) Munakata (1970) Munakata (1970) Mutation of 168TI; Munakata (1970) Transformation of 168TIM by D N A of GSY1027 a Transformation of 168TIM by D N A of her9 b Transformation of UVS10TIM by D N A of S7CH ° Transformation of UVS10TIM by D N A of S7CH c This laboratory This laboratory
B. amyloliquefaciens (formerly, B. megaterium) 203 203lys
lys
This laboratory
Intergenote strain Z5 H1 H5 T5
trp + (203, trp + (203, trp + (203, trp + (203,
Z5) H1) H5) T5)
This This This This
laboratory laboratory laboratory laboratory
Other strains listed in Table 2 are all derived by mutation of 168TI (Munakata 1970). Gene symbols: thy, trp, met, pur, his, arg, leu, and lys, are dependence, for growth, of thymine, tryptophan, methionine, adenine, histidine, arginine, leucine, and lysine, respectively. Symbols, sul, 1fro, and str, are resistance to sulfanyl amide, rifamycin or rifampicin, and streptomycin, respectively. Symbols, uvr and rec are ultraviolet light (UV) sensitivity and recombination deficiency, respectively. Phenotypes are shown by corresponding gene symbols with capitals replacing the first letters. Mit' (mitomycin C-insensitive) phenotype is determined by uvr + a b c
Given by H. Tanooka, National Cancer Center Research Institute, Tokyo Given by S. Okubo, Osaka University This laboratory
chromosome, which is termed as intergenote according to Wilson and Young (1972). The intergenote is designated, for example, trp + (203, Z5); the original and the hybrid strain numbers are shown in parenthesis. The longest hybrid portion, trp + (203, Z5), among various intergenote tested, appeared to cover at least several cistrons around trpC locus (Matsumoto and Saito, unpublished data).
DNA Preparation. D N A of intergenote strains was prepared by pH9-phenol method (Saito and Miura, 1963) with particular cautions, since the efficiency of intergenote transformation was quite variable according to differences in preparation. The preparation procedures included a step of RNase digestion between twice-repeated phenol treatment. The purified D N A sample was stored at 0 ° C and used within one month. When transformation was applied to make a new combination of markers, a crude D N A preparation dialyzed after the first phenol extraction was used for transformation.
Transformation Procedure. Recipient strains of B.subtilis Marburg were made competent by the method described by Bott and Wilson (1967) with minor modifications. At two and a half hours after cessation of logarithmic growth in Bott's competence medium, cells were collected and concentrated to 1/10 volume in the medium containing 15% glycerol to stock in liquid nitrogen. For use as recipient, the frozen cells were thawed at 37°C and diluted with 9 volumes of fresh Bott's competence medium. Maximal competence was usually attained between 60 and 120 min during incubation period at 37 ° C. The competent cells were incubated, unless otherwise stated, with 1 gg/ml of DNA for 30 rain and plated on selective media. Duplicate plates were usually used for scoring. The frequency of intergenote transformation (Trp +) was normalized by the transformation frequency of an unlinked homologous marker (mainly Thy+). This value, Trp+/Thy +, is termed as marker ratio.
Results Media. Spizizen's minimal medium was used as a basal medium for selection of prototrophic characters (Saito and Miura, 1963). Transformation of ultraviolet light (UV) sensitive to insensitive trait was selected and tested on nutrient agar containing 0.05 gg mitomycin C per ml. For selection of Str r trait, nutrient agar containing 500 gg streptomycin sulfate per ml was used.
Intergenote Transformation with V a r i o u s U V S e n s i t i v e R e c i p i e n t s Auxotrophic markers of B.subtilis 168TI (uvr + t h y t r p C 2 ) a n d a l s o o f v a r i o u s U V s e n s i t i v e m u -
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
231
Table 2. Effect of uvr mutations on the efficiency of intergenote transformation
Table 3. Intergenote transformation by DNA of strain Z5 with uvr + transformants of strain UVS10TIM as recipients
Donor
Recipient
Recipient Strain
(B.sub.)
