Research ArticleMicrobiology

Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation

See allHide authors and affiliations

Science Signaling  26 Jan 2016:
Vol. 9, Issue 412, pp. ra11
DOI: 10.1126/scisignal.aad0446
  • Fig. 1 Fic-1 affects plasmid DNA production in E. coli.

    (A) The effect of individual fic genes on the amount of plasmid DNA in E. coli. Plasmid DNA containing the indicated constructs isolated from saturated cultures of E. coli was resolved by agarose gel electrophoresis before (left panel) or after digestion with restriction enzymes Bam HI and Sal I (fic-1) or Xba I and Hind III (fic-2 and fic-3) (middle panel). In the middle panel, both bands represent the products of the digested plasmid DNA. Data are representative of three experiments. The intensity of the bands corresponding to the vector was measured from three independent experiments (right panel). ***P < 0.001. (B) The effect of mutating the Fic domain of Fic-1 on the amount of plasmid DNA in E. coli. Plasmid DNA containing wild-type fic-1 and fic-1 with a Fic domain mutation (Fic-1H135A) was isolated from saturated cultures of E. coli, subjected to restriction enzyme digestion, and separated by agarose gel electrophoresis (upper panel). Western blot analysis of isocitrate dehydrogenase (ICDH), a metabolic enzyme, served as a loading control. Data are representative of three experiments. The intensity of the bands corresponding to the vector was measured from three independent experiments (right panel). ***P < 0.001.

  • Fig. 2 Induction of cell filamentation by Fic-1.

    (A) Cells from E. coli transformants expressing SUMO-tagged Fic-1 or its H135A mutant grown for 16 hours on LB agar resuspended in phosphate-buffered saline (PBS) were fixed and stained with Hoechst. (B) Cells of P. fluorescens grown in LB with glucose were diluted in ABM medium containing 0.2% arabinose and grown for 12 hours. Cells were treated for imaging as described in (A). Images acquired with a fluorescence microscope were pseudocolored with IPLab software. (C and D) Cells grown as described in (A) and (B) were processed for SDS-PAGE. Proteins were probed for Fic-1 by immunoblotting. The metabolic enzyme ICDH was probed as a loading control (lower panels). Similar results were obtained from at least three independent experiments.

  • Fig. 3 The interactions between Fic-1 and GyrB.

    (A) Fic-1 interacts with GyrB as measured by a bacterial two-hybrid assay. E. coli strains derived from BTH101 harboring plasmids expressing the indicated fusion protein pairs were assayed for β-galactosidase activity indicative of protein-protein interactions. Experiments were performed in triplicate, and similar results were obtained from three independent experiments. V, control plasmid. (B) Interactions between GyrB and Fic-1 in vitro. GyrB-His6 was incubated with Affigels coated with Fic-1, Fic-1H135A, or BSA. After extensive washing, bound proteins eluted with SDS sample buffer were resolved by SDS-PAGE and detected by Coomassie brilliant blue staining. Ten percent of the GyrB used for the binding assay was loaded as reference. Data shown are representative of three experiments with similar results.

  • Fig. 4 Fic-1 AMPylates GyrB at Tyr109.

    (A) Identification of AMPylation of GyrB by MS. The upper panel is the extracted ion chromatograms of the unmodified and adenylylated versions of the peptide -F105DDNSYK110- derived from GyrB coexpressed with Fic-1 (left panel) or mutant Fic-1H135A (middle panel). The abundance of the randomly chosen peptide -G699LLEEDAFIER709- is almost identical in these two samples (right panel). The lower panel is the tandem mass spectrum of peptide -F105DDNSYK110-. The asterisks represent neutral losses of the adenosine moiety of the adenylylation modification. F, immonium ion of phenylalanine residue. (B) In vitro AMPylation of GyrB by Fic-1. After incubation at 35°C for 30 min, 32P-α-AMP–labeled signals and total proteins were detected by autoradiography for 30 sec (upper panel) or by staining (lower panel). (C) AMPylation of GyrB abolishes its ATPase activity. Samples from 30-min reactions were examined for ATP hydrolysis by measuring released phosphate (left panel). Induction of the SOS pathway by Fic-1 or Fic-1H135A was evaluated by the levels of LexA and RecA with ICDH as a loading control (middle panel). The band intensities of RecA (upper right) and LexA (lower right) were measured by LI-COR. All results are from three independent experiments. ***P < 0.0001.

  • Fig. 5 Self-AMPylation is important for the activity of Fic-1.

    (A) Self-modification by Fic-1 occurs on Tyr5. Fic-1 co-incubated with ATP was analyzed by MS after trypsin digestion. The upper panel is the extracted ion chromatograms of the unmodified and adenylylated versions of the tryptic peptide containing Tyr5. The abundance of the randomly chosen peptide -N179GVMEPMEQVFEK191- is almost identical in these two samples (right panel). The lower panel is the tandem mass spectrum of the AMPylated peptide -Y5GVGEDAYCYPGSTVLR21-. CAM, carbamidomethylation. (B) A mutation in Tyr5 abolishes self-AMPylation of Fic-1. Reactions containing the indicated proteins were incubated at 35°C for 30 min, AMPylation was detected by autoradiography for 2 min (upper panel), and total proteins were visualized by staining (lower panel). WT, wild type. (C) The Y5A mutation abolishes the ability of Fic-1 to inhibit plasmid DNA yield. DNA of plasmids carrying fic-1 or the indicated mutants isolated from identical amounts of E. coli cells was digested with restriction enzymes Bam HI and Sal I and was separated using agarose gels (upper panel). An identical set of samples was processed for immunoblotting to detect the levels of Fic-1 (lower panel). The images in (B) and (C) are representative of three independent experiments with similar results.

