Multiple Photon Microscopy for Bacterial Biofilm Plasmid Dynamics

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This project seeks to develop dynamic multiple photon laser microscopic methods to non-invasively quantify plasmid retention, transfer, and expression within model bacterial biofilms.Recent literature has confirmed that adherent bacteria are phenotypically different than their freely suspended counterparts. Being bound within a biofilm may also enhance the segregational stability, expression, and rate of conjugative transfer of plasmid-DNA between bacteria. This is important since plasmid-bearing bacteria, in a biofilm, may transfer genes and phenotypes (e.g., disinfectant and antibiotic resistance, xenobiotic degradation capacity) more readily to neighboring bacteria. To date, little quantitative data exists regarding plasmid retention, transfer, and expression in mixed culture biofilm ecosystems. Specific aims are to: (1) Develop recombinant bacterial species and recombinant plasmid(s) that will allow non-invasive microscopic determination of areal concentrations of all biofilm inhabitants including plasmid-bearing hosts, plasmid-free recipients, and transconjugants in a developing model biofilm; (2) Evaluate the segregational and structural stability of the transfer recombinant plasmid in developing pure cultures; (3) Refine existing non-invasive microscopic methods to quantify plasmid retention and transfer within developing binary-and tertiary culture biofilms; and (4) Quantify plasmid transfer kinetics and heterologous gene expression within a model tertiary culture biofilm community as a function of environmental conditions (nutrient concentration, O2 concentration, pH), donor species (gram positive vs. gram negative), and donor fluid phase concentration.