Dissertation
METABOLIC PROGRESSION DURING DEVELOPMENT OF SYMBIOTIC NITROGEN FIXATION IN THE MEDICAGO-SINORHIZOBIUM SYMBIOSIS AND THE ROLE OF HSLUV AND CLPXP PROTEASE MACHINERY
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/110859
Abstract
Symbiotic nitrogen fixation (SNF) describes a process in which atmospheric dinitrogen is reduced to ammonium through the coordinated interaction of bacteria with a host eukaryote. The majority of SNF occurs when soil bacteria, commonly called rhizobia, infect legumes and form root organs called nodules. The model symbionts Medicago truncatula and Sinorhizobium medicae form nodules containing developmentally distinct zones that spatially separate the metabolic progression from free-living individuals into nitrogen fixing partners. To understand the coordination of plant and bacterial metabolism within the nodule we used mass spectrometry to generate protein and metabolite profiles representative of these developmental zones, as well as profiles of whole-nodules and uninfected roots. Our approach identified 361 bacterial proteins and 888 plant proteins, as well as 160 metabolites, and confirms many previously characterized sub-nodule protein localization patterns. We used our comprehensive dataset to demonstrate how branches of metabolism are coordinated between symbionts and zones, focusing on central carbon, fatty acid, and amino acid metabolism. These data constitute the largest intra-nodule protein and metabolite resource to date.
Regulation of SNF involves the S. meliloti HslUV and ClpXP bacterial protease systems, which degrade targeted regulatory proteins and recycle damaged protein. Subjecting free-living Sinorhizobium to different stresses revealed via qPCR that clpXP and hslUV respond transcriptionally to heat, cold, elevated salt, and neomycin. We have demonstrated that deleting protease genes results in a less effective symbiosis, altered free-living growth rates, as well as significantly reduced exopolysaccharide and succinoglycan production. By tagging hslU, hslV, clpX, and clpP with an eGFP coding sequence at their 3’ ends, and expressing these constructs from their native promoter in S. meliloti, we were able to show that each protein was abundant in the different zones of the nodule during symbiosis with M. sativa. To determine novel protein-protein interactions, we used co-immunoprecipitation of the HslU-, HslV-, ClpX-, and ClpP-eGFP tagged proteins from S. meliloti in free-living conditions, and in symbiosis with M. sativa. These experiments revealed that HslUV and ClpXP interactions are significantly enriched for proteins associated with translation.
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Details
- Title
- METABOLIC PROGRESSION DURING DEVELOPMENT OF SYMBIOTIC NITROGEN FIXATION IN THE MEDICAGO-SINORHIZOBIUM SYMBIOSIS AND THE ROLE OF HSLUV AND CLPXP PROTEASE MACHINERY
- Creators
- Aaron Ogden
- Contributors
- Michael L Kahn (Advisor)Svetlana Yurgel (Committee Member)David Gang (Committee Member)B. Marcus Lange (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 156
- Identifiers
- 99900581710301842
- Language
- English
- Resource Type
- Dissertation