Article Figures & Data
Figures
- 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
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
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
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
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.
