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

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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.