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Title: Decoding the regulation of cryptic secondary metabolism in Burkholderia thailandensis
Authors: Mao, Dainan
Advisors: Seyedsayamdost, Mohammad R
Contributors: Chemistry Department
Keywords: bacterial virulence
cryptic secondary metabolism
mode of activation
Natural product
transcriptional regulator
Subjects: Chemistry
Issue Date: 2018
Publisher: Princeton, NJ : Princeton University
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.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog:
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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