Research ArticleMICROBIOLOGY

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism

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Science Advances  26 Feb 2020:
Vol. 6, no. 9, eaaz0260
DOI: 10.1126/sciadv.aaz0260
  • Fig. 1 Rapid disappearance of FtsA and FtsZ rings in Escherichia coli undergoing nitrosative stress.

    (A) Overview of E. coli cell division. Division starts with GTP-dependent assembly of the FtsZ ring, which is subject to both positive and negative regulation. (B) Representative micrographs of E. coli EC447 (ftsA-gfp) grown to midlog phase in LB-glycerol. Where indicated, the specimens were treated with 50 μM carbonyl cyanide-m-chlorophenylhydrazone (CCCP) or 750 μM spermine NONOate (sNO) for 2 min before fixation. The micrographs are representative of specimens from three experiments. (C) The percent of bacterial cells containing FtsA-GFP (green fluorescent protein) in septa or puncta was recorded in a total of 644 to 960 cells from three separate experiments. ***P < 0.001 compared to septa in control bacteria. (D) Organization of Z rings in FtsZ-GFP–expressing E. coli (EC448) treated for 1 to 30 min with 750 μM sNO. Untreated cells were used as controls.

  • Fig. 2 Effects of carbon source on early stages of cell division.

    (A and C) Bacterial growth was recorded by following optical density at 600 nm (OD600) after the cultures were treated with PAPA NONOate (pNO) or CCCP at the indicated times (arrows). Representative micrographs of E. coli EC448 (ftsZ-gfp) or EC447 (ftsA-gfp) grown in MOPS–casamino acids (CAA) (B) or MOPS-glucose (GLC) (D) minimal media containing isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce the gfp fusions. Where indicated, cultures were treated with 250 μM pNO or 50 μM CCCP for 5 min before fixation. The data are representative of three experiments.

  • Fig. 3 Carbon source modulates the energetics of bacterial cells undergoing nitrosative stress.

    Effect of NO on respiration (A) and PMF (B) of E. coli grown in MOPS-GLC or MOPS-CAA. O2 consumption was measured polarographically, whereas the PMF was estimated fluorometrically by the accumulation of 3,3′-dipropylthiadicarbocyanine iodide [DiSC3(5)]. Selected samples were treated for 5 min with 250 μM pNO, 750 μM sNO, or 50 μM CCCP. ****P < 0.0001 versus untreated controls, as calculated by one-way analysis of variance (ANOVA). A.U., arbitrary units. (C) Thin-layer chromatography (TLC) analysis of nucleoside triphosphates (NTPs) extracted from E. coli cells treated with sNO, pNO, or CCCP for 5 min. UTP, uridine 5′-triphosphate; CTP, cytidine 5′-triphosphate; PPi, inorganic pyrophosphate; ppGpp, guanosine 5′-diphosphate 3′-diphosphate. (D) ATP was measured in cytoplasmic extracts by luciferase-dependent chemiluminescence. ****P < 0.0001 versus untreated controls, as calculated by one-way ANOVA. (E) Localization of FtsZ-GFP was evaluated by fluorescence microscopy. **P < 0.001 versus wild-type (WT) control (Ctrl). The data in (A) are representative of three independent experiments, whereas the data in (B), (D), and (E) are means ± SD from four independent observations. Strain EC448 was used for (A), (C), and (D). Strain MC4100 was used for (B). Strains EC3749 and EC3751 were used for (E). ns, not significant.

  • Fig. 4 Inositol monophosphate dehydrogenase is a target of NO-mediated cytostasis.

