Identification of SCP2165, a new SCP2-derived plasmid of Streptomyces coelicolor A3(2)

AIMS: Characterization of SCP2165, a plasmid identified in the Gram-positive bacterium Streptomyces coelicolor A3(2). METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) of mycelia of a S. coelicolor strain embedded in low melting agarose revealed the presence of a plasmid. Restriction enzyme mapping and sequence analysis of a 2.1 kb fragment revealed that this plasmid could be SCP2. SCP2 and its spontaneous derivative SCP2* are self-transmissible plasmids and have chromosome mobilizing ability (c.m.a.). SCP2* has a c. 1000-fold increased c.m.a. compared with SCP2. Interestingly the plasmid, named SCP2165, shows a c.m.a. from 5x10(-2) to 1x10(-1) which is 50-100-fold higher than that described for crosses involving SCP2*. CONCLUSIONS: SCP2165 is a SCP2 derivative plasmid with the highest c.m.a. so far described for SCP2 derivative plasmids. PFGE, under conditions we used, seems to be a fast way to identify large circular plasmids in Streptomyces strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Further knowledge of the SCP2 family may allow the construction of improved SCP2-derived cloning vectors. SCP2165 could be a potential tool for conjugational transfer of gene clusters between different Streptomyces species.     

A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10-22

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.     

Structural peculiarities of the SCP2 plasmid of Streptomyces coelicolor A3(2)

Structural peculiarities of SCP2 plasmid isolated from different derivatives of Streptomyces coelicolor A3(2) were studied. By means of heteroduplexing 800 b.p. DNA insertion in SCP2 plasmid of S18-1 derivative was detected. The position of this insertion was localised on the SCP2 restriction map. A transposon-like structure with similar characteristics was detected and localised in different variants of SCP2 plasmid. This 600 bp DNA region is flanked by 20 bp inverted repeats and has the single EcoRI cleavage site. It is noted that the stretching of single-strand DNA circles of SCP2 plasmid was significantly less than that of single-strand DNA circles of pBR322 and PAS3; Tn9 plasmids. This may likely depend on the GC content of different plasmids.     

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19.     

Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

Streptomyces coelicolor A3(2) possesses two plasmids (SCP1 and SCP2) that act as sex factors. The plasmid deoxyribonucleic acid isolated from S. coelicolor A3(2) SCP1- strains A617 and A585 had the same molecular weight and endonuclease cleavage pattern as the SCP2 plasmid. The plasmidless strain S18 SCP2- was isolated from the A617 X A585 cross. SCP2 plasmid-containing strains acted as donors of chromosomal markers, whereas the plasmidless strain acted as recipient. The transfer of SCP2+ donor strain markers into the SCP2- recipient occurred at high frequencies (approximately 75%), was unidirectional, was initiated from a fixed region of the chromosome, and had the SCP2 fertility factor transferred first. The introduction of the SCP2 plasmid into a recipient strain greatly reduced the recombination frequency. These fertility properties differed from those previously reported, thereby suggesting that the SCP2 plasmid examined in this investigation may be an additional variant to those described in the literature. The SCP2 plasmid also regulated production of three antibacterial substances and conveyed resistance for S. coelicolor A3(2) strains against growth inhibition by one of them.     

Transformation and transfection of anthracycline-producing streptomycetes.

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

Transformation and transfection of anthracycline-producing streptomycetes

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

大肠杆菌-链霉菌高效接合载体的构建及其应用

以链霉菌质粒SCP2^*的衍生质粒pHJL400为基础,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒DGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化Escherichia coli ET12567(pUZ8002)后,与天蓝链霉菌(Streptomyces coelicolor A3(2))、除虫链霉菌(Streptomyces avermitilis)、变铅青链霉菌(Streptomyces lividans TK54)、毒三素链霉菌(Streptomyces toxytricini NRRL15443)、委内瑞拉链霉菌(Streptomyces．vertezuelae ISP5230)和红色糖多孢菌(Saccharopolypora erythraea)进行接合,发现本构建的pGH112与pKC1139相比,接合转移效率较高,稳定性好,而且宿主范围较广。把组成型启动子ermE^*与绿色荧光蛋白基因(gfp)克隆到本构建的pGH112,通过接合转移到链霉菌中,gfp获得表达,证明其可以用作基因接合转移的有效工具载体,这为研究链霉菌的基因功能创造了有利条件。     

Characterization of replication and conjugation of Streptomyces circular plasmids pFP1 and pFP11 and their ability to propagate in linear mode with artificially attached telomeres

Many Streptomyces species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb). We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360 bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 were inherited extremely unstably; par and additional loci were required for stable inheritance. Surprisingly, plasmids containing replication loci from pFP11 or Streptomyces circular plasmid SCP2 but not from pFP1, SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached. These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, for plasmid transfer, a major functional traB gene (encoding a DNA translocase typical for Streptomyces plasmids) as well as, surprisingly, a putative traA gene (encoding a DNA nickase, characteristic of single-stranded DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.     

