FEMS Microbiology Letters 155 (1997)
High efficiency intergeneric conjugal transfer of plasmid DNA from Esc’herichia coli to methyl DNA-restricting streptomycetes Fiona
Flett ‘, Vassilios
Many strcptomycetes, including S. ~or/i~o/o~ A3(2). possess a potent methyl-specific restriction system which can present an effcctivc barrier to the introduction of hcterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. ~oc/i~o/or and S. /i~~rt/trr~.r 66 using two C: c~olidonors: the standard, methylation proficient strain Sl7-I. and the methylation deficient donor. E:Tl2567(pUB307). We demonstrate that the mcthylation delicient donor can yield > IO’-fold more S’. CY~C~/~C~O/O~~ exconjugant5 than the standard donor. In the case of pSETl52 derivatives, which integrate into the host chromosome by site-specific recombination. up to IO% o f streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is cfticient enough to obtain exconjugants with ‘suicide’ delivery plasmids and thercforc provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes. and
possibly other bacteria.
In recent years there has been considerable interest in the use of intergeneric conjugation as a means of gene transfer. This technique enables one to construct recombinant plasmids in an experimentally tractable host such as E. co/i and then transfer them, by conjugation. into the required recipient. Since E. c~olilStrc~pto/~~~,~~~~.~ intergeneric conjugation was first reported by Mazodier et al. [I]. this method
* Corresponding author-. Tel.: +44 (161) 200 4183; Fax: +43 (I61 ) 2360409: E-mail: colin.smith(u~umist.ac uk ’ The first two author-s contributed Oi78-lOY7/Y7/Rl7.0(1
(’ 1997 Published
PII s 0 3 7 8 I 0 9 7 ( 9 7 ) 0 0 3‘12- 3
to this work.
has been used successfully with a number of streptomycete strains (e.g. [2-A]). Several versatile vectors which can be transferred from E. co/i to .Stwptotn~~ws spp. by conjugation have been developed [2,5,6]. Most of these vectors are non-replicative in streptomycetes but integrate into the chromosome to yield stable recombinant strains. thus avoiding the possible problems associated with multicopy plasmids. The vectors contain the oriT sequence from the IncP-group plasmid RK2 (also designated RPl/ RP4) but require the transfer functions to be supplied it7 trams by the E. coli donor strain. Str’cptom~ws c~wlic~olor A3(2) is genetically the best characterized actinomycete. One disadvantage of this strain is that in transformation studies it shows strong restriction of DNA isolated from
IZlsecierScience B.V. All IrIghtsreszr\cd
F. Fiett et al. IFEMS
Microbiology Letters 155 (1997) 223-229
standard strains of E. coli, whereas the closely related strain S. lividans 66 is largely non-restricting . This restriction is principally due to the presence of a methyl-specific restriction system in S. coelicolor A3(2) . MacNeil  found that several species of Streptomyces possess methyl specific restriction systems; moreover he demonstrated that DNA isolated from a methylation deficient E. coli strain could transform such strains efficiently. The aim of this study was to investigate the effect of the S. coelicolor methyl-specific restriction system on E. colilS. coelicolor intergeneric conjugations and to compare the donor efficiency of DNA methylation proficient and deficient E. coli strains.
2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions E. coli strain XLlBlue  was used as the general cloning host. Two strains of E. coli were used as donors in intergeneric conjugations : S 17- 1 [lo] which carries an integrated RP4 derivative and ET12567 (pUB307). ET12567 is a methylation defective strain (dam-13::Tn9, dcm-6, hsdkf) [ll]; pUB307, a derivative of RPl , was transferred into the strain by conjugation (this study). Two prototrophic strains were used as recipients: S. lividans 66 [ 131 and S. coelicolor A3(2) strain MT1 110 (isolated in this laboratory); the latter is an SCPll SCP2- isolate of the wild-type strain, 1147 (and is equivalent to M145) . The plasmids used in this study are listed in Table 1. E. coli strains were grown in LB broth and agar supplemented with antibiotics when required. To maintain plasmid selection in XLlBlue and S 17-1, apramycin (Melford Laboratories Ltd.) or ampicillin were used at a final concentration of 100 @ml. In addition, with ET12567(pUB307), kanamycin (50 ug/ ml) and chloramphenicol (25 &ml) were added to maintain the selection for pUB307 and the dam mutation respectively. The methods used for the cultivation of S. coelicolor and S. lividans were as described in . MS agar medium  was used for the growth of lawns of spores. The conjugation mixtures were plated on MS agar supplemented with 10 mM MgClz.
