Multiple Photon Microscopy for Bacterial Biofilm Plasmid Dynamics

该项目旨在开发动态多光子激光显微方法，以非侵入性地量化模型细菌生物膜内质粒的保留、转移和表达。最近的文献证实，粘附细菌在表型上不同于它们的自由悬浮对应物。结合在生物膜内也可以提高分离稳定性、表达和质粒dna在细菌之间的共轭转移率。这一点很重要，因为在生物膜中携带质粒的细菌可以更容易地将基因和表型（例如，消毒剂和抗生素耐药性，异种生物降解能力）转移给邻近的细菌。迄今为止，关于质粒在混合培养生物膜生态系统中的保留、转移和表达的定量数据很少。具体目标是：(1)开发重组细菌种类和重组质粒，使其能够在无创显微镜下测定所有生物膜居民的面积浓度，包括携带质粒的宿主、无质粒的受体和正在发育的模型生物膜中的转共轭物；(2)评估转移重组质粒在纯培养中的分离性和结构稳定性；(3)改进现有的非侵入性显微镜方法，以定量质粒保留和转移在发育的二、三级培养生物膜中；(4)量化质粒转移动力学和外源基因在模型三级培养生物膜群落中的表达，作为环境条件（营养浓度、O2浓度、pH）、供体物种（革兰氏阳性vs革兰氏阴性）和供体液相浓度的函数。

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.