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.     

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

尤马马杜拉放线菌染色体复制区的克隆及功能分析

尤马马杜拉放线菌(Actinomadura yumaensis)NRRL12515产生马杜拉霉素,用于防治禽类球虫病。试验以放线菌dnaA与dnaN基因保守区设计的简并引物进行PCR扩增,获得了包含尤马马杜拉放线菌的染色体复制区oriC的片段,并进行了序列分析和复制功能的研究。尤马马杜拉放线菌染色体的oriC全长为919碱基对,含有14个DnaA盒子和2个AT富含区,DnaA盒子的保守序列是(T/C)(T/C)GTCC(A/C)CA,与已发表的3个属的放线菌染色体oriC的序列特征不同。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制并以低拷贝方式遗传,表明这是一段有复制功能的序列。比较来自放线菌4个属的oriC,发现以oriC序列和以16S rRNA基因序列构建的进化树十分相似,表明oriC序列也可以体现放线菌物种之间的关系。     

Transposition of Tn4560 of Streptomyces fradiae in Mycobacterium smegmatis

Tn4560 (8.6 kb) was derived from Tn4556, a Tn3-like element from Streptomyces fradiae. It contains a viomycin resistance gene that has not been used previously for selection in mycobacteria. Tn4560, cloned in a Streptomyces plasmid, was introduced by electroporation into Mycobacterium smegmatis mc(2)155. Tn4560 transposed into the host genome: there was no obvious target sequence preference, and insertions were in or near several conserved open reading frames. The insertions were located far apart on different AseI macrorestriction fragments. Unexpectedly, the transposon delivery plasmid, pUC1169, derived from the Streptomyces multicopy plasmid pIJ101, replicated partially in M. smegmatis, but was lost spontaneously during subculture. Replication of pUC1169 probably contributed to the relatively high efficiency of Tn4560 delivery: up to 28% of the potential M. smegmatis transformants acquired a stable transposon insertion. The data indicated that Tn4560 may be useful for random mutagenesis of M. smegmatis.     

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.     

Integrated structures of the linear plasmid SCP1 in two bidirectional donor strains of Streptomyces coelicolor A3(2)

The linear plasmid SCP1 is integrated into the central region of the chromosome of Streptomyces coelicolor A3(2). The integrated structures of SCP1 in two bidirectional donor strains, 2612 and A634, were analyzed by cloning and sequencing of the junctions between the SCP1 DNA and the chromosomal DNA. In the NF (normal fertility) strain 2612, SCP1 is integrated in a right-handed direction into ORF-X at the left end of the IS cluster in AseI fragment E. An almost intact left end of SCP1 is retained, while the right terminal inverted repeat (TIR-R) of SCP1 and a 33-kb chromosomal DNA segment including the IS cluster are deleted. In the NF-like strain A634, SCPI is also integrated into AseI fragment E in a left-handed direction. The left junction is composed of IS466 with complete deletion of TIR-R of SCP1, and the right junction is located at the left end of IS468A* with half of TIR-L being deleted. During the integration event, a 5.4-kb chromosomal DNA segment including IS468A, IS468B, IS469 and IS466A was duplicated so that this sequence is now present on both sides of SCP1. Since 2612 and A634 exhibit a similar bidirectional gradient of gene transfer, it is surprising that their chromosomal structures are so different.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

Cloning of antibiotic resistance and nutritional genes in streptomycetes.

Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66 were adjusted to generate reproducibly a population of cloned genes likely to represent the entire genome. Factors which influence the recovery of viable transformants include: growth phase of the mycelium, ionic and osmotic characteristics of the medium during protoplast formation and transformation, and moisture content and protoplast density during regeneration. A modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes, using one of two plasmid vectors. Viomycin resistance genes on BamHI or PstI fragments were cloned from S. vinaceus genomic DNA into S. lividans, using the SLP1.2 vector. At least three different S. vinaceus BamHI fragments (1.9, 5.8, or 8.5 kilobases) confer viomycin resistance; only one PstI fragment (4.3 kilobases) was found. Recombinant plasmids were all able to produce lethal zygosis and to be transferred by conjugation within S. lividans. SCP2 was used to clone S. coelicolor A3(2) genes that "complemented" the auxotrophic mutation hisD3, argA1, or guaA1. Recombinant DNA technology can now be applied to economically and academically interesting problems unique to streptomycete molecular biology.     

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 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.