Novel genes required for proper cell envelope function in the plant symbiont Rhizobium leguminosarum

Vanderlinde, Elizabeth Margaret
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Faculty of Graduate Studies and Research, University of Regina

The cell envelope of Gram-negative bacteria is a complex structure that is critical for facilitating interactions between bacteria and their environment. The objective of this research was to identify and characterize novel genes required for proper cell envelope development in the legume symbiont Rhizobium leguminosarum. In R. leguminosarum there is a positive correlation between mutations that affect cell envelope structure and sensitivity to peptide-rich media; therefore, I implemented an innovative transposon mutagenesis approach to identify novel genes connected to the cell envelope by screening for mutants unable to grow on peptide-rich media, but unaffected in growth on minimal media. Of the 10 mutants isolated, 5 of the mutations were mapped to cell envelope components; therefore, the genetic screen was successful. Three mutants were chosen for further study because the mutations were in uncharacterized genes that affected the cell envelope function. One of the mutations was mapped to the putative ATP binding component of an ABC transporter. This mutant had significantly decreased levels of exopolysaccharide (EPS) compared to wild-type, and was also desiccation sensitive and unable to form biofilms. Another mutation mapped to the gene fabF2XL, which is required for synthesis of the unique lipid A very long chain fatty acid (VLCFA). The third mutation mapped to a conserved hypothetical membrane protein, RL3501, that is part of a four gene operon. Both of these genes were critical for maintaining membrane stability in the presence of detergents, hydrophobic antibiotics, and antimicrobial peptides. Mutation of fabF2XL or RL3501 also had a negative impact on plant infection, and expression of an outer membrane protein gene, ropB, indicating that they are crucial for proper cell envelope development in free-living conditions and during symbiosis. I also identified novel phenotypes associated with mutation of the sensor kinase chvG that regulates cell envelope structure. Collectively, the results of this study have identified new cell envelope genes that are critical for proper envelope function, and contributed to the development of a new model for cell envelope development in Rhizobium leguminosarum by revealing novel mechanisms for the regulation and interaction of key envelope components.

A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology University of Regina