Multiple Photon Microscopy for Bacterial Biofilm Plasmid Dynamics

该项目旨在开发动态多光子激光显微镜方法，以非侵入性地定量模型细菌生物膜中的质粒保留，转移和表达。最近的文献已经证实，粘附细菌与其自由悬浮的对应物在表型上不同。被结合在生物膜内还可以增强细菌之间质粒-DNA的分离稳定性、表达和接合转移速率。这是重要的，因为在生物膜中，携带质粒的细菌可以转移基因和表型（例如，消毒剂和抗生素抗性、异生物质降解能力）更容易被邻近的细菌感染。到目前为止，很少有定量数据存在关于质粒的保留，转移和表达在混合培养生物膜生态系统。 具体目标是：（1）开发重组细菌物种和重组质粒，其将允许在发育模型生物膜中非侵入性显微镜测定所有生物膜居民的面积浓度，包括携带质粒的宿主、无质粒的受体和转移接合物;（2）评估转移重组质粒在发育纯培养物中的分离和结构稳定性;（3）改进现有的非侵入性显微镜方法，以量化正在形成的二元和三元培养生物膜内的质粒保留和转移;和（4）定量作为环境条件的函数的模型三级培养物生物膜群落内的质粒转移动力学和异源基因表达（营养物浓度、O2浓度、pH）、供体物种（革兰氏阳性对革兰氏阴性）和供体流体相浓度。

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.