Title: Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States

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 ΔhslU, ΔhslV, ΔhslUV, and ΔclpP1 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 ΔhslUV and ΔclpP1 mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while ΔhslU and ΔclpP1 mutants were sensitive to kanamycin. All deletion mutants produced less exopolysaccharide and succinoglycan, as shown by replicate spot plating and calcofluor binding. We also generatedmore » 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.« less

Authors:

Ogden, Aaron J.  ^[1];

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McAleer, Jacqueline M. ^[1]; Kahn, Michael L. ^[1]; Becker, Anke ^[1]

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+ Show Author Affiliations

1. Washington State Univ., Pullman, WA (United States)

Publication Date:

2019-01-22

Research Org.:

Washington State Univ., Pullman, WA (United States)

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1614598

Grant/Contract Number:  

FG03-96ER20225

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Bacteriology

Additional Journal Information:

Journal Volume: 201; Journal Issue: 7; Journal ID: ISSN 0021-9193

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Microbiology; Medicago sativa; Sinorhizobium meliloti; ClpP; ClpX; HslU; HslV; protease; protein-protein interaction; symbiosis; symbiotic nitrogen fixation