2.2. DNA manipulation Procedures for standard DNA manipulations were as detailed in . The recombinant plasmids based on pSET151, pSET152 and pKC1132  were constructed in XLlBlue prior to their introduction into S17-1 and ET12567(pUB307) by transformation. E. coli competent cells were prepared as in . 2.3. Intergeneric conjugation procedure A culture of the donor E. coli was grown to an ODaos of 0.3-0.4. To overcome the problem of the slow growth of ET12567(pUB307) the inoculum was adjusted so that an overnight culture could be used directly without further subculture. The cells were pelleted by centrifugation, washed in LB, pelleted again and finally resuspended in a smaller volume of LB. Aliquots of a streptomycete spore suspension stored at -20°C were used as recipients. The required number (this varied from lo3 to 10’ per conjugation; see below) of viable spores of MT1 110 or S. lividans 66 were washed in LB, resuspended in S medium  and incubated at 50°C for 10 min to activate germination. Approximately 1Os E. coli donor cells were added to the prepared spores and the mixture was spread on two MS+10 mM MgCls agar plates. The viable count of the donor culture was determined by spreading samples on LB agar supplemented with the appropriate antibiotic(s). The conjugation plates were incubated for 1622 h at 30°C then most of the E. coli cells were washed from the surface using water and gentle agitation with a spreader. The surface of each plate was overlaid with 1 ml of water containing 500 ug nalidixic acid and 1 mg apramycin or, in the case of pDJ100, with 2.5 ml soft LB agar containing 500 l_rgnalidixic acid and 625 ug thiostrepton. The plates were then incubated for a further 5 days at 30°C and the exconjugant colonies counted. Each conjugation experiment was carried out three times. To estimate the frequency of occurrence of spontaneous apramytin or thiostrepton resistant mutants, control conjugation mixes were processed as above but without the addition of E. coli donor cells. 2.3.1. Donor:recipient ratio For each conjugation the initial number
of E. coli
donor cells was 10’ and the maximum number of recipient spores was 10’. However. for high efficiency conjugation events, this number of recipients produced a lawn of exconjugants which made it impossible to estimate the exconjugant frequency. Therefore the number of recipient spores was chosen so as to obtain discrete colonies on the conjugation plates. Routinely, each mating was set LIP with two different numbers of spores which differed by a factor of IO: the number of exconjugant colonies was found to increase in proportion to the number of spores used. Reduction in the initial number of donor cells by a factor of 10 did not alfect the exconjugant frequency markedly.
mosomal DNA was originally cloned by its ability to complement a ts DNA replication mutant of MTI 110 (F. Flett, D. Jungmann Campello, V. Mersinias and C.P. Smith, unpublished results). For this study. the 5.8 kb fragment and a central 2.1 kb R.vtEII segment of it (38-2) were subcloned into two classes of vector described by . One type of vector has the attachment site and integrase functions of the temperate phage 4rC31 and thus undergoes site-specific integration (pSETI52); the other type can only integrate as a result of homologous recombination between a cloned segment of DNA and the streptomycete chromosome (pSETI51 and pKClI32). The recombinant plasmids used in this study are listed in Table 1.
The dmtt and htt status of ETl2567(pUB307) donor cultures was confirmed routinely by AYhoIl .&YIRII digestion of plasmid DNA isolated from a sample of the donor culture.
3.2.1. Phsttiidr which intrgrcite rrt the &31 att site Two E. coli strains were used as donors in intergeneric conjugations: Sl7-1 and the methylation defective ET12567(pUB307). Donor cultures carrying pDJ50 or pDJ70 were mated with S. cwlicolor MT1 I IO and 5: /ividcrns 66. The results are shown in Table 2. Sl7-I efhciently transferred these plasmids to S. liCkttt.s. but when the recipient was S. c~oclicdot~ the exconjugant frequency was much lower: 2.8 X IO’-fold for pDJ50 and 3.2 X IO”-fold for pDJ70. When ET12567(pUB307) acted as donor the number of S. corlicolor exconjugants increased markedly. For pDJ70 the numbers of S. lividms and S. cm~licdor exconjugants were comparable at approximately IO- ’ per recipient; this represents a 5.2 X IO’-fold increase for S. coclic~olor and an 1Ifold increase for S. 1ividm.s when compared with the results obtained with Sl7-I as the donor. In
The thiostrepton resistant vector pSETI5I has the .YJ~IEreporter gene driven by the rrtnE promoter . To confirm that the thiostrepton resistant streptomycete colonies obtained from pDJ100 matings were true exconjugants, they were tested for the presence of the X.),/E gene as described in [ 181.
