Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces

pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.     

Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens

When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.     

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.     

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.     

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.     

Plasmid formation and its relation to the formation of spontaneously developing pocks in Streptomyces azureus ATCC 14921

Streptomyces azureus ATCC 14921 harboured a plasmid pSA1 together with its chromosomal integrated sequence (pSA1_int). The att P site on the plasmid was located at ca 170 bp Bam HI- Sph I fragment by site-specific integration. The free form was generated from the integrated sequence during the development of its host mycelia in the solid culture, but not in the liquid culture. The free form seemed to elicit the formation of spontaneously developing pocks on its host mycelia in the solid culture.     

Synthesis of Conceptually New,Potentially Anti-viral,Low-Toxity,Acid- and Enzyme-stable Furanoid and Pyranoid Nucleosides

A large (80 kbp) plasmid designated as pSLL was isolated from Streptomyces laurentii ATCC 31255 (strain P0). Another covalently closed circular plasmid, pSLS (16 kbp), was found in pSLL-cured PO cells (named PI), which spontaneously formed pocks on a solid medium. The frequency of the generation of pSLL-loss PI cells coordinated positively with the frequency of pock appearance. pSLS was also found as an integrated form in the chromosome of strains PO and Pl. The results indicated that strain PO harbored two plasmids, pSLL and integrated form pSLS, and pSLS seemed to be generated as a free-form plasmid with the loss of pSLL. It was also suggested that pSLL may suppress the excision of the integrated pSLS from the chromosome and that the free form of pSLS may participate in pock formation.     

Site-specific integration in Saccharopolyspora erythraea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101.

An 11.3-kilobase-pair plasmid, designated pSE101, exists in Saccharopolyspora erythraea NRRL 2338 as an integrated sequence (pSE101int) at a unique chromosomal location and in the free form in less than an average of 1 copy per 10 chromosomes. The plasmid sequence is missing from S. erythraea NRRL 2359. Restriction maps of the free and integrated forms of pSE101 showed point-to-point correspondence. Plasmid pECT2 was constructed by ligation of pSE101, pBR322, and the gene for thiostrepton resistance (tsr). When introduced by polyethylene glycol-mediated transformation into protoplasts of S. erythraea NRRL 2359, all thiostrepton-resistant regenerants examined were found to carry a single copy of pECT2 in the integrated state at a single chromosomal site. The chromosomal site of pECT2 integration in strain NRRL 2359 (attB) corresponded to the chromosomal location of pSE101int in strain NRRL 2338. The plasmid crossover site (attP) was mapped to the plasmid site that corresponded to the site of interruption of the plasmid sequence in the host carrying pSE101int, indicating that site-specific integrative recombination had occurred. An additional 2.8-kilobase-pair chromosomal sequence homologous to a segment of pSE101 was also observed in strains NRRL 2338 and NRRL 2359. After introduction of pECT2 into Streptomyces lividans, approximately half of the transformants examined were found to carry the plasmid as a stable, autonomously replicating element. The other half carried a single copy of pECT2 as an integrated sequence, but the location of pECT2int in Streptomyces lividans varied from one transformant to another. In each case, integrative crossover used the attP site. A model is proposed to account for the determination of the particular state of pSE101 in Streptomyces lividans.     

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.     

Efficiency of the transfer of a pSAM2-derivative plasmid between two strains of Streptomyces lividans in conditions ranging from agar slants to non-sterile soil microcosms

Abstract: The aim of this work was to determine the efficiency of the conjugative plasmid pTS130 to transfer in various environmental conditions between two strains of Streptomyces lividans . This plasmid is a derivative of the conjugative and integrative plasmid pSAM2 isolated originally from Streptomyces ambofaciens and capable of transfer to a large range of bacteria. Our results demonstrate the high frequency of the conjugation mechanism since more than 60% of the recipient cells developed on agar slants harbored the plasmid pTS130 (as evidenced by Southern hybridization with a pSAM2 derivative plasmid probe). When donor and recipient strains were inoculated into sterile and non-sterile soil microcosms, transconjugants were detected after two days of incubation in both cases. However, the number of donor, recipient and transconjugant cells were established at a lower level in the non-sterile soil than in the sterile soil experiments. Moreover, nutrient amendment of the sterile soil was found to increase the population levels of parental strains and transfer frequencies both significantly and simultaneously. On the other hand, modifying water potential of the soil microcosms did not result in affecting the establishment of the Streptomyces lividans cells or the transfer rate.     

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2

Summary Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.     

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.     

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).     

Structural analysis of plasmid and chromosomal loci involved in site-specific excision and integration of the SLP1 element of Streptomyces coelicolor.

SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes.