Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Latest Articles
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • Topics
    • Applied and Environmental Science
    • Clinical Science and Epidemiology
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Therapeutics and Prevention
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About mBio
    • Editor in Chief
    • Board of Editors
    • AAM Fellows
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
mBio
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • Topics
    • Applied and Environmental Science
    • Clinical Science and Epidemiology
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Therapeutics and Prevention
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About mBio
    • Editor in Chief
    • Board of Editors
    • AAM Fellows
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Research Article | Molecular Biology and Physiology

Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli

Suzy Bulot, Stéphane Audebert, Laetitia Pieulle, Farida Seduk, Emilie Baudelet, Leon Espinosa, Marie-Camille Pizay, Luc Camoin, Axel Magalon
Markus W. Ribbe, Editor
Suzy Bulot
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stéphane Audebert
bAix-Marseille Université, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Laetitia Pieulle
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Farida Seduk
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Emilie Baudelet
bAix-Marseille Université, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Leon Espinosa
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marie-Camille Pizay
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Luc Camoin
bAix-Marseille Université, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Axel Magalon
aAix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), IMM, Marseille, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Markus W. Ribbe
University of California, Irvine
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/mBio.01832-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Supplemental Material
  • FIG 1
    • Open in new tab
    • Download powerpoint
    FIG 1

    The sfGFP-labeled formate dehydrogenase complex concentrates at the cell poles under nitrate-respiring conditions. (A) Fluorescence images (top) and overlays of fluorescence and phase-contrast images (bottom) are shown for nitrate-respiring cells. (B) The histogram of the fluorescent cluster distribution across the transversal axis of nitrate-respiring LCB4215 cells is shown. Dashed lines delimit pole zones. Sixty-five percent of the clusters localize at the cell poles, and 99% of the cells exhibit polar clusters. The analysis was performed on 702 cells issued from three independent experiments. The fluorescence heat maps of the cells from each experiment are shown in Fig. S2.

  • FIG 2
    • Open in new tab
    • Download powerpoint
    FIG 2

    The membrane-associated nitrate reductase NarGHI has a specific interactome under nitrate-respiring conditions. (A) Volcano plot of proteins immunoprecipitated with NarGeGFP in nitrate- versus oxygen-respiring conditions using nanobodies directed against GFP. Volcano plot was constructed using log2 fold change and absolute log10 of the P values, enabling visualization of the relationship between fold change and statistical significance, respectively. The curves show the significance threshold established from the permutation-based FDR calculations (q value) (see Materials and Methods). The greater the difference between the group means (i.e., the enrichment) and the greater the absolute value of P (i.e., the reproducibility), the more the interactors move to the top right corner of the plot. Black dots located outside the curves represent proteins differentially found between the two conditions with q values below 1%, while gray squares between the two curves represent proteins that do not differ between conditions. Gray dots located outside the curves represent proteins that were identified to not be specific partners of the nitrate reductase (Table S1 and Table S2). Volcano plots showing NarGeGFP-specific interacting partners under each condition are presented in Fig. S3. (B) Heat map focusing on formate dehydrogenase (FdnGHI), nitrate/nitrite antiporter (NarK), cytoplasmic nitrite reductase (NirBD), NO reductase/transnitrosylase (Hcp) and its redox partner (Hcr), and NO reductase (Hmp). The normalized LFQ intensities using a Z-score (means centering the variable at zero and standardizing the variance) obtained for each biological replicate under oxygen (left)- or nitrate (right)-respiring conditions are shown according to the color gradient displayed on the right. Gray represents missing values (not identified proteins).

  • FIG 3
    • Open in new tab
    • Download powerpoint
    FIG 3

    Interactome of the nitrate reductase in the hcp-deficient strain. (A) Volcano plot of proteins immunoprecipitated with NarGeGFP versus untagged version of NarG under nitrate-respiring conditions. The volcano plot was constructed and displayed as described in the legend to Fig. 2. Black dots located outside the curves represent proteins differentially found between the two conditions with q values below 1% (Table S4). Proteins more enriched using the hcp strain are circled in red. (B) The two NO reductases, Hmp and NorV with its redox partner NorW, and YtfE (RIC) protein were found more abundantly associated with the nitrate reductase in the hcp strain. The heat map shows the Z-scored LFQ intensities obtained for each biological replicate in the wild-type strain (left) and in the hcp strain (right) grown under nitrate-respiring conditions according to the color gradient below.

  • FIG 4
    • Open in new tab
    • Download powerpoint
    FIG 4

    Inactivation of nirB and hcp induces a growth defect under nitrate-respiring conditions caused by elevated nitrosative stress. Generation times of strains JCB4011 (WT), LCB4136 (hcp), LCB4121 (nirB), LCB4137 (nirB hcp) grown under anaerobiosis in defined medium with glycerol as sole carbon source and nitrate as the sole electron acceptor without Casamino Acids (A) or supplemented with BCAA (B) are shown. Eight (A) and four (B) independent assays are, respectively, shown. Medians are represented by a horizontal line and averages by a red square. Statistical significance between the different strains was calculated using the Mann-Whitney nonparametric test: ***, P < 0.001; **, 0.001 < P < 0.01; ns, not significant (P > 0.05).