168TI
Number of transformants Genotype a x 10-3/ml
uvr +
Trp +
Thy +
380
260
Trp +
168TI
uvr +
KEI KE9 UVS10TI
uvrA 1 uvrA9 uvrAlO
UVS18TI UVS19TI UVS39TI UVS42TI UVS 100TI UVS102TI UVS191TI
uvrA18 uvrA19 uvrA39 uvrA42 uvrA 100 uvrAlO uvrA191
UVS46TI UVS105TI UVS108TI UVS 109TI UVSII4TI
uvrB46 uvrB10 uvrBl08 uvrBl09 uvrBI14
UVS 142TI uvr142 UVS80TI rec80 a
3400 330 110
0.11 0.05 0.07
40 240 31 24 9.8 2.0 8.0 2}. 460 18 9.0
380 450 350 85 53 160 72 100 240 4200 130 97
0.11 0.10 0.69 0.37 0.45 0.06 0.03 0.08 0.09 0.11 0.14 0.09
4.0 1.6 5.2 4.4 72 60 2.7
54 3I 41 93 210 230 14
0.07 0.05 0.13 0.05 0.34 0.26 0.19
1.4 0.71
Trp +
Trp +
Trp +
Thy +
Met +
Thy +
Met +
1.46
370 15 '7.5 43
Marker ratio
Thy +
SfrSmr (Intergenote) Z5:trp + (203, Z5)
Number of transformants x 10- 3/ml
28 0.05 6.5 0.11
Other genotypes are thy trpC2 for all recipients
tants ( u v r t h y trpC2) were t r a n s f o r m e d by D N A of the intergenote strain Z5 ( t h y + t r p + (203, Z5)) (Table 2). The efficiency of intergenote t r a n s f o r m a t i o n with u v r + recipient, expressed as ratio of ]-rp + (intergenote marker) to T h y + ( h o m o l o g o u s marker) was 0.05-0.11, which was significantly lower t h a n the corr e s p o n d i n g m a r k e r ratio of h o m o l o g o u s t r a n s f o r m a tion, 1.46. A m o n g U V sensitive m u t a n t s , m a n y were similar to u v r + recipients, whereas U V S 1 0 T I a n d U V S 1 1 4 T I exhibited e n h a n c e d efficiency of the intergenote transf o r m a t i o n . The t r a n s f o r m a t i o n experiments were usually repeated more t h a n twice a n d a part of the results are s h o w n as triplicate data in T a b l e 2. Alt h o u g h the t r a n s f o r m a t i o n efficiency with the same cross varied considerably, the e n h a n c e m e n t of intergen o t e - t r a n s f o r m a t i o n efficiency by the two u v r m u t a tions was obvious. The f l u c t u a t i o n was due m a i n l y to v a r i a t i o n in the c o m p e t e n c e developed in recipient cells.
Parents : 168TIM UVS10TIM
44 42
1300 110
2100 250
0.03 0.38
0.02 0.17
uvr + transformants :" W-10 W-16 W-19 W-21 W-23
16 16 11 14 6
330 320 440 460 230
590 550 640 600 380
0.05 0.05 0.03 0.03 0.03
0.03 0.03 0.02 0.02 0.02
a uvr + transformants were selected as mitomycin C-insensitive transformants (Mitr)
Genetic Relation of the uvrAlO
Mutation
to t h e E n h a n c e m e n t
of Intergenote- Transformation
Efficiency
The u v r A l O m u t a t i o n in the strain U V S 1 0 T I M was t r a n s f o r m e d to u v r + by D N A of 168TI at 0.05 gg/ml. U n d e r these conditions, t r a n s f o r m a t i o n of single genes is p r e d o m i n a n t a n d multiple t r a n s f o r m a t i o n is rare. The u v r + t r a n s f o r m a n t s were selected on plates c o n t a i n i n g m i t o m y c i n C a n d then the isolates were tested for U V sensitivity. I n t e r g e n o t e t r a n s f o r m a t i o n by D N A of strain Z5 (trp + (203, Z5)) was p e r f o r m e d with the five u v r + isolates ( t h y trpC2 m e t l 4 ) as recipients (Table 3). All exhibited the i n t e r g e n o t e - t r a n s f o r m a t i o n efficiency