  • Fig. 6 Inhibition of the AMPylation activity of Fic-1 by AntF.

    (A) Dose-dependent inhibition of Fic-1 by AntF. The indicated amounts of His6-SUMO-AntF were added to a series of identical reactions containing Fic-1. After 30 min of incubation, equal amounts of a mixture containing GyrB and 32P-α-ATP were added, and the reactions were allowed to proceed for 30 min at 35°C. 32P-α-GyrB and total proteins were detected by autoradiography (upper panel) and Coomassie blue staining (lower panel). (B) Quantification of 32P-α-GyrB signals. The strength of autoradiography signals from three independent experiments done under the same conditions was measured and analyzed by ImageJ. **P < 0.01.

  • Fig. 7 Expression of Fic-1 in P. fluorescens strains and induction of cell filamentation by Fic-1 in E. coli defective in sulA and recA.

    (A) Bacterial strains were grown in LB to an optical density at 600 nm (OD600) of 2.4, and protein samples prepared from 300-μl cultures were resolved by SDS-PAGE. Fic-1 was probed with a specific antibody. A protein nonspecifically recognized by the antibody at about 30 kD served as a loading control. Note that Fic-1 is detectable only in the WT. (B) Morphology of cells expressing Fic-1. Cells of transformants grown for 16 hours on LB agar resuspended in PBS were fixed and stained with Hoechst. Images acquired with a fluorescence microscope were pseudocolored with IPLab software. Images shown are representative of three experiments with similar results. (C) Distribution of cells with different lengths. The length of 500 cells was measured from each of three samples, and their distribution was plotted. Data shown are representative from three independent experiments.

  • Fig. 8 A model of Fic-1–mediated induction of bacterial cell filamentation and its regulation.

    Fic-1 and the α-inhibitor AntF form a dynamic complex under normal conditions. Inducing signals from the environment activate a cascade that leads to the production of a sequestering protein (Seq) that competes for AntF or the activation of a protease that degrades the α-inhibitor. Freed or activated Fic-1 then inactivates GyrB by AMPylation, leading to the induction of SulA and the formation of filamentous cell. Alternatively, AMPylated GyrB may induce cell filamentation through an SOS response–independent pathway (dashed arrows).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/412/ra11/DC1

    Fig. S1. Diagrams of the three fic genes present in P. fluorescens strain 2P24.

    Fig. S2. Alignment of Fic proteins similar to Fic-1 from other bacteria.

    Fig. S3. Fic-1 affects the DNA yield of plasmids from different incompatibility groups.

    Fig. S4. Mutants of Fic-2 and Fic-3 defective in the intramolecular inhibitory motif did not affect plasmid DNA yield.

    Fig. S5. Fic-1 inhibits the growth of both E. coli and P. fluorescens.

    Fig. S6. Fic-1 AMPylates the N-terminal domain of GyrB (1–200).

    Fig. S7. Fic-1 AMPylates GyrB from P. fluorescens on Tyr111.

    Fig. S8. The Fic-1Y5A mutant is defective in self-AMPylation and has low activity against GyrB.

    Fig. S9. AntF inhibits the activity of Fic-1 by direct interactions.

    Table S1. Proteins analyzed for interactions with Fic-1.

    Table S2. Bacterial strains, plasmids, and primers used in the study.

    Reference (60)

  • Supplementary Materials for:

    Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation

    Canhua Lu, Ernesto S. Nakayasu, Li-Qun Zhang,* Zhao-Qing Luo*

    *Corresponding author. E-mail: zhanglq{at}cau.edu.cn (L.-Q.Z.); luoz{at}purdue.edu (Z.-Q.L.)

    This PDF file includes:

    • Fig. S1. Diagrams of the three fic genes present in P. fluorescens strain 2P24.
    • Fig. S2. Alignment of Fic proteins similar to Fic-1 from other bacteria.
    • Fig. S3. Fic-1 affects the DNA yield of plasmids from different incompatibility groups.
    • Fig. S4. Mutants of Fic-2 and Fic-3 defective in the intramolecular inhibitory motif did not affect plasmid DNA yield.
    • Fig. S5. Fic-1 inhibits the growth of both E. coli and P. fluorescens.
    • Fig. S6. Fic-1 AMPylates the N-terminal domain of GyrB (1–200).
    • Fig. S7. Fic-1 AMPylates GyrB from P. fluorescens on Tyr111.
    • Fig. S8. The Fic-1Y5A mutant is defective in self-AMPylation and has low activity against GyrB.
    • Fig. S9. AntF inhibits the activity of Fic-1 by direct interactions.
    • Table S1. Proteins analyzed for interactions with Fic-1.
    • Table S2. Bacterial strains, plasmids, and primers used in the study.
    • Reference (60)

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.58 MB


    Citation: C. Lu, E. S. Nakayasu, L.-Q. Zhang, Z.-Q. Luo, Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation. Sci. Signal. 9, ra11 (2016).

    © 2016 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article