actinorhodin and avermectin

A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.

Topics: Actins; Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Binding Sites; DNA-Binding Proteins; Feedback, Physiological; Gene Expression Regulation, Bacterial; Gene Order; Helminth Proteins; Ivermectin; Molecular Sequence Data; Mutation; Oligomycins; Phenotype; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Streptomyces coelicolor

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-andconstitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the lowproducer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

Topics: Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Regulator; Ivermectin; Streptomyces

A transcriptional activator for actinorhodin biosynthesis, AtrA, was previously characterized in Streptomyces coelicolor A3(2), and an orthologue of atrA, named aveI, is identified in the Streptomyces avermitilis NRRL8165 genome (Uguru et al., Mol Microbiol, 58:131-150, 2005). In this study, genetic and functional characterization of aveI gene was reported. Deletion of aveI gene led to increased biosynthesis of avermectin B1a by about 16-fold. The increased synthesis of avermectin B1a was suppressed by complementation with either aveI gene or its orthologue gene atrA from S. coelicolor, suggesting AveI and AtrA shared the similar functionality and were negative regulators for avermectin biosynthesis in S. avermitilis. However, when aveI was introduced into S. coelicolor on a multi-copy plasmid, the production of actinorhodin was significantly increased, indicating that aveI had a positive effect on actinorhodin biosynthesis in S. coelicolor, the same as its orthologue atrA. Electrophoretic mobility shift assays revealed AveI can bind specifically to the promoter region of actII-ORF4 in vitro but not that of aveR. Although its mechanism still needs to be defined, the species-differential regulation by the same regulator may represent an example of the evolutional strategy that enables bacteria to adapt the existing molecular machinery to a variety of functionalities for growth and survival.

Topics: Anthraquinones; Bacterial Proteins; Gene Expression; Gene Expression Regulation, Bacterial; Ivermectin; Polyketide Synthases; Promoter Regions, Genetic; Repressor Proteins; Sequence Deletion; Streptomyces; Transcription Factors