similar to that of the p a r e n t a l strain 168TI, indicating a close relation between u v r A l O a n d the e n h a n c e m e n t . Table 3 includes also t r a n s f o r m a t i o n frequencies of a n o t h e r h o m o l o g o u s marker, Met + . The m a r k e r ratio, T r p + / M e t +, also indicates the inefficiency of intergenote t r a n s f o r m a t i o n , as does the ratio, T r p + / Thy ÷ . Reciprocally to the cross described above, the strain 168TIM was t r a n s f o r m e d to U V sensitive by D N A of strain KE181 or KE511 ( s t r A 4 u v r A l O ) . Since direct selection of U V sensitive t r a n s f o r m a n t s was difficult, d o u b l e t r a n s f o r m a t i o n of s t r A a n d u v r A was carried out with D N A at a s a t u r a t i n g c o n c e n t r a tion (5 gg/ml). U n d e r these conditions, d o u b l e transf o r m a t i o n of two u n l i n k e d m a r k e r s occurs frequently. T h u s Str r t r a n s f o r m a n t s were p r i m a r i l y selected a n d a m o n g these isolates, U V sensitive t r a n s f o r m a n t s were detected as m i t o m y c i n sensitive (Mit s) clones.
232
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
Table 4. Intergenote transformation by DNA of strain Z5 with
Table 5. Efficiency of transformation by various intergenote DNA
uvrAlO transformants of strain 168 TIM as recipients Donor Recipient
Parents : 168TIM UVS10TIM
Recipient
b/a
Number of transformants x 10 3/ml
Marker ratio
168TIM
Trp +
Trp ÷
Ttp +
Thy +
Met +
Number of transformants x 10 ~/ml
Number of (a) transformants Trp + x 10 3/ml
(b) Trp +
Trp + Thy +
Thy + Trp + Thy +
Thy +
0.02 0.18
0.01 0.09
380
260
1.5
200
97
2.1
1.4
250 160 140 160
0.39 0.22 0.14 0.04
120 85 20 21
94 51 23 53
1.3 1.7 0.87 0.40
3.3 7.7 6.2 10
14 13
Thy +
570 74
Met +
1100 150
B. sub tiffs ." SfrSmr
uvrAlO transformants (I):" S-2 40 180 S-5 4.5 28 S-11 120 780 S-12 150 580 S-13 90 340 S-14 79 330
1400 1300 760 710
0.22 0.16 0.15 0.26 0.26 0.24
0.09 0.12 0.12 0.11
uvrA 10 transformants (II) :a S-3 60 230 S-4 180 560 S-22 96 430 S-23 53 140 S-39 120 320 S-40 130 560
660 930 840 380 870 980
0.26 0.32 0.22 0.38 0.38 0.23
0.09 0.19 0.11 0.14 0.14 0.13
a uvrA10 donors were strains KE181 for (I) and KE511 for (II). uvrA 10 transformants were obtained as double transformants, Mit t Strr; the latter was used for primary selection
Intergenote: T5 H5 H1 Z5
97 35 19 5.8
168 i
10 10
~10
10 s o
I n t e r g e n o t e t r a n s f o r m a t i o n o f the U V sensitive isolates as r e c i p i e n t by D N A o f s t r a i n Z5 is s h o w n in T a b l e 4. I n t h e s e cases also, t h e e n h a n c e m e n t o f interg e n o t e - t r a n s f o r m a t i o n f r e q u e n c y was f o u n d to be c l o s e l y a s s o c i a t e d w i t h t h e U V sensitivity. E x a m i n a t i o n o f uvr + r e v e r t a n t s f r o m t h e s t r a i n U V S 1 0 , if t h e r e v e r s i o n o c c u r r e d , w o u l d o f f e r a g o o d e v i d e n c e for the g e n e t i c i d e n t i t y o f t h e t w o traits. W h e n , h o w e v e r , m o r e t h a n 1011 cells o f U V S 1 0 T I M were plated on the medium containing mitomycin C, n