Characterizing gene networks involved in cell envelope development in Rhizobium leguminosarum.

豆科植物包括几种重要的粮食作物，如大豆和扁豆，并具有与固氮细菌（统称根瘤菌）共生的能力，其中细菌生活在根瘤中并将大气中的氮还原为氨。固定氮被植物吸收以满足其对氮的需求。根瘤菌成功在豆科植物根际定殖并适应根际定殖和植物结瘤过程中所遇到的胁迫，根瘤菌的遗传特性和生物学机制是必要的。细菌的细胞包膜是一种众所周知的细胞特征，它对细菌在环境中的生存和宿主生物的定植过程非常重要。它是细胞与其环境之间的第一个接触点，也起着维持细胞完整性和保护细胞免受有毒物质侵害的作用。尽管包膜在根际生存和植物结瘤中起着重要作用，但在根瘤菌中，参与细胞包膜适应的遗传网络只被部分描述。我的实验室研究豆科根瘤菌（豌豆和扁豆的共生体）作为模型物种来表征革兰氏阴性菌包膜发育过程中涉及的遗传网络。在此更新申请中，学员将专注于识别新的包膜调节网络，并确定这些网络如何与已知的调节系统（如细胞周期调节因子CbrA）集成。该研究计划将利用我们在下一代测序应用方面的能力，例如RNA-Seq来表征保守的DNA基序，这些基序是包膜基因调控的重要顺式元件，并询问由关键细胞包膜基因突变引起的转录变化。我们最近开发了一种用于根瘤菌高通量转座子突变筛选（INSeq）的遗传工具。我们将使用该工具作为RNA-Seq实验的补充，以鉴定在细胞包膜功能和适应中重要的新基因，特别是在对植物来源的抗菌肽的反应中。INSeq方法的高通量筛选特性（可以筛选数百万转座子突变体），以及我们最近完成的核心必需基因参考库，这些参考库是从以前的INSeq实验中开发出来的，将极大地促进我们确定根际和植物定植过程中参与细胞包膜适应的新基因的目标。结合RNA-Seq实验，我们将能够在一种农业上重要的细菌物种中产生一个更精细的细胞包膜调节网络模型。

Legume plants include several important food crops such as soybean and lentils, and have the ability to exist in symbiosis with nitrogen fixing bacteria (collectively termed rhizobia), whereby the bacteria live in root nodules and reduce atmospheric nitrogen to ammonia. The fixed nitrogen is assimilated by the plant to meet its nitrogen requirements. Fundamental discovery-based research is necessary to unravel the genetic traits and biological mechanisms that are important for rhizobia to successfully colonize the legume rhizosphere and adapt to stresses encountered during rhizosphere colonization and plant nodulation. The bacterial cell envelope is a well-known cellular feature that is important to the survival of bacteria in the environment and during colonization of host organisms. It is the first point of contact between a cell and its environment, and also functions to maintain cell integrity and protects the cell from toxic substances. The genetic networks involved in cell envelope adaptation are only partially described in rhizobia, despite the envelope's importance in rhizosphere survival and plant nodulation. My lab studies Rhizobium leguminosarum (a symbiont of peas and lentils) as a model species to characterize genetic networks involved in cell envelope development in Gram negative bacteria. In this renewal application the student trainees will focus on identifying new envelope regulatory networks, and determine how these networks are integrated with known regulatory systems such as the cell cycle regulator, CbrA. The research plan will leverage our capacity in next generation sequencing applications, such as RNA-Seq to characterize conserved DNA motifs that are important cis elements for regulation of envelope genes, and interrogate transcriptional changes caused by mutations to key cell envelope genes. We have recently developed a genetic tool for high throughput transposon mutagenesis screening (INSeq) in rhizobia. We will use this tool as a complement to the RNA-Seq experiments to identify novel genes important in cell envelope function and adaptation, particularly in response to plant derived antimicrobial peptides. The high throughput screening nature of the INSeq approach (millions of transposon mutants can be screened), and our recently completed reference library of core essential genes, developed from previous INSeq experiments, will greatly facilitate our goal of identifying new genes involved in cell envelope adaptation during rhizosphere and plant colonization. Combined with the RNA-Seq experiments we will be able to generate a more refined model of cell envelope regulatory networks in an agriculturally important bacterial species.