Characterization of novel genes involved in cell envelope function in Rhizobium leguminosarum

Neudorf, Kara Dawn
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Faculty of Graduate Studies and Research, University of Regina

The complex structure of the rhizobial Gram-negative cell envelope is a crucial component for both survival in the environment, as well as for establishing a successful symbiosis with its legume host. The objective of this research was to identify and characterize novel genes involved in cell envelope development in Rhizobium leguminosarum. Previous studies have identified a positive correlation between cell envelope defective mutants and an inability to grow on peptide-rich media, as well as minimal media supplemented with 3 mM glycine. Identifying novel genes involved in cell envelope function through the use of transposon mutagenesis, and screening for growth sensitivity to peptide-rich media, has been previously successful. Many of these genes are annotated as hypothetical genes of unknown function and it is unclear of how they are integrated in a cell envelope development gene network. Therefore I was interested in trying to identify functional partners to these genes. This study was done through the use of a suppressor mutation strategy, by selecting for spontaneous mutations that restored growth on peptide-rich media to the wild-type phenotype. Various mutants in genes of both known function and unknown function, previously isolated as sensitive to growth on complex media, were selected for isolation of suppressor mutants. Suppressor frequencies were obtained to provide insight into the complexity involved in restoring the growth phenotype. In addition, suppressors were characterized using phenotypic assays to assess cell envelope function. Suppressors were isolated for a mutant containing a transposon insertion in the gene fabF2XL (fabF2XL mutant), which is required for synthesis of the lipid A very long chain fatty acid (VLCFA). Secondly, suppressor mutants were isolated for a mutant disrupted in the putative carboxy-terminal processing protease gene (ctpA). The third suppressor isolation was done using a mutant containing a disruption to the sensor kinase chvG, which is involved in regulation of cell envelope genes. Lastly, suppressors were isolated from various mutants with in-frame deletions in a four-gene operon RL3499- RL3502 (cmdA-cmdD), which has a demonstrated role in proper cell envelope function. All suppressors were phenotypically characterized and two suppressors from the cmd operon mutants (38cmdA-S and 38cmdB-S) were selected for further genotypic analysis. A transposon mutagenesis of a suppressor strain that restored growth in a 38cmdB background (38cmdB-S), implicated the locus RL0936 to be involved in the restored growth phenotype pathway. A transposon mutagenesis to the suppressor in the 38cmdA background (38cmdA-S) did not identify a locus required for the restored growth, but instead identified two uncharacterized genes (RL4613 and RL0188) independently required for growth on complex, peptide rich media. RNA-Seq data provided multiple potential genes involved in compensating for a mutation in cmdB, and has provided predicted operon structures that can provide insight into future experiments. Collectively, the results of this study have highlighted the complexity of the genes involved in cell envelope development, and have identified putative new genetic networks that may be linked to cell envelope development.

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. xxi, 228 p.