o r e v e r t a n t c o l o n y a p p e a r e d e v e n a f t e r i n c u b a t i o n for s e v e r a l days. T h e f a i l u r e to f i n d r e v e r s i o n o f t h e U V sensitivity o f U V S 1 0 T I M , leaves a q u e s t i o n w h e t h e r the t r a i t is a c t u a l l y d e t e r m i n e d by a single g e n e o r not. T h e t r a n s f o r m a t i o n f r e q u e n c i e s o f u v r A l O to uvr + a n d o f the r e c i p r o c a l c r o s s w e r e likely to be t h o s e o f single events, b u t f u r t h e r e x a m i n e d as f o l l o w s . T h e s t r a i n U V S 1 0 T I M was t r a n s f o r m e d w i t h D N A o f R S L at the s a t u r a t i n g c o n c e n t r a t i o n o f D N A (5 g g / m l ) . P r i m a r i l y M e t + t r a n s f o r m a n t s were selected and then they were tested for the unselected m a r k e r s : T h y +, T r p + a n d M i t r ( m i t o m y c i n C insensitivity). T h e f r e q u e n c i e s o f d o u b l e t r a n s f o r mants among Met + isolates: Thy+Met+/Met+, Trp+Met+/Met + and MitrMet+/Met + were 4.3%, 5 . 2 % a n d 2 . 3 % , r e s p e c t i v e l y . S i n c e T h y ÷, M e t ÷ a n d
UVS10TIM
_
$19
I
I
$80
I
i
01
gy0 x.-
i---
10"
0.01
'
0.1
'
0.01 0.1 1 10 H1 DNA / u g / m [
1
10
Fig. 1. Transformation frequency of homologous marker and intergenote marker. Transformation frequency of homologous marker (Thy+: - - o - - ) and intergenote marker (Trp+: o--) plotted as a function of DNA concentration. Donor was strain HI: tip + (203, H1). Recipients were 168TIM (uvr + rec+), uvsl0TIM (uvrAlO), UVS19TIM (uvrA19), and UVSSOTI (recSO), indicated in the figures as 168, S10, S19, and $80, respectively
T r p + t r a n s f o r m a t i o n has b e e n k n o w n to be single events, t h e s i m i l a r i t y o f f r e q u e n c i e s suggests t h a t every trait is d e t e r m i n e d by a single gene.
T r a n s f o r m a t i o n by D N A C o m p r i s i n g o f Various L e n g t h o f H e t e r o l o g o u s R e g i o n Intergenote DNA prepared from different clones of p r i m a r y t r a n s f o r m a n t s f r o m the h e t e r o s p e c i e s t r a n s -
K. Matsumoto et al. : D N A Repair and lntergenote Transformation in B. subtilis
0.5 0.4 ...~0.3 ~0.2 v O
,.0.1 x..
2~
//
0.05 I
0.01
I
I
I
0.1 1 10 Z5 DNA ,,ug/m[
Fig. 2. Dependence of the marker ratio on D N A concentration and on the recipient strain. D o n o r was strain Z5: trp + (203, Z5). Recipients were 168TIM (uvr+): - - o and UVS10TIM (uvrA10): --0--
formation was examined for transformation efficiency (Table 5). The individual intergenote D N A exhibited unique efficiency with 168TIM as recipient. The difference in frequencies appeared to reflect the length of heterologous portion in the intergenotes. Transformation experiments with closely linked markers suggested that the lower the frequency the longer the heterologous portion (Saito and Matsumoto, unpublished results). With UVS10TIM as recipient the transformation efficiecies of intergenote D N A were enhanced and the magnitude of enhancement (column b/a in Table 5) appeared to be related to the length of the heterologous portion in the intergenote.
