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，以非侵入性地量化质粒在模型细菌生物膜中的保留、转移和表达。最近的文献证实，附着的细菌与自由悬浮的细菌具有不同的表型。结合在生物膜内也可以提高细菌间质粒-DNA的分离稳定性、表达和接合转移的速度。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，消毒剂和抗生素的耐药性、外来生物降解能力)转移给邻近的细菌。到目前为止，关于混合培养生物膜生态系统中的质粒保留、转移和表达的定量数据很少。具体目的是：(1)开发重组细菌物种和重组质粒(S)，它将能够无创地显微测定所有生物膜居民的区域浓度，包括携带质粒的宿主、无质粒受体和转接子在正在形成的模型生物膜中；(2)评估转移的重组质粒在正在形成的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方法，以量化正在形成的二元和三级培养生物膜中的质粒保留和转移；(4)定量研究了第三代培养生物膜群落中外源基因的表达与环境条件(营养浓度、氧气浓度、pH值)、供体种类(革兰氏阳性与革兰氏阴性)和供体液相浓度之间的关系。

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.