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Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces
F Boccard1, T Smokvina, J L Pernodet
1Laboratoire de Biologie et Génétique Moléculaire, URA CNRS D 0086, Orsay, France.
Abstract:
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.
Insights
The pSAM2 plasmid efficiently integrates into Streptomyces genomes at specific attB sites. This site-specific integration mechanism is conserved across multiple unrelated Streptomyces species, indicating a broad host range for pSAM2.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Plasmids and Mobile Genetic Elements
Background:
- The pSAM2 plasmid is found integrated in Streptomyces ambofaciens genomes and as a free replicon.
- Efficient and specific integration of pSAM2 into new Streptomyces hosts was observed.
Purpose of the Study:
- To characterize the mechanism and host range of pSAM2 site-specific integration.
- To identify conserved sequences involved in pSAM2 integration across different Streptomyces strains.
Main Methods:
- Plasmid transfer experiments into Streptomyces lividans and Streptomyces ambofaciens DSM40697.
- DNA hybridization and sequence analysis to identify attachment (att) sites and conserved regions.
- Characterization of integration events in unrelated Streptomyces strains, including Streptomyces griseofuscus.
Main Results:
- Specific integration of pSAM2 occurred efficiently in S. ambofaciens and S. lividans.
- A conserved 58-bp att sequence (attP on pSAM2, attB on host DNA) mediates integration.
- Integration sites in different strains shared homology, particularly around an open reading frame (ORF A) near attB.
- Site-specific integration was demonstrated in three unrelated Streptomyces strains, including S. griseofuscus.
Conclusions:
- pSAM2 utilizes a site-specific integration mechanism involving conserved att sequences in Streptomyces.
- The identified integration mechanism exhibits a wide host range within the Streptomyces genus.
- Conserved regions, including ORF A, likely play a role in the integration process.