Project description:Activation of cryptic biosynthetic gene clusters (BGCs) in actinomycetes often requires strong and reliable promoters, yet native promoters from rare actinomycetes remain scarce. Here, we used transcriptome analysis of Actinoplanes siamensis TBRC 4237 to identify highly expressed genes and selected 22 upstream regions as candidate constitutive promoters. Their activities were evaluated in Streptomyces coelicolor M1146 using indigoidine synthetase and latex-clearing protein (LCP) as heterologous reporters. Thirteen promoters drove indigoidine production at levels exceeding those of kasOp* and ermEp*, the widely used strong constitutive promoters in Streptomyces. Several promoters also yielded robust LCP activity, with promoter 650A achieving the highest expression across both reporter systems. Motif analysis showed no clear correlation between predicted -10/-35 elements and promoter strength, indicating additional regulatory features. These natural promoters provide valuable genetic parts for heterologous expression, BGC activation, and pathway refactoring in actinomycetes.
Project description:Lincomycin is a lincosamide antibiotic that forms cross-links within the peptidyl transferase loop region of the 23S rRNA of the 50S subunit of the bacterial ribosome, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains. We aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes. Therefore, the dose-dependent response of lincomycin on gene expression of the model actinomycetes Streptomyces coelicolor A3(2) and possible relationships to secondary metabolism have been investigated.
Project description:DNA replication is a fundamental process in biology with initiation marking its key, first step. In bacteria, DNA replication is initiated by the DnaA protein. DnaA exhibits multidomain architecture, consisting of an N-terminal domain I, linker region, AAA+ family ATPase cassette, and C-terminal DNA-binding motif. Taxon-specific regulatory functions are primarily coordinated by the DnaA domain I, which exhibits substantial sequence variation across bacteria. Notably, although the DnaA domain I has been shown to be essential, its contributions to initiation are not completely understood. Importantly, studies have indicated a role for DnaA domain I dimerization in the cooperative assembly of the initiation complex at the origin. However, the mechanism(s) and molecular basis of DnaA domain I dimerization have proved elusive. Here, we report structures of the DnaA domain I from ten Actinobacterial species. Strikingly, all structures reveal the same, unique dimer, and key elements which support DnaA domain I self-interaction are broadly conserved across the class Actinomycetes. Further, a suite of biochemical oligomerization assays and HDX-MS studies support the structural dimer. These findings suggest weak dimerization, as mediated by the DnaA domain I, acts a fine-tuned trigger in cooperative oriC assembly and that this is a broadly conserved biological mechanism for replication initiation in the class Actinomycetes.