Regulation of Secondary Metabolism by Low Doses of Antibiotics in Burkholderia thailandensis

Abstract

Microbial secondary metabolites, or natural products, have served as a critical source for modern drug development. Recent genome analyses show the majority of biosynthetic gene clusters (BGCs), which are responsible for secondary metabolite production, remain unexpressed under normal culture conditions. Investigation into these “cryptic” BGCs and deciphering the regulatory mechanism could shed light on exploring strategies to discover new natural products.We addressed this question in the Gram-negative model bacterium Burkholderia thailandensis. Previous studies identified elicitors with distinct modes of action, including the antibiotics trimethoprim, β-lactams, and fluoroquinolones. We first characterized the mechanism underlying the stimulatory effect of trimethoprim and found low doses induced global changes in the transcriptome, proteome, and metabolomes. Low-dose trimethoprim treatment affected methionine biosynthesis, leading to the accumulation of the precursor homoserine, which in turn activated the silent malleilactone BGC through the pathway-specific regulator MalR. We next characterized the signaling pathway for β-lactam piperacillin. The stimulatory activity of piperacillin, and a family of β-lactams, correlated with its growth-inhibitory effect. Specifically, piperacillin induced production of reactive oxygen species (ROS), leading to DNA damage and the oxidative stress response, which through OxyR activated the silent malleilactone BGC, again via MalR. Our results, therefore, established a link between the oxidative stress response and secondary metabolism in B. thailandensis. Additionally, we explored the mechanism of induction of fluoroquinolones. Fluoroquinolones induce DNA damage and the SOS response in B. thailandensis. The SOS and oxidative stress responses partially explained fluoroquinolones’ elicitation. Subsequent transcriptomic studies revealed a novel transcriptional regulator related with oxidative stress is also involved in the regulatory network. Finally, we employed transposon mutagenesis and a high-throughput reporter guided screening approach to identify regulators of secondary metabolism. We find that the phage growth limitation (Pgl) system and the tight adherence (Tad) system genes are involved in secondary metabolism regulation, while future investigation is undergoing.