Bourassa, Dianna Viola
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Nitrogen fixation is a biological process essential to life. Atmospheric nitrogen (N2) must be converted to an available form such as ammonia (NH3) so that nitrogen is available for use in biological processes. The symbiotic relationship of legume plants and rhizobia bacteria plays a crucial role in this process. The complex interaction between the plant and bacteria from the first recognition and interaction through the fully functional nodule filled with nitrogen-fixing bacteroids involves many factors from both the plants and bacteria. This work takes an in depth look at an unusual very long chain fatty acid (27-hydroxyoctacosanoic acid, VLCFA) bound to the lipid A portion of the lipopolysaccharide (LPS) found in the Rhizobium leguminosarum cell membrane. Two genes required for the biosynthesis and transfer of the VLCFA (acpXL acyl carrier protein and lpxXL acyl transferase) were mutated producing Rlv22 (acpXL-), EL196 (acpXL-, lpxXL-), and EL197 (effective lpxXL-). The effects on LPS structure, bacterial phenotype, and symbiotic proficiency were examined. Both acpXL and lpxXL mutants were unable to add the VLCFA to the lipid A. In the acpXL mutant a shorter acyl chain (palmitic or stearic) was transferred in place of the VLCFA. However, in strains without lpxXL neither the VLCFA nor the shorter acyl chain was added. It was also noted that for both parent and mutant strains nodule bacteroids tended to have LPS with longer acyl chains than the laboratory-cultured bacteria. The acpXL and lpxXL mutants also affected bacterial phenotype. Mutants had diminished membrane integrity, altered sensitivity to cationic peptides, and diminished relative levels of cyclic β-glucans. When the host plant (Pisum sativum, Pea) was inoculated with parent and mutant strains changes to the symbiotic phenotype was also observed. Host root infection was less efficient and infected plant nodule cells were disorganized. Bacteroids formed atypical multiply branched structures with multiple bacteroids per symbiosome. The bacteroids were not packed as tightly into the nodule plant cells, and were senescing earlier than normal. The main purpose of symbiosis, nitrogen fixation, was also detrimentally affected. Clearly this unusual VLCFA plays an important role in the symbiotic relationship between Rhizobium and pea plants.