2020 Virtual AIChE Annual Meeting

(330d) Revealing Genetic Functions during Bacterial Adhesion and Biofilm Formation Using Barcode Transposon Mutant Library

Authors

Song, F. - Presenter, Lawrence Berkeley Nat Lab
Illouz, S., University of California Berkeley
Arkin, A. P., University of California, Berkeley
Deutschbauer, A. M., Lawrence Berkeley National Lab
Many bacteria live associated with material surfaces. This sessile mode of bacterial cells is phenotypically different from their planktonic counterparts, and often lead to chronic infections in humans and biofouling in water pipes. However, the genetic functions and regulations in bacterial adhesion and biofilm formation remain largely unknown. There are multiple difficulties with gaining a comprehensive molecular understanding of these processes. First, many genes are involved in bacterial adhesion and biofilm formation, and the regulatory pathways change at different steps. Second, the genetic functions are dependent on the genetic background of the host strain, the material surface properties, and environmental factors. Thus, the bacterial adhesion and biofilm formation of one bacteria on one material are often not the same as the bacterial adhesion and biofilm formation of another bacteria on another material. To have a systematic understanding of bacterial adhesion and biofilm formation, a high-throughput assay is required. Here, we developed a high-throughput screening assay to reveal the functions of 4000 genes during bacterial adhesion and biofilm formation in 2 days, using a barcoded transposon mutant library.

To demonstrate the method, we used an E. coli K-12 barcoded transposon mutant library as the model strain and polydimethylsiloxane (PDMS) as model material to test bacterial adhesion and biofilm formation under various conditions. By tracking the mutation fitness on surfaces, this method successfully identified the overall genetic functions in different steps of biofilm formation. For example, lipopolysaccharides (LPS) synthesis pathway and TolA-TolQ-TolR complex are important for the bacterial adhesion. In particular, short LPS decreases the bacterial adhesion. These results are consistent with previous reports based on both the classical Derjaguin-Landau-Verwey-Overbeek theory prediction and the experimental observation. Moreover, the genetic functions of early stage biofilm formation are different from those during adhesion. Type I fimbriae and flagella are the key players for E. coli staying and growing on PDMS during early biofilm formation. Loss of type I fimbriae causes low biofilm formation compared to wild-type cells; fimbriae promotion significantly increases the biofilm formation. But this phenomenon does not occur in the bacterial adhesion. Similarly, although the mutations of LPS and TolA-TolQ-TolR complex exhibit defects in adhesion, most of them do not show defects in early biofilm formation, highlighting that genetic functions change during different processes and suggesting that bacterial adhesion inhibition may not be successful to inhibit biofilm formation. In addition to these genes, multiple hypothetical proteins were identified to influence biofilm formation as well.

This high-throughput method is a powerful tool for systematically revealing the genetic functions in bacterial adhesion and early biofilm formation. Ultimately, by measuring the genetic functions in various bacteria, materials, and environmental factors, we expect to discover the fundamental principles of bacterial adhesion and biofilm formation.