A 5.8 kb B~rlrHl DNA fragment
of MTI 110 chro-
used in this study
S.8 kh R~/,,IHI fragment,’
pDJ70 pVM382 pUB307
is a 5.8 kb &m~Hl
5.X kb Bu/,rHI
2.1 kh B.s/EII
of MTI I IO chromosomnl
38-2 is a 2.1 kh B.stEII fragment
F. Flett et al. I FEMS Microbiology
Letters lS5 (1997)
Table 2 Comparison of E. coli strains S17-1 and ET12567(pUB307) as donors in intergeneric and S. lividans 66: plasmids which integrate via site-specific recombination E. coli donor”
s17-1 s17-1 ET12567(pUB307) ET12567(pUB307) s17-1 s17-1 ET12567(pUB307) ET12567(pUB307)
pDJ50 pDJ50 pDJ50 pDJ50 pDJ70 pDJ70 pDJ70 pDJ70
of recipient [email protected]
108 105 104 10s 10s 10s 10s 10s
S. lividans S. coelicolor S. lividans S. coelicolor S. lividans S. coelicolor S. lividans
with S. co&color
3.2.2. Plasmids which integrate by homologous recombination E. coli S17-1 and ET12567(pUB307)
were used as donors of two plasmids which would integrate into the streptomycete chromosome by homologous recombination: pDJ100, which has an insert of 5.8
Table 3 Comparison of E. coli strains S17-1 and ET12567(pUB307) and S. lividans 66: plasmids which integrate via homologous
2). When ET12567(pUB307) was the donor of pDJ100, MT1110 exconjugants were obtained at a frequency of 1.8 x 10e5, representing an increase in frequency of > 1.2 X lo3 over that observed with S17-1 as donor. Even with pVM382, which has only 2.1 kb homologous DNA insert, exconjugants of both S. lividans and MT1110 were obtained when
E. coli donor”
s17-1 s17-1 ET12567(pUB307) ET12567(pUB307) s17-1 s17-1 ET12567(pUB307) ET12567(pUB307)
pDJlO0 pDJlO0 pDJlO0 pDJlO0 pVM382 pVM382 pVM382 pVM382
10’ 10’ 106 10s 10s 10s 10s
S. coelicolor S. lividans S. coelicolor S. lividans S. coelicolor S. lividans
Values represent the
kb, and pVM382, which has an insert of 2.1 kb. The results are given in Table 3. When S17-1 was the used as the donor, no MT1110 exconjugants were obtained. With S. lividans, the exconjugant frequency was 1.2 X lop5 for pDJlO0 and 51 X lo-’ for pVM382; these frequencies are 2.1 X 103- and 6.8 x 105-fold lower respectively than those obtained for the equivalent att site integration events (Table
as donors in intergeneric recombination
9.0x 10-7 2.5 x 10-s 9.3 x 10-s 1.1 x 10-t 2.1 x10-s 6.8 x 1O-3 1.1x10-r 7.7 x 10-s
“The same number of donor cells (lOs) was used in each experiment. bThe number of recipient spores was chosen to obtain discrete colonies on the conjugation plates. ‘The quotient of the number of exconjugant colonies divided by the number of recipient spores used in the experiment. average frequencies from two or three independent experiments.
the case of pDJ50 the exconjugant frequency for MT1110 showed a 104-fold increase over that obtained with S17-1 as donor but it was l%-fold lower than that observed with S. lividans as recipient. We have also tested several other pSET152-based plasmids in this system (data not shown) and in all cases the use of ET12567(pUB307) as donor produced an increase in S. coelicolor exconjugant frequency of > lo4 relative to that from S17-1.
of recipient sporesb
with S. coelicolor MT1 110
< 1 x 10-8 1.2x 10-s
1.8x 1O-5 9.2x 10-s < 1 x10-s 5 1 x10-s 2x 10-7
1.1 x 10-7
“The same number of donor cells (108) was used in each experiment. “The number of recipient spores was chosen to obtain discrete colonies on the conjugation plates. ‘The quotient of the number of exconjugant colonies divided by the number of recipient spores used in the experiment. average frequencies from two or three independent experiments.
