Agents Provocateur: Exploiting bacterial biofilm stimulation to identify bioactive small molecules

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Most bacteria grow as surface-associated biofilms, with reduced susceptibility to antibiotics and disinfectants. Biofilms are important in industrial, agricultural, and medical settings, prompting searches for ways to modulate their formation. While searching for biofilm inhibitors, we identified diverse molecules that stimulated Pseudomonas aeruginosa biofilm formation at sub-inhibitory concentrations. We hypothesize that biofilm stimulation by toxic molecules is a defensive response. We will pursue two objectives: 1) Determine how P. aeruginosa perceives and responds to sub-inhibitory levels of noxious molecules. Sub-inhibitory concentrations of diverse antibiotics, detergents, and even some antidepressants, anti-emetics, and anti-cancer drugs stimulate biofilm formation of many bacterial species. Whether each evokes a unique response or if they converge on a common damage or stress response is unknown. A graduate student will: a) grow an ordered P. aeruginosa PA14 transposon mutant library as biofilms with stimulatory molecules to identify mutants unable to respond to uncover the pathways involved; b) grow a P. aeruginosa lux promoter reporter library as biofilms with/without stimulators to identify those promoters whose activity is altered; and c) perform transcriptional profiling (RNAseq) of biofilms grown with/without sub-inhibitory concentrations of select stimulators, to compare patterns of gene expression. Select promoter subsets that are reliably up-regulated by biofilm stimulators will be further developed as tools (Obj. 2). 2) Use the biofilm-stimulation phenotype to uncover new physiology and chemistry. Informed by the pathways identified in Obj 1, a postdoctoral fellow will a) develop and validate transcriptional reporters coupled to promoters that are activated by biofilm-stimulatory molecules. These tools will be used to ask b) at what concentration of a given molecule do we see promoter activation compared with the stimulation response? Do bacteria that are resistant to stimulators (and the damage they cause) respond in the same way as a susceptible strain? Are mutants that are hyper-biofilm formers pre-activated' for the pathways of interest? We will also leverage our validated reporters in a complementary ORF-funded project, Solving the antibiotic resistance crisis', to uncover new molecules present at sub-IC in complex mixtures. Significance: This work will uncover the basic mechanisms by which bacteria detect and respond to toxic molecules with increased biofilm formation, a common phenotype, and will point to new and better ways to manipulate biofilm development. The results will prove informative for scenarios in which increased biofilm formation may be desirable e.g. in wastewater treatment, extraction of minerals from mine tailings, development of pre- and probiotics, or plant root colonization for growth promotion.