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 and legume plant 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. 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 HslUV and ClpXP1 under free-living conditions and in symbiosis with , we generated Δ, Δ, Δ, and Δ knockout mutants. The shoot dry weight of plants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of the Δ and Δ mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while Δ and Δ 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 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. 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., and , 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 HslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation.