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

Abstract

ABSTRACT Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteriaSinorhizobium melilotiand legume plantMedicago sativais 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. melilotistress 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 ofS. melilotiHslUV and ClpXP1 under free-living conditions and in symbiosis withM. sativa, we generated ΔhslU, ΔhslV, ΔhslUV, and ΔclpP1knockout mutants. The shoot dry weight ofM. sativaplants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of the ΔhslUVand ΔclpP1mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while ΔhslUand ΔclpP1mutants 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 inS.more » 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. IMPORTANCESymbiotic 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 sativaandS. 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 thatS. melilotiHslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation.« less

Authors:
ORCiD logo; ; ;
Publication Date:
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:
Journal Article: 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

Citation Formats

Ogden, Aaron J., McAleer, Jacqueline M., Kahn, Michael L., and Becker, Anke. Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States. United States: N. p., 2019. Web. doi:10.1128/jb.00498-18.
Ogden, Aaron J., McAleer, Jacqueline M., Kahn, Michael L., & Becker, Anke. Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States. United States. doi:10.1128/jb.00498-18.
Ogden, Aaron J., McAleer, Jacqueline M., Kahn, Michael L., and Becker, Anke. Tue . "Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States". United States. doi:10.1128/jb.00498-18. https://www.osti.gov/servlets/purl/1614598.
@article{osti_1614598,
title = {Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States},
author = {Ogden, Aaron J. and McAleer, Jacqueline M. and Kahn, Michael L. and Becker, Anke},
abstractNote = {ABSTRACT Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteriaSinorhizobium melilotiand legume plantMedicago sativais 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. melilotistress 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 ofS. melilotiHslUV and ClpXP1 under free-living conditions and in symbiosis withM. sativa, we generated ΔhslU, ΔhslV, ΔhslUV, and ΔclpP1knockout mutants. The shoot dry weight ofM. sativaplants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of the ΔhslUVand ΔclpP1mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while ΔhslUand ΔclpP1mutants 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 inS. 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. IMPORTANCESymbiotic 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 sativaandS. 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 thatS. melilotiHslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation.},
doi = {10.1128/jb.00498-18},
journal = {Journal of Bacteriology},
issn = {0021-9193},
number = 7,
volume = 201,
place = {United States},
year = {2019},
month = {1}
}

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Works referenced in this record:

An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in Sinorhizobium meliloti
journal, March 2007

  • Sauviac, Laurent; Philippe, Heinui; Phok, Kounthéa
  • Journal of Bacteriology, Vol. 189, Issue 11
  • DOI: 10.1128/JB.00175-07

STRING v10: protein–protein interaction networks, integrated over the tree of life
journal, October 2014

  • Szklarczyk, Damian; Franceschini, Andrea; Wyder, Stefan
  • Nucleic Acids Research, Vol. 43, Issue D1
  • DOI: 10.1093/nar/gku1003

Activation of the Cpx regulon destabilizes the F plasmid transfer activator, TraJ, via the HslVU protease in Escherichia coli
journal, February 2008


Global Transcriptional Response of Bacillus subtilis to Treatment with Subinhibitory Concentrations of Antibiotics That Inhibit Protein Synthesis
journal, May 2005


Trigger factor and DnaK cooperate in folding of newly synthesized proteins
journal, August 1999

  • Deuerling, Elke; Schulze-Specking, Agnes; Tomoyasu, Toshifumi
  • Nature, Vol. 400, Issue 6745
  • DOI: 10.1038/23301

Antimicrobial Nodule-Specific Cysteine-Rich Peptides Induce Membrane Depolarization-Associated Changes in the Transcriptome of Sinorhizobium meliloti
journal, August 2013

  • Tiricz, Hilda; Szűcs, Attila; Farkas, Attila
  • Applied and Environmental Microbiology, Vol. 79, Issue 21
  • DOI: 10.1128/AEM.01791-13

The binary protein-protein interaction landscape of Escherichia coli
journal, February 2014

  • Rajagopala, Seesandra V.; Sikorski, Patricia; Kumar, Ashwani
  • Nature Biotechnology, Vol. 32, Issue 3
  • DOI: 10.1038/nbt.2831

Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins
journal, February 1997

  • Randall, L. L.; Topping, T. B.; Hardy, S. J. S.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 3
  • DOI: 10.1073/pnas.94.3.802

AAA+ Proteases: ATP-Fueled Machines of Protein Destruction
journal, July 2011


Proteomic Discovery of Cellular Substrates of the ClpXP Protease Reveals Five Classes of ClpX-Recognition Signals
journal, March 2003


Transfer-messenger RNA-SmpB Protein Regulates Ribonuclease R Turnover by Promoting Binding of HslUV and Lon Proteases
journal, August 2012

  • Liang, Wenxing; Deutscher, Murray P.
  • Journal of Biological Chemistry, Vol. 287, Issue 40
  • DOI: 10.1074/jbc.M112.375287

Oxidative Burst in Alfalfa- Sinorhizobium meliloti Symbiotic Interaction
journal, January 2001

  • Santos, Renata; Hérouart, Didier; Sigaud, Samuel
  • Molecular Plant-Microbe Interactions, Vol. 14, Issue 1
  • DOI: 10.1094/MPMI.2001.14.1.86

Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease.
journal, January 1996


The time course of the transcriptomic response of Sinorhizobium meliloti 1021 following a shift to acidic pH
journal, January 2009

