The Pathway-Specific Regulator ClaR of Streptomyces clavuligerus Has a Global Effect on the Expression of Genes for Secondary Metabolism and Differentiation

Streptomyces clavuligerus claR::aph is a claR-defective mutant, but in addition to its claR defect it also carries fewer copies of the resident linear plasmids pSCL2 and pSCL4 (on the order of 4 x 10(5)-fold lower than the wild-type strain), as shown by qPCR. To determine the function of ClaR without potential interference due to plasmid copy number, a new strain, S. clavuligerus Delta claR::aac, with claR deleted and carrying the wild-type level of plasmids, was constructed. Transcriptomic analyses were performed in S. clavuligerus Delta claR::aac and S. clavuligerus ATCC 27064 as the control strain. The new Delta claR mutant did not produce clavulanic acid (CA) and showed a partial expression of genes for the early steps of the CA biosynthesis pathway and a very poor expression (1 to 8%) of the genes for the late steps of the CA pathway. Genes for cephamycin C biosynthesis were weakly upregulated (1.7-fold at 22.5 h of culture) in the Delta claR mutant, but genes for holomycin biosynthesis were expressed at levels from 3- to 572-fold higher than in the wild-type strain, supporting the observed overproduction of holomycin by S. clavuligerus Delta claR::aac. Interestingly, three secondary metabolites produced by gene clusters SMCp20, SMCp22, and SMCp24, encoding still-cryptic compounds, had partially or totally downregulated their genes in the mutant, suggesting a regulatory role for ClaR wider than previously reported. In addition, the amfR gene was downregulated, and consequently, the mutant did not produce aerial mycelium. Expression levels of about 100 genes in the genome were partially up-or downregulated in the Delta claR mutant, many of them related to the upregulation of the sigma factor-encoding rpoE gene.