Lidamycin Produce Preliminary Study Atra Homologous Gene And Its Function In Bacteria

Lidamycin, a novel enediyne antitumor antibiotic produced by S. globisporus C-1027, is composed of a chromophore and an apoprotein. Lidamycin biosynthetic gene cluster has been cloned, sequenced and characterized. Based on the bioinformatic analysis, at least five genes encode putative regulatory genes, i.e., sgcR1, sgcR2, sgcR3, sgcR and sgcM. However, there is no report on the regulatory gene of Lidamycin biosynthesis outside the biosynthetic gene cluster. In 2005, Uguru et al. described the identification and characterization of a transcriptional factor, designated AtrA, that regulated transcription of actâ…¡-orf4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. After that, an orthologue of atrA in S. griseus and S. avermitilis were identified in succession, which played a positive role in the production of streptomycin and a negative role in the production of avermectin B1a respectively. These results suggest the possibility of existance of a putative orthologue of atrA in S. globisporus C-1027.First of all, the expression of S. coelicolor AtrA in S. globisporus C-1027 resulted in a DNA binding-dependent reduction in lidamycin production, which suggested that the orthologue of atrA in S. globisporus C-1027 may have a regulatory role in the lidamycin biosynthesis. Then, the atrA gene and flanking gene fragments were successfully amplified with the primers designed by the alignment of amino acid sequences of AtrA and flanking proteins of distinct Streptomyces. The alignment of amino acid sequences suggests, S. globisporus C-1027 AtrA shares highly conservative sequences with other homologous AtrA proteins, for the percentage of indentity ranging from 65% to 87% and the percentage of similarity ranging from 70% to 89%.To investigate the roles of atrA involved in the biosynthetic pathway of lidamycin, gene inactivation experiment was conducted. An apramycin resistant vector pKC1139 was used for constructing disruption plasmid that consisted of a fragment of atrA left side flanking region, a thiostrepton-resistance gene (tsr), and another fragment of atrA right side flanking region. The recombinant plasmid was transferred into S. globisporus C-1027 through conjugation between E. coli ET12567/pUZ8002 and S. globisporus C-1027. The transconjugatants that had undergone gene replacement via double cross-over were resistant to thiostrepton and sensitive to apramycin. Disruption of atrA was confirmed by PCR and Southern blot analysis of restriction enzyme digested total genomic DNA. atrA disruption mutant was obtained and named S. globisporus atrAKO. Fermentation broth of S. globisporus atrAKO was collected for bioassay against B. subtilis. The antibacterial activity of S. globisporus atrAKO was lower compared with the antibacterial activity of S. globisporus C-1027. This result suggested that atr A played a positive role in regulating the lidamycin biosynthesis.