    (A) Total GuaB protein in E. coli MC4100 expressing pWSK29::guaB-3xFLAG (strain AV17035) grown in MOPS-GLC or MOPS-CAA was measured by Western blot. Where indicated (+), the cells were treated with 250 μM pNO. The DnaK chaperone was used as internal control. GuaB/DnaK ratios were calculated by densitometry. (B) The redox state of GuaB cysteine residues was determined by Western blotting after alkylation of thiol groups with AMS. Oxidized cysteine residues are not amenable to derivatization with AMS, and thus, the higher the number of reduced cysteine residues in GuaB, the higher the molecular weight. (C) IMPDH enzymatic activity in cytoplasmic extracts of log-phase E. coli grown in MOPS-GLC or MOPS-CAA media. Some cultures were treated with increasing concentrations of pNO for 5 min before determination of enzymatic activity. IMPDH enzymatic activity, corrected for protein concentrations, is expressed as mean ± SD; n = 4. (D) IMPDH activity of recombinant GuaB protein after treatment with pNO. (E) TLC analysis of nucleotides extracted from E. coli MC4100 expressing pBAD18 (strain AV17086) or pGUAB (strain AV17072) grown in MOPS supplemented with CAA and 0.2% l-arabinose. Bacteria were treated with 0 to 100 μM pNO for 1 min before extraction. Autoradiogram is representative of three independent experiments. (F) Quantification of the GTP pool by ImageJ analysis (N = 3). (G) The localization of FtsZ-GFP in log-phase E. coli EC448 grown in MOPS-CAA media containing 10 μM IPTG and 0.2% arabinose. Where indicated, the specimens were treated with 150 μM pNO. The micrographs are representative of specimens from four to six experiments. (H) The percentage of cells with FtsZ rings was quantified from cells imaged in (G). (I) Model for NO-dependent inhibition Z ring formation in respiring E. coli. Nitrosylation of quinol oxidases (Qnl Ox) poisons respiration, collapses the PMF, and prevents ATP synthesis via oxidative phosphorylation and, thus, conversion of GDP to GTP. Concomitantly, NO inhibits GTP synthesis by modifying cysteine residues in IMPDH, the first committed step for de novo GTP synthesis. ***P < 0.001; ****P < 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/9/eaaz0260/DC1

    Fig. S1. Nitric oxide causes rapid disassembly of the divisome in E. coli grown in LB.

    Fig. S2. SPOR domains are retained at the division site in E. coli undergoing nitrosative stress.

    Fig. S3. Nitrosative stress destroys FtsZ rings in Salmonella Typhimurium and B. subtilis.

    Fig. S4. NO causes rapid disassembly of FtsZ rings in E. coli strains deficient in the SOS response, Min system, or stringent response.

    Fig. S5. Nitrosative stress delocalizes E. coli divisome proteins when amino acids but not glucose serve as the source of carbon and energy.

    Fig. S6. Energy source determines whether CCCP delocalizes FtsZ and FtsA in E. coli.

    Fig. S7. Influence of glucose media on the localization of FtsA in NO-treated E. coli.

    Fig. S8. IMPDH expression in NO-treated E. coli.

    Table S1. Strains used in this study.

    Table S2. Plasmids and oligonucleotide primers used in this study.

    Table S3. Oligonucleotide primers.

    References (4162)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Nitric oxide causes rapid disassembly of the divisome in E. coli grown in LB.
    • Fig. S2. SPOR domains are retained at the division site in E. coli undergoing nitrosative stress.
    • Fig. S3. Nitrosative stress destroys FtsZ rings in Salmonella Typhimurium and B. subtilis.
    • Fig. S4. NO causes rapid disassembly of FtsZ rings in E. coli strains deficient in the SOS response, Min system, or stringent response.
    • Fig. S5. Nitrosative stress delocalizes E. coli divisome proteins when amino acids but not glucose serve as the source of carbon and energy.
    • Fig. S6. Energy source determines whether CCCP delocalizes FtsZ and FtsA in E. coli.
    • Fig. S7. Influence of glucose media on the localization of FtsA in NO-treated E. coli.
    • Fig. S8. IMPDH expression in NO-treated E. coli.
    • Table S1. Strains used in this study.
    • Table S2. Plasmids and oligonucleotide primers used in this study.
    • Table S3. Oligonucleotide primers.
    • References (4162)

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