EDGE CMT: RUI: Roles of Toxin-Antitoxin Systems in Biofilm Formation and Horizontal Gene Transfer in the genus Variovorax

这一建议将解决细菌种群中DNA运动与称为生物膜的复杂多细胞结构形成之间的联系。这个项目的重点是评估这些元素在重要的土壤细菌Variovorax paradoxus中的作用。这种细菌在细菌种群与植物根系的关联以及许多其他土壤过程中起着至关重要的作用。生物膜内的细胞将遗传物质释放到环境中，将生物膜的形成和环境中的DNA转移联系在一起。这种物质构成了生物膜结构的一部分，也是遗传变异的潜在来源。生物膜内的细菌被裂解，释放出包括DNA在内的细胞内容物，这些细胞内容物随后成为结构的一部分。细菌细胞间DNA的移动是了解细菌群体如何对环境变化作出反应的关键因素。这些过程被认为在来自不同环境的许多细菌物种中是相互关联的，并且在这里提出的工作机制可能广泛地负责连接这两种表型。利用尖端的DNA和RNA测序方法，结合实验室实验，包括许多本科生在内的研究人员旨在将特定遗传元件的结构和功能与生物膜的形成以及遗传物质通过生物膜基质在细胞之间的转移联系起来。通过基于课程的本科研究经验，学生将有机会制定自己的研究问题，执行最先进的基因组测序和生物信息学分析，并为同行评审出版物的撰写做出贡献。最近完成的基因组组装表明，Variovorax分离物具有多种基因组结构，包括染色体、染色粒和质粒。这些基因组在所有三种类型的复制子中包括许多假定的毒素-抗毒素系统，根据表达水平和位置，这些系统被提出作为成瘾或抗成瘾模块。这被假设与生物膜的形成有关，通过一个控制生物膜形成过程中转录物稳定性的全球调控系统。另一种RNA降解体成分在Variovorax paradoxus EPS生物膜中特异性高表达，这可能会降低TA系统RNA的稳定性，从而促进细胞裂解，释放对致密生物膜形成和水平基因转移至关重要的eDNA。差异表达的TA操纵子是生物膜形成和水平基因转移的潜在决定因素。Variovorax属的TA系统将在计算机上进行鉴定，表达将使用RNA-seq进行评估，成瘾模块功能将使用严格调控的表达系统进行测试。饱和诱变（RB-TnSeq）将用于鉴定影响TA系统调节质粒稳定性功能的辅助基因。作为抗成瘾模块的染色体TA系统将与使用mini-Tn7集成在单拷贝中的TA系统进行类似的评估。将使用标记菌株检查TA系统功能、生物膜形成和HGT之间的联系。这些实验将证明，由TA系统毒素活性产生的eDNA是必要和充分的，以允许在Variovorax生物膜中进行自然转化。他们还将表明，这些表型通过依赖于生物膜生长的RNA稳定性的改变是密不可分的。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This proposal will address the connection between the movement of DNA in bacterial populations and the formation of complex multi-cellular structures called biofilms. This project is focused on evaluating the role of these elements in the important soil bacterium Variovorax paradoxus. This bacterium plays a crucial role in the association of bacterial populations with plant roots, as well as in many other soil processes. Biofilm formation and DNA transfer in the environment are connected by the release of genetic material into the environment by cells within the biofilm. This material forms part of the structure of the biofilm, as well as a potential source of genetic variation. The bacteria within the biofilm are lysed to release their cellular contents, including their DNA, which then becomes part of the structure. The movement of DNA between bacterial cells is critical factor in understanding how bacterial populations respond to changes in their environment. These processes are thought to be connected in many bacterial species from diverse environments, and the mechanism proposed to be at work here may be widely responsible for connecting the two phenotypes. Using cutting edge DNA and RNA sequencing approaches, combined with laboratory experiments, the researchers, which include many undergraduates, aim to functionally link the structure and function of specific genetic elements to the formation of biofilms and the transfer of genetic material between cells via the biofilm matrix. Via course based undergraduate research experiences, students will have the opportunity to formulate their own research questions, perform state of the art genomic sequencing and bioinformatic analyses, and contribute to the writing of peer-reviewed publications. Recently finished genome assemblies show that Variovorax isolates have diverse genomic architectures including chromosomes, chromids, and plasmids. These genomes include numerous putative Toxin-Antitoxin systems in all three types of replicon, that are proposed to function as addiction or anti-addiction modules depending on expression level and location. This is hypothesized to be connected with biofilm formation through a global regulatory system controlling transcript stability during biofilm formation. An alternative RNA degradosome component is highly expressed specifically in Variovorax paradoxus EPS biofilms, which is proposed to decrease TA system RNA stability to promote cell lysis, releasing eDNA critical for dense biofilm formation and horizontal gene transfer. Differentially expressed TA operons are potential determinants of both biofilm formation and horizontal gene transfer. The TA systems in the genus Variovorax will be identified in silico, expression will be evaluated using RNA-seq, and addiction module function will be tested using tightly regulated expression systems. Saturation mutagenesis (RB-TnSeq) will be used to identify accessory genes that influence the function of TA systems in regulating plasmid stability. Chromosomal TA systems as anti-addiction modules will be evaluated similarly with TA systems integrated in single copy using mini-Tn7. The connection between TA system function, biofilm formation, and HGT will be examined using marked strains. These experiments will demonstrate that eDNA generation by TA system toxin activity is necessary and sufficient to allow for natural transformation in Variovorax biofilms. They will also show that these phenotypes are inextricably linked through biofilm growth dependent alteration of RNA stability.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.