Values represent the
was the donor.
at a low
4. Discussion This study has shown that the methylation specific restriction system of S. co&olor A3(2) has the effect of drastically reducing the number of exconjugants obtained from an intergeneric conjugation when the donor E. coli is methylation proficient. Thus, transfer of DNA by conjugation from a methylation proficient strain of E. coli does not evade the S. coelico/o~ restriction barrier. Use of the methylation deficient donor ET12567(pUB307) overcomes this problem to such an extent that with certain plasmids (e.g. pDJ70) there was no significant difference in the exconjugant frequency obtained with S. codicolor compared with that for the non-restricting host. S lividms. Thus, with plasmids which integrate at the oC31 att site, an exconjugant frequency of 10 ’ can be achieved. The use of ETI2567(pUB307) or equivalent strains may be more widely applicable since several Strc~ptottt~ws species have been shown to possess a methyl-specific restriction system . For example, we have been able to transfer such pSETl52 derivatives to the industrially important S c1~wuligeru.susing this donor strain, while it was not possible to obtain exconjugants using the Sl7-I donor (F. Amini and C.P. Smith, unpublished data). To achieve gene replacements and disruptions, vectors which integrate via homologous recombination between the chromosome and a cloned fragment of DNA are required. Since this type of recombination occurs at a much lower frequency than that mediated by site-specific recombination (Tables 2 and 3) and is dependent on the length of the homologous DNA, it is particularly important to have an efficient conjugation system. The use of ET12567(pUB307) as donor fulfils this requirement, making it possible to obtain exconjugants which are the result of homologous recombination between the chromosome of a methyl DNA-restricting streptomycete and DNA fragments of 2 kb (Table 3 and data not shown). Intergeneric conjugation has previously been used to generate gene disruptions in streptomycetes which do not possess methyl DNA restriction systems [ 19,201.
The results of matings between S. lividms and the two E coli donor strains suggest that ET12567(pUB307) is a somewhat more efficient donor than Sl7-I. This might be due to the autonomous state of RPl in the former strain and to the copy number of I-2 rather than the single integrated state of the plasmid in S17-1. An alternative explanation is that the more vigorous growth of S17-I may cause the donor to rapidly reach stationary phase or to overgrow the recipient before streptomycete/E. co/i mating junctions have formed. Although the use of a self-transmissible RPI plasmid in the donor could be perceived as a disadvantage, we have found no evidence, by Southern analysis, that pUB307 sequences are maintained in the streptomycete exconjugants, either in an autonomous state or integrated into the chromosome (data not shown). A derivative of ET12567 has recently been constructed which carries the RK2 derivative, pUZ8002, which is not selftransmissible but can mobilise other plasmids efticiently (M.S.B. Paget. J.W. Wilson, and D.H. Figurski, personal communication). The number of exconjugants obtained from ETl2567(pUB307)/MTlllO matings was found to vary depending on the insert in pSET152 (Table 2). MTI 110 yielded more exconjugants than S. hi&m with pDJ70 but approximately 12-fold less than S. li~~itlmwwith the larger plasmid, pDJ50. It is possible that this difference is attributable to the presence of a ‘classical’ restriction site in the latter which is specific for a S. coc~/irdor endonuclease, distinct from the methyl specific restriction system under consideration in this report. The existence of an additional restriction system within S. corlicolor was previously suggested from the observation that SCP2* derivatives isolated from S. /i~iu’~zmstransform S. corlicolot very poorly (D.J. Lydiate, personal communication). There are several examples where the presence of a restriction system in the recipient is found to have a marked effect on the conjugal transfer of plasmids from E. coli to other bacteria (e.g. [21-231). The 12fold reduction in S. coelicolor exconjugant frequency observed with pDJ50 would be consistent with that reported for other intergeneric conjugation systems (that is, - 15-fold reduction per restriction site, e.g. ). In contrast. other studies have shown that some bacterial (including streptomycete) restriction systems are circumvented by conjugal transfer of
I? Flett et al. IFEMS Microbiology
DNA ([2,4]; literature cited in ). This is thought to be because conjugation involves the transfer of ss DNA which is generally refractory to restriction endonuclease attack and which, after synthesis of the complementary strand, will be host-modified. In the case of methyl-specific restriction systems found in S. coelicolor and other streptomycetes  the foreign methyl-modified DNA is restricted whereas the non-modified host DNA is not. In this case, after introduction of the ss DNA by conjugation, the resulting ds DNA will be hemi-methylated and thus potentially still subject to restriction. Moreover, there is recent evidence from transformation studies that the methyl specific restriction system of S. coelicolor cannot be evaded by the use of ss DNA . This would explain the great improvement in the conjugation frequency reported here with the methylation defective ET12567(pUB307) donor.
Acknowledgments We thank Keith Chater for comments on the manuscript and thank Douglas MacNeil and Wolfgang Wohlleben, respectively, for providing E. coli strains ET12567 and S17-1. This work was partly supported by a grant from the BBSRC (to C.P.S.).
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Letters 155 (1997)
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Trleu-Cow. lin. and
Gram-positive Chater. facilitates
a multiple of