  • Hellweg, Christoph; Pühler, Alfred; Weidner, Stefan
  • BMC Microbiology, Vol. 9, Issue 1
  • DOI: 10.1186/1471-2180-9-37

Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021
journal, January 2013

  • Schlüter, Jan-Philip; Reinkensmeier, Jan; Barnett, Melanie J.
  • BMC Genomics, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2164-14-156

A Rhizobium leguminosarum AcpXL Mutant Produces Lipopolysaccharide Lacking 27-Hydroxyoctacosanoic Acid
journal, March 2003


Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii
journal, July 2011

  • Hamilton, T. L.; Ludwig, M.; Dixon, R.
  • Journal of Bacteriology, Vol. 193, Issue 17
  • DOI: 10.1128/JB.05099-11

Co-translational Binding of GroEL to Nascent Polypeptides Is Followed by Post-translational Encapsulation by GroES to Mediate Protein Folding
journal, June 2006

  • Ying, Bei-Wen; Taguchi, Hideki; Ueda, Takuya
  • Journal of Biological Chemistry, Vol. 281, Issue 31
  • DOI: 10.1074/jbc.M603091200

Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.
journal, September 1985

  • Leigh, J. A.; Signer, E. R.; Walker, G. C.
  • Proceedings of the National Academy of Sciences, Vol. 82, Issue 18
  • DOI: 10.1073/pnas.82.18.6231

A Large Scale Analysis of Protein-Protein Interactions in the Nitrogen-fixing Bacterium Mesorhizobium loti
journal, January 2008


Heat shock transcription factor δ32 is targeted for degradation via an ubiquitin-like protein ThiS in Escherichia coli
journal, April 2015

  • Xu, Xibing; Niu, Yulong; Liang, Ke
  • Biochemical and Biophysical Research Communications, Vol. 459, Issue 2
  • DOI: 10.1016/j.bbrc.2015.02.087

The Ribosome as a Hub for Protein Quality Control
journal, February 2013


Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.
journal, April 1995

  • Soupene, E.; Foussard, M.; Boistard, P.
  • Proceedings of the National Academy of Sciences, Vol. 92, Issue 9
  • DOI: 10.1073/pnas.92.9.3759

Large-scale identification of protein-protein interaction of Escherichia coli K-12
journal, May 2006


Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005

  • Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
  • Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
  • DOI: 10.1038/nbt1172

Next-Generation Annotation of Prokaryotic Genomes with EuGene-P: Application to Sinorhizobium meliloti 2011
journal, April 2013


Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins
journal, August 2004

  • Ferbitz, Lars; Maier, Timm; Patzelt, Holger
  • Nature, Vol. 431, Issue 7008
  • DOI: 10.1038/nature02899

CbrA Is a Stationary-Phase Regulator of Cell Surface Physiology and Legume Symbiosis in Sinorhizobium meliloti
journal, June 2006

  • Gibson, Katherine E.; Campbell, Gordon R.; Lloret, Javier
  • Journal of Bacteriology, Vol. 188, Issue 12
  • DOI: 10.1128/JB.01923-05

Co-translational Involvement of the Chaperonin GroEL in the Folding of Newly Translated Polypeptides
journal, January 2005

  • Ying, B. -W.; Taguchi, H.; Kondo, M.
  • Journal of Biological Chemistry, Vol. 280, Issue 12
  • DOI: 10.1074/jbc.M500364200

Fiji: an open-source platform for biological-image analysis
journal, June 2012

  • Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin
  • Nature Methods, Vol. 9, Issue 7
  • DOI: 10.1038/nmeth.2019

Aminoglycosides: Perspectives on Mechanisms of Action and Resistance and Strategies to Counter Resistance
journal, December 2000


Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection
journal, August 2009


Proteomic Profiling of ClpXP Substrates after DNA Damage Reveals Extensive Instability within SOS Regulon
journal, April 2006


Interaction network containing conserved and essential protein complexes in Escherichia coli
journal, February 2005

  • Butland, Gareth; Peregrín-Alvarez, José Manuel; Li, Joyce
  • Nature, Vol. 433, Issue 7025
  • DOI: 10.1038/nature03239

Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins
journal, April 2009


Sinorhizobium meliloti dctA Mutants with Partial Ability To Transport Dicarboxylic Acids
journal, January 2005


An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing
journal, February 2014

  • Roux, Brice; Rodde, Nathalie; Jardinaud, Marie-Françoise
  • The Plant Journal, Vol. 77, Issue 6
  • DOI: 10.1111/tpj.12442

The degradation of RcsA by ClpYQ(HslUV) protease in Escherichia coli
journal, March 2016


OxyR-Dependent Transcription Response of Sinorhizobium meliloti to Oxidative Stress
journal, January 2018

  • Lehman, Alisa P.; Long, Sharon R.
  • Journal of Bacteriology, Vol. 200, Issue 7
  • DOI: 10.1128/JB.00622-17

SecA mediates cotranslational targeting and translocation of an inner membrane protein
journal, September 2017

  • Wang, Shuai; Yang, Chien-I; Shan, Shu-ou
  • Journal of Cell Biology, Vol. 216, Issue 11
  • DOI: 10.1083/jcb.201704036

Polypeptide Flux through Bacterial Hsp70
journal, June 1999