Sansanmycin Biosynthesis Gene Cluster Of Cloning And Biosynthesis Regulate Gene Ssaa Regulatory Mechanism Research

Sansanmycins, isolated from Streptomyces sp. SS, are members of a uridyl peptide antibiotic family including closely related pacidamycins, napsamycins and mureidomycins. Sansanmycins were reported to exert antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa, and more interesting, Mycobacterium tuberculosis H37RV and multidrug-resistant M. tuberculosis strains. The biosynthetic gene clusters for pacidamycin and napsamycin have been cloned, and most of the pacidamycin biosynthetic genes have been characterized. However, there were no reports about the transcriptional regulation of the uridyl peptide antibiotics prior to the present work.The sansanmycin biosynthetic gene cluster (ssa) was identified in Streptomyces sp. SS by a combination of genome mining approach and conventional probing and cosmid sequencing, comprised of25ORFs showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster. The structure homology search of SsaA using the online program HHpred revealed that it combined an N-terminal forkhead-associated (FHA) domain with a C-terminal helix-turn-helix DNA-binding motif, suggesting it might be a pathway-specific regulator for sansanmycin biosynthesis. To investigate the contribution of ssaA to the regulation of sansanmycin biosynthesis, the coding region of ssaA was cloned into plasmid pL646under the control of a strong constitutive promoter ermE*p, and the recombinant plasmid were introduced into Streptomyces sp. SS by conjugation. Bioassay and HPLC analyses revealed that overexpression of ssaA increased sansanmycin production, suggesting ssaA is a positive regulator for sansanmycin biosynthesis. To confirm the role of ssaA in sansanmycin biosynthesis, the ssaA disrupted mutant SS/AKO was obtained via PCR-targeting method. Bioassay and HPLC analyses revealed that disruption of ssaA completely abolished sansanmycin production, which was restored by introducing intact ssaA into SS/AKO. These results comfirmed that ssaA is indeed a pivotal positive regulator for sansanmycin biosynthesis. Quantitative RT-PCR was performed to investigate the relative mRNA level o sansanmycin biosynthetic genes in SS/AKO. The results showed that the relative mRNA level of five structural genes, ssaH, ssaN, ssaP, ssaX and ssaC was decreased, indicatinj that ssaA could positively regulate the sansanmycin biosynthesis by controlling the expression of the structural genes of ssa cluster.SsaA was overexpressed in E. coli BL21(DE3) as His10-tagged protein. EMSA result: showed that His10-SsaA bound to several promoter regions of the ssa cluster, including ssaHp, ssaN-Pp, ssaU-Ap, ssaC-Dp and ssaWp, suggesting that SsaA controlleo sansanmycin biosynthesis by directly binding to the promoter regions of biosynthetio genes. The binding sites of SsaA in ssa promoter regions were identified by DNase footprinting analysis, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA-binding sequence, GTMCTGACAN2TGTCAGKAC, which consisted of two9-bp inverted repeats (IRs) separated by a2-bp linker. The identified SsaA consensus binding site was validated by mutation or deletion of the most conserved CTGAC sequence in IRs.EMSA results showed that SS-A or SS-H inhibited the band-shifting of ssaC-Dp anc ssaU-A-lp caused by SsaA in a concentration-dependent manner. SPR analysis results showed that a concentration-dependent inhibition of SsaA binding to ssaC-Dp-3fragment immobilized on an SA sensor chip was readily observed when the amount of SS-A was increased. All these results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. These results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. Further more, SPR analysis results showed that SsaA could direct interact with SS-A or SS-H. These results indicated that SsaA may strictly control the production of sansanmycins at transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end-products.In conclusion, in this work, the biosynthetic gene cluster of uridyl peptide antibiotic sansanmycins was identified for the first time. It has comfirmed that SsaA is a novel class of pathway-specific transcriptional activator of sansanmycin biosynthesis, and that the end products can bind to and change the activity of SsaA to autoregulate the antibiotic production.