Annick Friedmann

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.

KorSA from the Streptomyces Integrative Element pSAM2 Is a Central Transcriptional Repressor: Target Genes and Binding Sites

pSAM2, a 10.9-kb mobile integrative genetic element from Streptomyces ambofaciens, possesses, as do a majority of Streptomyces conjugative plasmids, a kil-kor system associated with its transfer. The kor function of pSAM2 was attributed to the korSA gene, but its direct role remained unclear. The present study was focused on the determination of the KorSA targets. It was shown that KorSA acts as a transcriptional repressor by binding to a conserved 17-nucleotide sequence found upstream of only two genes: its own gene, korSA, and pra, a gene positively controlling pSAM2 replication, integration, and excision. A unique feature of KorSA, compared to Kor proteins from other Streptomyces conjugative plasmids, is that it does not directly regulate pSAM2 transfer. KorSA does not bind to the pSAM2 genes coding for transfer and intramycelial spreading. Through the repression of pra, KorSA is able to negatively regulate pSAM2 functions activated by Pra and, consequently, to maintain pSAM2 integrated in the chromosome.

Functional analysis of the Streptomyces ambofaciens element pSAM2

pSAM2 is an 11-kb element integrated in the Streptomyces ambofaciens ATCC23877 genome and found additionally as a free replicon present at several copies per chromosome in strain JI3212, the derivative of ATCC23877 isolated after uv irradiation. In spite of its small size, this element specifies numerous functions including maintenance, site-specific integration, self-transmissibility, pock formation, and mobilization of chromosomal markers. After transfer of the free form of pSAM2 to Streptomyces lividans, the free and the integrated forms coexist. A functional map of pSAM2 was deduced from phenotypes exhibited in S. lividans by numerous deletion or insertion derivatives. In addition to the previously characterized regions sufficient for site-specific integration we have shown that separate regions are involved in either plasmid maintenance as a free molecule, plasmid transfer, and pock formation. Transfer of pSAM2 could depend on its ability to be maintained in a free form, since plasmids deficient in this function are transferred at very low frequency. Deletions of some regions of the plasmid are lethal for the plasmid or the host, but if some other regions are deleted simultaneously, transformants can be obtained.

Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens.

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2 which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.

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

SummaryStreptomyces 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).

Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens.

pSAM2 is integrated into the Streptomyces ambofaciens chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) site. The 43 kDa integrase protein encoded by pSAM2 catalyses this recombination event. Tools have been developed to study site-specific recombination in Escherichia coli. In vivo studies showed that a 360 bp fragment of attP is required for efficient site-specific recombination and that int can be provided in trans. pSAM2 integrase was purified and overexpressed in E. coli and Int binding at the attP site was studied. DNaseI footprinting revealed two sites that bind integrase strongly and appear to be symmetrical with regard to the core site. These two P1/P2 arm-type sites both contain a 17 bp motif that is identical except at one position, GTCACGCAG(A/T)TAGACAC. P1 and P2 are essential for site-specific recombination.