Examination of the Regulation of Biofilm-Specific Antibiotic Resistance Genes in Pseudomonas Aeruginosa

Upon contact with a surface, the bacterium Pseudomonas aeruginosa forms a bacterial community encased in an exopolysaccaride matrix. One difference between this structure, known as a biofilm, and free-swimming cells is its ability to be 10 to 1,000 times more resistant to antibiotics. This increased resistance is partially due to the presence of six biofilm-specific antibiotic resistance genes. Three of these genes, ndvB, PA1874-77 and tssC1 have been characterized in terms of their role in antibiotic resistance, while the other three have not been fully characterized. However, all six genes are more highly expressed in biofilm cells as compared to planktonic cells and contribute to a biofilm-specific antibiotic resistance phenotype. Previous in silico analysis found a 22 base pair consensus sequence upstream in the promoters of all six biofilm-specific antibiotic resistance genes. It is hypothesized that this motif controls the biofilm-specific expression of these genes. Results from a DNA-affinity chromatography experiment suggest that several proteins from a biofilm extract bound the 22bp motif of ndvB and PA1874-77. However, one major band was consistently binding the 22bp motif. It is predicted that the regulatory protein that binds to the 22bp motif will play a prominent role in conferring a biofilm-specific antibiotic resistance phenotype. This protein was identified as a putative transcriptional regulator. Further experimentation using this protein in the binding reaction found that this interaction may not be specific to the 22bp motif and further investigation is warranted. A second separate but related research goal was to analyze temporal expression of ndvB using promoter-fluorescent protein constructs. The reason for this study was to examine the effects of heterogeneity within biofilms on expression of biofilm-specific antibiotic resistance genes. Using fluorescence microscopy it was found that planktonically, ndvB begins to be expressed at an OD600 of 2.0, an OD600 that corresponds to early stationary phase. In biofilms ndvB expression begins following monolayer formation. These initial discoveries have highlighted the usefulness of this experimental system and laid the groundwork for a more comprehensive study of spatial and temporal expression of all six biofilm-specific antibiotic resistance genes in Pseudomonas aeruginosa