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ABSTRACT Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteria Sinorhizobium meliloti and legume plant Medicago sativa is carried out in specialized root organs called nodules. During nodule development, each symbiont must drastically alter their proteins, transcripts, and metabolites in order to support nitrogen fixation. Moreover, bacteria within the nodules are under stress, including challenges by plant antimicrobial peptides, low pH, limited oxygen availability, and strongly reducing conditions, all of which challenge proteome integrity. S. meliloti stress adaptation, proteome remodeling, and quality control are controlled in part by the large oligomeric protease complexes HslUV and ClpXP1. To improve understanding of the roles of S. meliloti HslUV and ClpXP1 under free-living conditions and in symbiosis with M. sativa , we generated Δ hslU , Δ hslV , Δ hslUV , and Δ clpP1 knockout mutants. The shoot dry weight of M. sativa plants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of the Δ hslUV and Δ clpP1 mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while Δ hslU and Δ clpP1 mutants were sensitive to kanamycin. All deletion mutants produced less exopolysaccharide and succinoglycan, as shown by replicate spot plating and calcofluor binding. We also generated endogenous C-terminal enhanced green fluorescent protein (eGFP) fusions to HslU, HslV, ClpX, and ClpP1 in S. meliloti . Using anti-eGFP antibodies, native coimmunoprecipitation experiments with proteins from free-living and nodule tissues were performed and analyzed by mass spectrometry. The results suggest that HslUV and ClpXP were closely associated with ribosomal and proteome quality control proteins, and they identified several novel putative protein-protein interactions. IMPORTANCE Symbiotic nitrogen fixation (SNF) is the primary means by which biologically available nitrogen enters the biosphere, and it is therefore a critical component of the global nitrogen cycle and modern agriculture. SNF is the result of highly coordinated interactions between legume plants and soil bacteria collectively referred to as rhizobia, e.g., Medicago sativa and S. meliloti , respectively. Accomplishing SNF requires significant proteome changes in both organisms to create a microaerobic environment suitable for high-level bacterial nitrogenase activity. The bacterial protease systems HslUV and ClpXP are important in proteome quality control, in metabolic remodeling, and in adapting to stress. This work shows that S. meliloti HslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation.
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This content will become publicly available on August 10, 2026
Stachydrine Catabolism Contributes to an Optimal Root Nodule Symbiosis Between Sinorhizobium meliloti and Medicago sativa
Sinorhizobium meliloti forms a robust N2-fixing root-nodule symbiosis with Medicago sativa. We are interested in identifying the minimal symbiotic genome of the model strain S. meliloti Rm1021. This gene set refers to the minimal genetic determinants required to form a robust N2-fixing symbiosis. Many symbiotic genes are located on the 1,354 kb pSymA megaplasmid of S. meliloti Rm1021. We recently constructed a minimalized pSymA, minSymA2.1, that lacked over 90% of the pSymA genes. Relative to the wild-type, minSymA2.1 showed a reduction in M. sativa shoot biomass production and nodule size with an increase in total nodule number. Here we show that the addition of either the stachydrine (stc) or trigonelline (trc) catabolism genes from pSymA to minSymA2.1 restores nodule size and total nodule number to levels indistinguishable from the wild-type but does not restore reduced shoot biomass production. In the context of the complete Rm1021 genome, removing the stc genes reduced nodule size and increased total nodule number while removal of the trc genes alone had no apparent effect. Together, these observations implicate stachydrine catabolism as an important determinant of root nodule symbiosis between S. meliloti and M. sativa while trigonelline catabolism seems to contribute in a more conditional manner, in the context of the minimized genome. These findings highlight the minimal symbiotic genome as a tool for investigating the impact individual genetic determinants have in conferring an optimal symbiosis. Factors whose impact, in the context of a complete genome, may be hidden or dampened due to redundancies.
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- Award ID(s):
- 2243818
- PAR ID:
- 10633784
- Publisher / Repository:
- APS Molecular Plant-Microbe Interactions
- Date Published:
- Journal Name:
- Molecular Plant-Microbe Interactions®
- ISSN:
- 0894-0282
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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