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

EDGE CMT：RUI：毒素-抗毒素系统在 Variovorax 属生物膜形成和水平基因转移中的作用

• 批准号: 2201665
• 负责人: Paul Orwin
• 金额: $ 69.27万
• 依托单位: University of the Pacific
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2025-09-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2201665&HistoricalAwards=false
• 关键词: EDGE; CMT; RUI; Roles; Toxin

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.

这项提案将解决细菌群体中DNA的移动和称为生物膜的复杂多细胞结构的形成之间的联系。本项目的重点是评估这些元素在重要的土壤细菌Variovorax paroxus中的作用。这种细菌在细菌种群与植物根部的联系以及在许多其他土壤过程中发挥着至关重要的作用。生物膜的形成和环境中的DNA转移是通过生物膜内的细胞将遗传物质释放到环境中而联系在一起的。这种材料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，释放出它们的细胞内容物，包括它们的DNA，然后成为结构的一部分。DNA在细菌细胞之间的移动是理解细菌群体如何对环境变化做出反应的关键因素。这些过程被认为与来自不同环境的许多细菌物种有关，而这里提出的作用机制可能是连接这两种表型的广泛原因。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从功能上将特定遗传元件的结构和功能与生物膜的形成和通过生物膜基质在细胞之间转移遗传物质联系起来。通过基于课程的本科生研究经验，学生将有机会制定他们自己的研究问题，执行最先进的基因组测序和生物信息学分析，并为撰写同行评议的出版物做出贡献。最近完成的基因组组装表明，Variovorax分离株具有不同的基因组结构，包括染色体、染色体和质粒。这些基因组在所有三种类型的复制子中都包括许多假定的毒素-抗毒素系统，根据表达水平和位置，这些复制子被认为是成瘾或抗成瘾模块。这被认为与生物膜的形成有关，通过一个全球调控系统控制生物膜形成过程中转录的稳定性。另一种RNA降解体成分在Variovorax paradoxus EPS生物膜中高度特异表达，它被认为降低TA系统RNA的稳定性，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系统将在电子计算机中鉴定，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。饱和突变(RB-TnSeq)将被用来鉴定影响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整合成单一拷贝的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，TA系统产生的EDNA毒素活性是必要的，也是允许Variovorax生物膜自然转化的充分条件。他们还将表明，这些表型通过生物膜生长依赖于RNA稳定性的改变而密不可分地联系在一起。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。