Genetics and Regulation of Bacterial Biofilms

Bacterial biofilm formation, the construction of dense, protective, multicellular communities, is a widely conserved behavior. In some bacteria, such as the Gram-positive model organism Bacillus subtilis, the genetics controlling biofilm formation are well understood. In other bacteria, however, including the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, the identities or roles of many biofilm genes remain unknown. Importantly, many proposed applications of biofilm research, particularly in the medical field, require knowledge not only of biofilm assembly but also of biofilm disassembly, the latter being a recent and underdeveloped area of study. It was previously reported that B. subtilis biofilms disassemble late in their life cycle due to the incorporation of four D-amino acids (D-leucine, D-methionine, D-tryptophan, and D-tyrosine, or D-LMWY) into peptidoglycan. It was further argued that D-LMWY specifically inhibits and disassembles the biofilms of diverse bacterial species, including B. subtilis and P. aeruginosa. Here I present a contrasting report. I describe how what had been perceived as D-LMWY-mediated biofilm inhibition is actually D-tyrosine-mediated toxicity. B. subtilis is sensitive to growth inhibition by D-tyrosine due to the absence of D-tyrosyl tRNATyr deacylase (Dtd), an enzyme that prevents the misincorporation of D-tyrosine and other D-amino acids into nascent proteins. By repairing the gene for Dtd, I was able to render B. subtilis resistant to both growth inhibition and biofilm inhibition by D-tyrosine and D-LMWY. In parallel, I recovered spontaneous mutants of B. subtilis that survive in the presence of D-LMWY. These isolates harbored mutations in pathways that regulate tRNATyr charging. Three of these mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNATyr synthetase (TyrRS), while a separate mutation improved the stereoselectivity of TyrRS. I concluded that these spontaneous D-LMWY resistance mutations were compensating for the absence of Dtd. In addition to my research on B. subtilis biofilm regulation, I demonstrated a new, non-destructive screening approach for identifying P. aeruginosa biofilm genes. Using this screen, I was able to recover a wide range of known biofilm genes as well as the new biofilm gene candidates ptsP, PA14_16550, and PA14_69700. These three genes are the focus of an ongoing study dedicated to characterizing P. aeruginosa biofilm formation, particularly as it relates to the secondary messenger cyclic di-GMP. In summary, this dissertation covers aspects of biofilm formation and dispersal in two bacterial species. My work offers mechanistic insight into D-amino acid resistance, resolves the relationship between D-amino acids and biofilms, and establishes a new tool for understanding the complexities of biofilm genetics and regulation.Biology, Molecular and Cellula