Article Figures & Data
Figures
- FIG 1
PdcA enzymatic activity is controlled by regulatory domains in vitro and in vivo. (A) Schematic of the PdcA derivatives used in these experiments with the corresponding symbols and colors used in panels B to D. The EAL domain of PdcA contains a substitution of valine for alanine in the EAL motif, noted here as EVL. (B) Percentages of initial c-di-GMP substrate hydrolyzed by full-length PdcA, PdcA(GA), PdcAΔPAS, and PdcA-EAL, determined at 1-min intervals after initiation of the reaction upon addition of the substrate. Open circles indicate values for buffer-only negative controls. The data shown are mean values and standard deviations from three [WT and PdcA(GA)] or two (PdcAΔPAS and PdcA-EAL) separate protein purifications. (C) PDE activity, expressed as a percentage of the starting c-di-GMP hydrolyzed at 1 min with increasing concentrations of GTP. The data shown are mean values and standard deviations. The activity of full-length PdcA (blue) and PdcAΔPAS (purple) at a given GTP concentration was compared to the activity of the same protein in the absence of GTP by one-way analysis of variance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; asterisks are color coded according to comparison). The activities of PdcA(GA) and PdcA-EAL in the presence of GTP were compared to their activities in the absence of GTP by one-way analysis of variance and were not statistically significantly different at any of the GTP concentration tested (not shown). (D) C. difficile strains with (left to right) the vector (v), pPdcA, pPdcA(GA), pPdcAΔPAS, or pPdcA-EAL were assayed for motility in BHIS-Tm with 0.3% agar supplemented with 0 or 5 μg/ml nisin. Motility diameters were measured after 72 h at 37°C. Data were analyzed by two-way analysis of variance and Bonferroni's multiple-comparison test comparing values to the average vector control value (**, P < 0.01).
- FIG 2
CodY regulates pdcA transcription. Transcript levels of the genes indicated in WT JIR8094 (erythromycin-sensitive derivative of C. difficile strain 630) and an isogenic codY-null strain in exponential phase (black) and stationary phase (gray) were measured by qRT-PCR. The data were analyzed by the ΔΔCT method as described in Materials and Methods, here with exponential-phase WT cells as the reference condition. The mean values and standard deviations from three biological replicates are shown. The data were analyzed by unpaired t test comparing the exponential- and stationary-phase transcript levels of each gene (*, P < 0.05; ***, P < 0.001).
- FIG 3
CodY binds directly to the pdcA promoter region. Purified six-histidine-tagged C. difficile CodY was tested for the ability to bind the pdcA promoter region with EMSAs. Serial 2-fold dilutions of CodY were incubated with the ilvC promoter region previously shown to be directly bound by CodY (A) or the pdcA promoter region (B). As a negative control, a 133-bp V. cholerae DNA fragment was included in each binding reaction mixture (asterisks). The concentrations of CodY used were (left to right) 0, 7.2, 3.6, 1.8, 0.9, 0.45, 0.23, 0.12, and 0.06 μM. In each reaction mixture and in the electrophoresis buffer, isoleucine, leucine, and valine were added to promote CodY binding. A representative of three independent experiments is shown. The values to the left are molecular sizes in kilodaltons.
- FIG 4
pdcA affects biofilm formation but not swimming motility or global c-di-GMP levels of C. difficile. (A) c-di-GMP in the cytoplasm of the 630 WT and pdcA::catP mutant C. difficile strains was quantified by UPLC-MS and normalized to the total cellular protein level. The mean values and standard deviations of six biologically independent samples are shown. (B) Swimming motility through BHIS–0.3% agar was measured at 24, 48, and 72 h. The mean values and standard deviations of six biologically independent samples are shown. (C) Biofilm formation assayed by crystal violet staining after 24 h of growth. The mean values and standard deviations of five biologically independent samples are shown. **, P < 0.01 by unpaired t test. (D, E) Complementation analysis of biofilm formation after 24 h of growth. pRT1099 is the vector control, pRT1214 encodes PdcA, and pRT1662 encodes a catalytically inactive form of PdcA. (D) Representative image of crystal violet-stained biofilms. (E) Quantification of biofilm biomass by crystal violet staining. The mean values and standard deviations of three biologically independent samples are shown. **, P < 0.05 by one-way analysis of variance and Dunnett's multiple-comparison test.
- FIG 5
pdcA influences C. difficile toxin production and cytopathicity. (A) TcdA production by 630/pRT1099 (vector control), pdcA::catP/pRT1099 (vector control), pdcA::catP/pRT1214 (complementation with pdcA WT allele), and pdcA::catP/pRT1662 (complementation with pdcA E479A mutant allele). Strains were grown for 16 h in TY medium, and lysates were probed for TcdA by Western blot analysis. Samples were normalized by adjustment to the OD of the culture. The data are expressed as a percentage of the 630/pRT1099 value in each respective experiment. The mean values and standard errors from four independent assays are shown. Data were analyzed by one-way analysis of variance and Tukey's multiple-comparison test (**, P < 0.01; ****, P < 0.0001 compared to 630/pRT1099; #, P < 0.01 for the comparisons indicated). A representative Western blot analysis for TcdA is shown at the top. (B) Relative viability of MDCK-LA cells after incubation with supernatants from 630 WT and pdcA::catP mutant strain stationary-phase cultures. Data shown are mean values and standard deviations of three biological replicates and were analyzed by two-way analysis of variance and Dunnett's posttest (n.t., not treated; n.s. not significant; *, P < 0.05; ***, P < 0.001). (C) Cytopathicity of 630 WT and pdcA::catP mutant strain supernatants against MDCK-LA cells after 24 h of incubation. Scale bars, 10 μm. (D) Effects of the 630 WT and pdcA::catP mutant strains on the actin cytoskeleton (red). TIRF microscopy shows stress fibers at the basolateral cell surface. Epifluorescence shows punctate microvilli at the apical surface. Scale bars, 10 μm.
- FIG 6
Transcriptional regulation in the pdcA::catP mutant. (A) Relative transcript levels of tcdA, flgB, and fliC in WT (black) and pdcA::catP mutant (gray) cells during exponential growth. (B) Relative transcript levels during stationary phase. The mean values and standard deviations of five biological replicates are shown. The data were analyzed by unpaired t test (**, P < 0.05).
- FIG 7
GTP levels inversely regulate the production and activity of PdcA enzymes. At very low cytoplasmic GTP levels, the activity level of existing PdcA proteins will be low, but CodY will be deactivated to permit additional pdcA transcription. At intermediate concentrations, both pdcA transcription and PdcA activity will be responsive to GTP fluctuations. At high GTP levels, pdcA transcription will be inhibited but existing PdcA will be very active.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1. Strains and plasmids used in this study. Table S2. Oligonucleotides used in this study. Fig. S1. Construction of the pdcA::catP mutant. Fig. S2. Putative CodY binding site upstream of pdcA. Fig. S3. Complementation of toxin and flagellar gene expression during growth in stationary phase.
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- Supplemental file 1 -