Effect of DNA Concentration on the Efficiency of Intergenote Transformation Transformation of both intergenote and homologous markers was examined at various D N A concentra-
233
tions with 168TI and three UV sensitive mutants as recipient (Fig. 1). The transformation frequency increased with D N A concentration but exhibited saturation at almost definite concentration level, except for the case of UVS80TI recipient. The strain UVS80TI requires a much higher concentration of D N A for saturation than other recipients. This phenomenon appears to be a common feature of recombination deficient mutants ofB.subtilis (Hoch et al., 1967), and suggests the enhanced inactivation of transforming D N A as a result of defects in Rec functions. Figure 2 illustrates the marker ratio of intergenote marker (Trp +) to homologous marker (Thy +) plotted as a function of D N A concentration. Difference between the recipients, 168TIM and UVS10TIM, is obvious. With 168TIM as recipient, the marker ratio is markedly reduced with decrease of D N A concentration, whereas UVS10TIM exhibits fairly constant marker ratio at every concentration tested. These results suggest the presence of a DNA-inactivating function which acts especially on the intergenote transformation; the uvrAlO mutation may diminish this function.
Effect of uvrAlO Mutation on Heterospecies Transjormation In contrast to the intergenote transformation, when D N A of B.amyloIiquefaciens 203 was used to transform UVS10TIM, transformation frequency to Trp ÷ was not enhanced by uvrA10 at all (Table 6). Another marker purB6 was also examined, since this marker was known to be transformed by the heterologous D N A with appreciably higher frequency comparable to the intergenote transformation of trp marker (Saito et al., 1973). Although we expected the enhancement of Pur + transformation frequency with uvrAlO recipient, the results were again negative (Table 6) suggest-
Table 6. Effect of uvrAlO on the heterospecies transformation by D N A of B. amyloliquejaciens 203 Donor
Number of transformants/0.1 mI Trp +
Thy *
Put +
Recipient a
SfrSmr (B. subtilis) 203lys (B. amyloliquefaciens 203) ~
Trp +
Thy +
Pur +
Recipient a
168TI M
I01
UVS 10TI M
UVS 10TA
(uvr +trpC2 thy)
(uvr +purB6)
(uvrA 10 trp C2 thy)
(uvrA 10 purB6)
35,000 6
210,000 8,100
56,000 4
210,000 2,600
Relevant genotypes are shown in parentheses
26,000 1
30,000 1
234
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis
ing some difference between heterospecies transformation and intergenote transformation.
Discussion Intergenote strains can be constructed by integration of the heterospecies D N A segment into B.subtilis chromosome. When such intergenote D N A was used to transform B.subtilis, recombinant D N A having a mismatched region between double stranded portions would be formed. If the donor D N A strand of the mismatched region were preferentially corrected, it would result in the inefficiency of intergenote transformation as observed in this study. In transformation of Pneumococcus, a selective excision repair system was postulated to act on low efficiency markers (Ephrussi-Taylor and Gray, 1966). Guild and Shoemaker (1974) reported that competition by nonisogenic but homologous D N A leads to a rise in the number of transformants of the low efficiency marker, suggesting the similarity in recognition of nonisogenic D N A and of low-efficiency marker DNA. A mechanism which asymmetrically corrects a mismatched region was found in B.subtilis by transfection of heteroduplex SPP1 D N A constructed in vitro with wild and mutant phage (Spatz and Trautner, 1970). The similar correction mechanism was posturated in transformation system of B.subtilis to account for the formation of pure clones of transformants on nonselective media, because, if heteroduplex D N A persisted, not pure but mixed clones would be formed (Bresler et al., 1971), The data presented in this paper suggest the presence of a correction mechanism for intergenote transformation. A m o n g sixteen UV sensitive mutants tested, two exhibited enhanced efficiencies of intergenote transformation. The mutations, uvrAlO and uvrB114, relating to the enhancement, locate at two unlinked loci concerning with the host-cell reactivation (Munakata, 1977). Thus it is likely that the correction of the mismatched region may be carried out by a function related to excision repair of UV damage in DNA. However, the genetic relationship is yet uncertain since other uvr mutations including extensively deleted mutations, uvrA191 and uvrBl09, have no effect on the enhancement. In Escherichia coli, Nevers and Spatz (1975) reported that the correction of heteroduplex D N A of phage lambda was deficient in uvrD and uvrE mutants. The frequency of heterospecies transformation was not enhanced by the uvrA10 mutation. Since the efficiency of heterospecies transformation is generally more than two orders lower than that of intergenote transformation, an additional mechanism may be
concerned with the former inefficiency. Although host-controlled restriction had been suspected to operate in the heterospecies transformation, Trautner et al. (1974) reported that a restriction-modification system of B.subtilis R, which was active on both phages and transducing DNA, was inert for transforming DNA. This result seems to be reasonable, since transforming D N A integrates as a single strand into the host chromosome already modified. Thus the inefficiency of heterospecies transformation may be due mainly to the low efficiency in pairing of D N A strands. In heterospecies transformation of Pneumococcus by Streptococcus D N A , the transformation frequency rised with D N A concentration increasing up to a higher concentration level than the saturation level of homospecies transformation (Chen and Ravin, 1966). The shift of saturation concentration was explained as a result of internal inactivation of heterospecies DNA. Effect of D N A concentration on the marker ratio of intergenote to homologous markers, as described in the present paper, suggests the selective inactivation ofintergenote DNA, resulting in the inefficiency as observed. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Japan.
