Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

Abstract

Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the clt cluster encoding cyclothiazomycin from Streptomyces hygroscopicus 10-22 was revealed by genome sequencing in S. hygroscopicus 5008. Genes SHJG8833 and SHJG8837 of the cluster and flanking gene SHJG8838 were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that SHJG8833, but not SHJG8837 or SHJG8838, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of SHJG8833 was located to a thymidine 54 nt upstream of the start codon. Inactivation of SHJG8833 abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing SHJG8833 into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from SHJG8826 to SHJG8832, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons, SHJG8826-27 and SHJG8828-32. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of SHJG8826 and SHJG8828 and sequences internal to SHJG8826 and SHJG8829, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the clt cluster.