Molecular Genetics Of Genomic Island Containing DNA Sulfur Modification System In Streptomyces Lividans

The physical map for S. lividans 66 was not easily achieved due to its degradation in electrophoresis, especially in PFGE. In this research, the physical map for S. lividans 1326 was achieved based on that for ZX1, a DNA stable mutant, on which a ca. 90 kb AseI fragment was absent accompanied with changes for another two AseI fragments. Using the southern blot, we determined that these DNA pattern changes were caused by the insertion of the ca. 90kb between AseI-Aa (1800 kb) and AseI-Ab (900 kb) in S. lividans 66. Further analysis on the deletion and fusion sequences harbored on the clones obtained by Ailing Li reveals that there are 15 bp direct repeats at both end of the genomic island in S. lividans 66. After precise deletion and fusion of the genomic island mediated by the 15 repeat in ZX1, only one 15 bp was remained and its 1 kb flanking sequence shared 100% identity to corresponding sequence in S. coelicolor M145. The insertion site is the last five 15 coding sequence of SCO5998.In previous reports, the actinomycetes resistance geneφHAU3R and the dnd gene cluster was present on this genomic island. Put together all above the comparative analysis, this 90 kb DNA sequence was implied to be acquired by the S. lividans via gene horizontal transfer. The complete sequence (92,770 bp) of a genomic island (GI) named SLG from Streptomyces lividans 66 was determined. Its overall G+C content was 67.8%, lower than those of three sequenced Streptomyces genomes. Among eighty-five predicted open reading frames (ORFs) in SLG, twenty-two ORFs showed little homology with previously known proteins. SLG displays a mosaic structure composed of four modules, indicative of multiple recombination events in its formation. Spontaneous excision and circularization of SLG was observed, and the excision rate appeared to be induced at least five-fold by MNNG exposure. Using constructed mini-islands of SLG, we demonstrated that Slg01, a P4-like integrase, was sufficient to promote SLG integration, excision and circularization. Eleven counterpart dnd clusters, which also mapped to GIs in ten chromosomes and a plasmid, were found in taxonomically unrelated bacterial species from various geographic niches. Additionally, ca. 10% of actinomycetes were found to possess a dnd cluster in a survey involving 74 strains. Comparison of dnd clusters in the twelve bacteria strongly suggests that these dnd-bearing elements might have evolved from a common ancestor similar to plasmid-originated chromosome II of Pseudoalteromonas haloplanktis TAC125. By the resistant marker labeling in different places on SLG, we tried reconstitution of the SLG inter-species or intra-species transfer in standard laboratory condition. These attempts were all turned out to be failures, suggesting that SLG might lost the capability for gene transferring during the long periods of evolution. This was consistent with the observation that no intact conjugation genes was detected and SLG mosaic structure.Finally, we demonstrate that there exists an strong restriciton system to the DNA sulfur modification in S. coelicolor M145.