Supplemental Material

  • Figures
  • FIG S1

    The sfGFP-labeled formate dehydrogenase complex is active and stable. (A) The activity of the FdnGHI complex is unaffected by the sfGFP fusion. Formate::PMS/DCPIP oxidoreductase activity assays were performed on membrane prepared from LCB4200 (untagged version) and LCB4215 (tagged version) cells grown under nitrate-respiring conditions. The activities are expressed in μmol of CO2 produced min−1 mg−1 of proteins. (B) Western blot using antibodies directed against GFP shows stable expression of FdnI-sfGFP chimera. Whole-cell extract was prepared from LCB4215 cells grown under nitrate-respiring conditions to mid-exponential phase. Download FIG S1, TIF file, 0.4 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    The sfGFP-labeled formate dehydrogenase complex concentrates at the cell poles under nitrate-respiring conditions. The heat maps built from three independent experiments are shown. From left to right, 259, 297, and 146 cells were analyzed, respectively. The mean fluorescence density is represented by a color gradient shown to the right of each heat map. Download FIG S2, TIF file, 1.4 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    The membrane-associated nitrate reductase NarGHI has a specific interactome under nitrate-respiring conditions. (A and B) Volcano plots of proteins immunoprecipitated with NarGeGFP versus the untagged version of NarG under nitrate-respiring (A) or oxic (B) conditions. The volcano plot was constructed and displayed as described in the legend to Fig. 2. Black dots located outside the curves represent proteins differentially found between the two conditions with q values below 1% (Table S1). (C) Partners of the nitrate reductase under nitrate-respiring conditions no longer interact with the complex in anoxic fermentation. Shown is a heat map focusing on the 9 proteins identified in Fig. 2 displaying the Z-scored LFQ intensity obtained for each biological replicate using untagged (left) or tagged (right) version of the nitrate reductase under anoxic fermentative conditions (Table S3). Gray represents missing values (not identified proteins). Download FIG S3, TIF file, 1.8 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S4

    A similar nitrate reductase interactome is obtained in the absence of cross-linking. The experiment was done exactly as for Fig. S3A, but the cross-linking step was avoided. The volcano plot of proteins immunoprecipitated with NarGeGFP versus the untagged version of NarG under nitrate-respiring conditions is shown. The volcano plot was constructed and displayed as described in the legend to Fig. 2, except the threshold was fixed by an enrichment greater than 4-fold and a P value of <0.05. Proteins identified in the legend to Fig. 2 are represented by labeled black dots. Download FIG S4, TIF file, 2.1 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    List of identified proteins immunoprecipitated with NarGeGFP versus untagged version of NarG under nitrate-respiring (Fig. S3A) or oxic (Fig. S3B) conditions. Download Table S1, XLSX file, 0.01 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    List of identified proteins immunoprecipitated with NarGeGFP under nitrate- versus oxygen-respiring conditions (Fig. 2). Download Table S2, XLSX file, 0.01 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    List of LFQ intensities for all identified partners of NarGeGFP and full data set. Download Table S3, XLSX file, 1.7 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S5

    The subcellular organization of the nitrate reductase is unchanged in the hcp-deficient strain. (A) Fluorescence images (top) and overlays of fluorescence and phase-contrast images (bottom) are shown for nitrate-respiring cells. (B) The histogram of the fluorescent cluster distribution across the transversal axis of nitrate respiring LCB3063 cells harboring pVA70GFP is shown. Dashed lines delimit pole zones. The analysis was performed on 797 cells issued from three independent experiments. (C) The heat map shows the mean fluorescent profiles from 797 cells. The fluorescence density is represented by a color gradient shown on the right side. Download FIG S5, TIF file, 1.6 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    List of identified proteins immunoprecipitated with NarGeGFP versus the untagged version of NarG under the nitrate-respiring condition but in the absence of hcp (Fig. 3). Download Table S4, XLSX file, 0.01 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S5

    Strain, plasmid, and oligonucleotide list. Download Table S5, DOCX file, 0.02 MB.

    Copyright © 2019 Bulot et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

PreviousNext
Back to top
Download PDF
Citation Tools
Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli
Suzy Bulot, Stéphane Audebert, Laetitia Pieulle, Farida Seduk, Emilie Baudelet, Leon Espinosa, Marie-Camille Pizay, Luc Camoin, Axel Magalon
mBio Oct 2019, 10 (5) e01832-19; DOI: 10.1128/mBio.01832-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this mBio article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli
(Your Name) has forwarded a page to you from mBio
(Your Name) thought you would be interested in this article in mBio.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli
Suzy Bulot, Stéphane Audebert, Laetitia Pieulle, Farida Seduk, Emilie Baudelet, Leon Espinosa, Marie-Camille Pizay, Luc Camoin, Axel Magalon
mBio Oct 2019, 10 (5) e01832-19; DOI: 10.1128/mBio.01832-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

cellular respiration
fluorescence microscopy
metalloprotein
nitric oxide

Related Articles

Cited By...

About

  • About mBio
  • Editor in Chief
  • Board of Editors
  • AAM Fellows
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Author Warranty
  • Article Types
  • Ethics
  • Contact Us

Follow #mBio

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Online ISSN: 2150-7511