IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPα plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 ± 0.2) × 10⁻² S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPα plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPα plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained. The text was submitted by the authors in English.     

Intergeneric conjugation between Escherichia coli and Streptomyces species.

We have constructed Escherichia coli-Streptomyces shuttle plasmids which are capable of conjugal transfer from E. coli to Streptomyces spp. These plasmids contained the pBR322 and pIJ101 origins of replication and the RK2 (IncP) origin of transfer. The transfer of plasmid was specifically dependent the presence of a 760-base-pair, cis-acting, oriT-containing fragment and on RP4 (IncP) functions supplied in trans. Conditions of mating and selection of exconjugants were analyzed with Streptomyces lividans as recipient. Plasmid transfer to other Streptomyces species was also demonstrated.     

The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans.

Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Construction of two stable bifunctional plasmids for Streptomyces spp. and Escherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.     

质粒RSF1010从大肠杆菌到稀有放线菌头状链轮丝菌和星状诺卡氏菌的接合转移

１９８７年,ＴｒｉｅｎＣｕｏｔ等人［１］证明穿梭质粒可以在革兰氏阴性的大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）和多种革兰氏阳性细菌之间发生接合转移。在这种转移中质粒需具备大肠杆菌的复制起始位点,同时又具备革兰氏阳性细菌的广宿主范围复制起始位点。转…     

质粒RSF1010从大肠杆菌到稀有放线菌头状链轮丝菌和星状诺卡氏菌的？…

RSF1010 is a naturally occurring Escherichia coli broad host-range plasmid about 8.7 kb in size. It can be mobilized at high frequency between different gram-negative bacterial species when transfer functions are available in trans. Following the pioneering work of conjugational transfer of RSF1010 from E. coli to Streptomyces lividans and Mycobacterium smegmatis, the transfer of this plasmid by conjugation from E. coli S17.1 tp two gram-positive rare actinomycetes, Nocardia asteroides 3927 and Streptoverticillum caespitosus ATCC27422 was first time reported in this study. Southern blot analysis of the total DNA extracted from the actinomycetes' exconjugants proved that RSF1010 had been transferred from E. coli into the two new hosts and maintained staby in the exconjugants. Meanwhile, partial deletions of RSF1010 replicon loosing its antibiotics resistance makers were readily detected in E. coli. The implenmentation of this observation was discussed.     

Development of a system of plasmid transfer in the strains Streptomyces avermitilis ATCC 31272 and Saccharopolyspora erythraea ATCC 11635 by the method of intergeneric conjugation

A new method of plasmid DNA transfer from the donor strain Escherichia coli S17-1 to the erythomycin-producing strain Saccharopolyspora erythraea and avermectin-producing strain Streptomyces avermitilis via intergeneric conjugation was proposed. The optimal parameters of the method were chosen for increasing the efficiency of crosses and ensuring easily reproducible results. The behavior of the multicopy plasmid pPM803 and the integration vector pTO1 along with a number of new plasmids specially created by us, was examined in these strains. A new plasmid vector (pSI60) capable of integrating into the chromosome of actinomycetes at the integration site of the temperate actinophage phi C31 was constructed. This vector possesses unique sites convenient for cloning and may be stably maintained in exconjugants of S. avermitilis and in the model strain Streptomyces lividans. The gene encoding resistance to spectinomycin and streptomycin was cloned into the vector pSI60 in this strain. For cloning in strain Sac. erythraea, vectors pSI261-280, which integrate into the chromosome via homology with the cloned DNA and can be stably maintained in exconjugants, were constructed.     

High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes

Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1. and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 10⁴-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with 'suicide' delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.     

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.     

Five genes involved in self-transmission of pSN22, a Streptomyces plasmid.

An 11-kbp multicopy plasmid, pSN22, was isolated from Streptomyces nigrifaciens SN22. pSN22 is self-transmissible (conjugative), is maintained stably in S. lividans, and forms pocks in a wide range of Streptomyces strains. Mutational analyses showed that a fragment of pSN22 contained five genes involved in plasmid transfer and pock formation. traB was essential for plasmid transfer. traA was required for pock formation, but not for plasmid transfer. spdA or spdB were concerned with pock size; mutations in these genes decreased pock size. The fifth gene, traR, could be deleted together with other genes to give nontransmissible plasmids, but plasmids with insertions or deletions only within traR became nonviable. traR is probably needed to counterbalance the lethal effects of another plasmid gene. Transfer of pSN22 promoted the cotransfer of nontransmissible plasmids and enhanced chromosome recombination between the host and recipient strains, suggesting that plasmid transfer accompanies cytoplasmic mixing.     

Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis.

The plasmid RSF1010 belongs to a class of plasmids (IncQ) that replicate in a range of bacterial hosts. Although non-self-transmissible, it can be mobilized at high frequency between different gram-negative bacterial species if transfer functions are supplied in trans. We report the transfer of RSF1010 by conjugation from Escherichia coli to the gram-positive actinomycetes Streptomyces lividans and Mycobacterium smegmatis. In its new hosts, the plasmid was stable with respect to structure and inheritance and conferred high-level resistance to streptomycin and sulfonamide. This is the first reported case of conjugative transfer of a naturally occurring plasmid between gram-negative and gram-positive bacteria.     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.     

Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.     

Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis.

Streptomyces avermitilis contains a unique restriction system that restricts plasmid DNA containing N6-methyladenine or 5-methylcytosine. Shuttle vectors isolated from Escherichia coli RR1 or plasmids isolated from modification-proficient Streptomyces spp. cannot be directly introduced into S. avermitilis. This restriction barrier can be overcome by first transferring plasmids into Streptomyces lividans or a modification-deficient E. coli strain and then into S. avermitilis. The transformation frequency was reduced greater than 1,000-fold when plasmid DNA was modified by dam or TaqI methylases to contain N6-methyladenine or by AluI, HhaI, HphI methylases to contain 5-methylcytosine. Methyl-specific restriction appears to be common in Streptomyces spp., since either N6-methyladenine-specific or 5-methylcytosine-specific restriction was observed in seven of nine strains tested.