Decoding the regulation of cryptic secondary metabolism in Burkholderia thailandensis

Abstract

For decades, bacterial secondary metabolites have served as a major source of drugs and drug

leads. Recent genome analyses have indicated that the majority of biosynthetic gene clusters

(BGCs), which are responsible for production of these metabolites, are not expressed under

normal laboratory conditions. These so-called “cryptic” gene clusters represent an untapped

source of potential therapeutics and a better understanding of how they are silenced would have

a profound impact on the development of effective strategies to discover new natural products.

We addressed this question in the Gram-negative model bacterium Burkholderia thailandensis

using genetic, transcriptomic, metabolomics, and chemical approaches. We discovered a

quorum-sensing-regulated LysR-type transcriptional regulator, which we named ScmR, as a

global regulator of secondary metabolism and a repressor of numerous BGCs. Transcriptionally

and metabolically, the scmR deletion mutant displayed a hyperactive phenotype relative to wild

type and overproduced a number of compound families. Phenotypically, the scmR deletion

mutant displayed aberrant colony morphology, enhanced biofilm formation, and increased

virulence against Caenorhabditis elegans. A model for how the interplay of ScmR with quorum

sensing and pathway-specific transcriptional regulators coordinately silences cryptic metabolites

is proposed.

Extracellular small molecules, mainly sub-inhibitory concentrations of antibiotics such as

trimethoprim (Tmp), b-lactams, and fluoroquinolones, have been found to be effective inducers

of cryptic BGCs. We investigated the mechanism underlying this phenomenon and found that

exposure of B. thailandensis to low doses of Tmp transcriptionally activated BGCs through

inhibition of folate pathway and quorum sensing. Tmp treatment also led to increased ribosome

biogenesis and amino acid and NTP pools. Together, our studies indicate that Tmp

transcriptionally, translationally, and metabolically bolsters natural product biosynthesis.

Finally, to explore other important genes involved in natural product regulation, we employed

a transposon (Tn) mutagenesis approach coupled with reporter-guided screening to activate the

cryptic malleilactone gene cluster in B. thailandensis. We find that disruption of pyrF, the gene

encoding the orotidine 5’-phosphate decarboxylase, results in activation of malleilactone

biosynthesis. These results highlight the complex regulatory pathways that silence BGCs. Future

application of this Tn-guided discovery approach in other organisms promises to unearth new

cryptic natural products.