Characterization of the Sinorhizobium meliloti HslUV and ClpXP protease systems in free-living and symbiotic states.

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 in free-living conditions and in symbiosis with M. sativa, we generated Δ hslU, Δ hslV, Δ hslUV, and Δ clpP1 knockout mutants. 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 Δ hslUV and Δ clpP1 suggest HslUV and ClpP1 were involved in adapting to heat stress, 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 eGFP fusions to HslU, HslV, ClpX and ClpP1 in S. meliloti Using anti-eGFP antibodies, native coimmunoprecipitation experiments of proteins from free-living and nodule tissues were performed and analyzed by mass spectrometry. The results suggest HslUV and ClpXP were closely associated with ribosomal and proteome quality control proteins, and 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 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. Bacterial protease systems, HslUV and ClpXP are important in proteome quality control, 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.