UNCOVERING THE ROLE OF OXIDATIVE STRESS IN MODULATING BACTERIAL SECONDARY METABOLISM

Abstract

Microorganisms produce an extraordinary diversity of secondary metabolites that have served as drugs for decades. These compounds are synthesized by dedicated biosynthetic gene clusters (BGCs), sets of contiguous genes that facilitate co-regulation and identification. Most BCGs are considered ‘silent’ as they are minimally expressed or transcribed in standard laboratory growth conditions leading to negligible compound amounts. Tackling this problem at the regulatory level allows for a greater understanding of the connections between signal inputs and outputs of secondary metabolites. To that end, we investigated the regulatory mechanisms underlying silent BGCs with two disparate Gram-negative bacterial strains, Burkholderia thailandensis, a model strain for the pathogen Burkholderia pseudomallei, and Phaeobacter inhibens, which participates in a unique symbiosis with the microalga Emiliani huxleyi. For B. thailandensis, we have previously screened large libraries of bioactive compounds that identified multiple drug classes as broad natural product elicitors. Herein, we have characterized the stimulatory influence of the phenothiazine antipsychotics and found a focused response in the transcriptome and secondary metabolome. Phenothiazine treatment elicited production of the cryptic polyketide malleicyprol through the oxidative stress response regulator, OxyR, and a two-component system. New elicitors that were missed in our previous screens, the polymyxins, were revealed by our work with the phenothiazines. We characterized the effect of these antimicrobial peptides and found that the oxidative stress response through OxyR was the main regulatory actor that led to malleicyprol biosynthesis. A byproduct of these studies was the fortuitous discovery of novel cryptic metabolites from B. thailandensis including 4-Hydroxy-3-methyl-2(1H)-quinolone and N-acetyl-bactobolin analogs that were elicited by the phenothiazines. Finally, we examined the dynamic phases of the symbiosis between P. inhibens and E. huxleyi through the inducing effects of the phenylpropanoid p-coumaric acid (pCA). Transcriptional, genetic, and biochemical analyses revealed thay marginally toxic pCA leads to production of the algaecidal roseobacticides through generation of reactive oxygen species. These results have revealed the immense influence that oxidative stress has on bacterial secondary metabolism. We hope to investigate these pathways in other species to elucidate a potential universal regulatory mechanism that may aid in uncovering the wealth of cryptic natural products encoded in bacteria.