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.     

Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens

pSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single-stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling-circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M-O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.     

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.     

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.     

Genetic analysis in Streptomyces ambofaciens

A chromosomal linkage map of ten markers was established for Streptomyces ambofaciens by the four-factor cross method and allele-gradient analysis. Mutants were obtained by nitrous acid treatment as well as UV mutagenesis. The fertility of crosses was enhanced over 100-fold by pSAM2, a plasmid present in some strains of S. ambofaciens, and over 1000-fold by the conjugative plasmid pIJ303.     

The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer

pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single-stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA. This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.     

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.     

The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes.

Summary The pSAM2 element ofStreptomyces ambofaciens integrates site-specifically in the genome of differentStreptomyces species by recombination between a 58 by sequence common to the plasmid (attP) and the chromosome (attB). Southern hybridization analysis showed that sequences similar to the pSAM2attB site were found in otheractinomycetes (Mycobacterium,Nocardia,Micromonospora) as well as unrelated bacteria (Bacillus circulans,Escherichia coli,Clostridium botulinum,Bordetella pertussis, andLegionella pneumophila). Hybridizing fragments fromB. circulans andMycobacterium tuberculosis were cloned and sequenced. Comparison of these sequences with the sequence of the integration zone ofS. ambofaciens revealed a conserved region of 76 by which overlapped with theattB site. This conserved sequence was similar to theSalmonella typhimurium andE. coli tRNA _inf1 ^suppro genes as well as a number of eucaryotic tRNA genes and had a proline-tRNA-like cloverleaf structure. Furthermore, theStreptomyces lividans attB site of theStreptomyces glaucescens element pIJ408 was also found to overlap a potential tRNA gene (tRNA^thr). We note here that these two putative tRNA genes as well as those which overlap theattB site of the elements SLP1 ofStreptomyces coelicolor and pMEA100 ofNocardia mediterranei all contain the site where integrative recombination takes place. These presumptive actinomycete tRNA genes lack the 3 terminal CCA sequence found in most procaryotic tRNA genes.     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.     

Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity.

DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.     

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.