Identification of a butenolide signaling system that regulates nikkomycin biosynthesis in Streptomyces

Abstract

Butenolides are an emerging family of signaling molecules in Streptomyces. They control complex physiological traits, such as morphological differentiation and antibiotic production. However, how butenolides regulate these processes is poorly investigated because of obstacles in obtaining these signaling molecules. This study reports the identification of a butenolide-type signaling system for nikkomycin biosynthesis in Streptomyces ansochromogenes with distinct features. We identified a gene cluster, sab, consisting of three genes, sabAPD, for butenolide biosynthesis and two regulator genes, sabR1 and sabR2, and characterized three butenolides (SAB1,-2, and-3) by heterologous expression of sabAPD. sabA disruption abolished nikkomycin production, which could be restored by the addition of SABs or by deletion of sabR1 in ⌬sabA. Electrophoretic mobility-shift assays and transcriptional analyses indicated that SabR1 indirectly represses the transcription of nikkomycin biosynthetic genes, but directly represses sabA and sabR1. In the presence of SABs, the SabR1 transcriptional regulator dissociated from its target genes, verifying that SabR1 is the cognate receptor of SABs. Genome-wide scanning with the conserved SabR1-binding sequence revealed another SabR1 target gene, cprC, whose transcription was strongly repressed by SabR1. Intriguingly, CprC positively regulated the pleiotropic regulatory gene adpA by binding to its promoter and, in turn, activated nikkomycin biosynthesis. This is the first report that butenolide-type signaling molecules and their cognate receptor SabR1 can regulate adpA via a newly identified activator, CprC, to control nikkomycin production. These findings pave the way for further studies seeking to unravel the regulatory mechanism and functions of the butenolide signaling system in Streptomyces.

Regulation of Antibiotic Production by Signaling Molecules in Streptomyces

“…It is therefore necessary to identify more hormone-like signaling molecules. Currently, several approaches have been used to discover new signaling molecules in Streptomyces, including deletion of gene encoding repressor of signaling molecule biosynthetic gene (Sidda et al, 2016), bioassay using disruptants of known signaling molecule biosynthetic genes as indicator strains (Thao et al, 2017;Nguyen et al, 2018), and heterologous expression of putative signaling molecule biosynthetic genes in model microorganisms (Wang et al, 2018). These approaches will speed up the process of identifying new signaling molecules, and their signal transduction pathways.…”

“…Representative γ-butenolides include two butenolides (SRB1 and SRB2) from Streptomyces rochei (Arakawa et al, 2012;Arakawa, 2014), the avenolide from Streptomyces avermitilis (Kitani et al, 2011), four butenolides from Streptomyces albus J1074 (Nguyen et al, 2018), and three butenolides (SAB1-3) from Streptomyces ansochromogenes (Wang et al, 2018; Figure 1). Of special note is the avenolide that is necessary for triggering avermectin production in S. avermitilis (Kitani et al, 2011).…”

“…Within the gene cluster, sabA encodes an AfsAlike enzyme, whereas sabP and sabD encode phosphatase and dehydrogenase enzymes, respectively. Heterologous expression of sabAPD in E. coli and Streptomyces led to the identification of three novel butenolides (SAB1, 2, and 3) (Wang et al, 2018). Since there are only limited numbers of natural GBLs and γ-butenolides identified in Streptomyces, these studies provide effective strategies for the discovery of new signaling molecules, which are normally produced in very small quantities.…”

“…CSR activator gene sanG and biosynthetic genes of the sanF-X operon (Figure 2; Pan et al, 2009;Wang et al, 2018). Of the three GBL receptors in S. avermitilis, AvaR1 acts as an avenolide receptor to control avenolide and avermectin production by directly repressing the transcription of aco, aveR, its own gene and the other two GBL receptor homologous genes (avaR2 and avaR3) (Figure 3; Zhu et al, 2017).…”

“…Of the three GBL receptors in S. avermitilis, AvaR1 acts as an avenolide receptor to control avenolide and avermectin production by directly repressing the transcription of aco, aveR, its own gene and the other two GBL receptor homologous genes (avaR2 and avaR3) (Figure 3; Zhu et al, 2017). ScbR2, JadR2, AvaR2 and SabR2 were identified as pseudo GBL receptors (Xu et al, 2010;Zhu et al, 2016;Wang et al, 2018), and the roles of these pseudo GBL receptors in the regulation of antibiotic biosynthesis will be discussed in a subsequent section. AvaR3 contains an extra 75amino acid stretch that is not present in typical GBL receptors, and promotes avermectin production through a yet-unknown regulatory mechanism (Miyamoto et al, 2011).…”