References Bott, K.F., Wilson, G.A. : Development of competence in Bacillus subtilis transformation system. J. Bact. 94, 562-570 (1967) Bresler, S.E., Kreneva, R.A., Kushev, V.V.: Molecular heterozygores in Bacillus subtilis and their correction. Molec. gen Genet. 113, 204-213 (1971) Chert, K.C., Ravin, A.W. : Heterospecifictransformation in Pneumococcus and Streptococcus. I. Relative efficiency and specificity of DNA helping effect. J. molec. Biol. 22, 109-121 (1966) Ephrussi-Taylor, H., Gray, T.C.: Genetic studies of recombining DNA in pneumococcaltransformation. J. gen. Physiol. 49, Part 2, 211-231 (1966) Guild, W.R., Shoemaker, N.B.: Intracellular competition for a mismatch recognition system and marker-specific rescue of transforming DNA from inactivation by ultraviolet irradiation. Molec. gen. Genet. 128, 291-300 (1974) Hoch, J., Barat, M., Anagnostopoulos, C.: Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J. Bact. 93, 1925-1937 (1967) Ikeda, Y., Saito, H., Miura, K., Takagi, J., Aoki, H. : DNA base composition, susceptibility to bacteriophages and interspecific transformation as a criteria for classification in the genus Bacillus. J. gen. appl. Microbiol. 11, 181-190 (1965) Marmur, J., Seaman, E., Levine, J.: Interspecific transformation in Bacillus. J. Bact. 85, 461-467 (1963) Munakata, N. : Repair of ultraviolet-induced damage in transforming DNA of Bacillus subtilis. Jap. J. Genet. 45, 1-9 (1970) Munakata, N.: Mapping of the genes controlling excision repair of pyrimidin photoproducts in Bacillus subtilis. Molec. gen. Genet. 156, 49-54 (1977)
K. Matsumoto et al. : DNA Repair and Intergenote Transformation in B. subtilis Nevers, P., Spatz, H.Ch. : Escherichia coli mutants uvrD and uw'E deficient in gene conversion of 2-heteroduplexes. Molec. gen. Genet. 139, 233 243 (1975) Saito, H., Miura, K.: Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. biophys. Acta (Amst.) 72, 619 629 (1963) Saito, H., Takahashi, H., Ikeda, Y. : Gene conservation in Bacillus subtilis. In: Genetics of industrial microorganisms, Vol. 1 (Van~k, Z., Ho~t'filek, Z., Cudliu, J., eds.), pp. 89-94. Prague: Academia 1973 Spatz, H.Ch., Trautner, A.: One way to do experiments on gene conversion? Transfection with heteroduplex SPP1 DNA. Molec. gen. Genet. 109, 84 106 (1970)
235
Trautner, T.A., Pawlek, B., Bron, S., Anagnostopoulos, C.: Restriction and modificaton in Bacillus subtilis. Molec. gen. Genet. 131, 181 191 (1974) Wilson, G.A., Young, F.E. : Intergenote transformation of Bacillus subtilis genospecies. J. Bact. 111, 705 716 (1972)
Communicated
by T.Yura
Received December 20, 1977
