Article Figures & Data
Figures
- FIG. 1.
cdpA encodes a c-di-GMP PDE. (A) The domain structure of the VC0130 protein, CdpA, is shown. CdpA is a three-domain protein containing an N-terminal domain of unknown function (COG3287), a GGDEF, and an EAL domain. The divergent amino acid sequences present in cdpA at the GG(D/E)EF and EAL motifs are indicated in the relevant domains. The amino acids bounding each domain are noted below. (B) Lysates from AC1817 [VieA], AC1835 [VieA(E170A)], AC2376 [CdpA], and AC2642 [CdpA(AVGAW)] were assayed for protein expression by Western blot analysis. The WT and respective mutant proteins were comparably expressed. No His6-tagged protein was detected in vector-only controls (not shown). (C) Lysates containing the indicated proteins as well as buffer- and vector-only controls were tested for the ability to hydrolyze radiolabeled c-di-GMP. The reactions were analyzed by TLC; products and corresponding Rf values are indicated on the right.
- FIG. 2.
The degenerate GGDEF domain of CdpA is required for optimal PDE activity of the EAL domain. Altered PDE activity of the mutated proteins was determined based on their effects on in vitro c-di-GMP-regulated phenotypes in the cdpA strain, namely, motility and biofilm production. (A) The mean diameters of motility are given in mm for strains containing empty vector, pCdpA, pCdpAΔEAL, or pAVGAW (CdpA containing a mutated GGDEF motif). The medium contained 50 μM IPTG to induce gene expression and Km to maintain the plasmids. (B) The amount of biofilm formed by the above-described strains grown under static conditions in the presence of 50 μM IPTG is shown. In both figures, asterisks indicate significant changes from WT. The pCdpA and pAVGAW strains had significantly different motility (P = 9 × 10−5) and biofilm formation (P = 6 × 10−4) levels.
- FIG. 3.
CdpA regulates a subset of c-di-GMP-regulated processes. (A) CdpA negatively regulates biofilm production. Shown are the mean A570 values reflecting crystal violet staining of the biofilms formed by WT, the cdpA strain, and the complemented cdpA(pCdpA) strain. The amount of IPTG present in the medium is indicated above each sample. All strains showed significant differences in biofilm formation from WT by Student's t test (P < 0.05). (B) CdpA does not regulate the motility of V. cholerae. Motility was assessed in soft agar medium and is presented as the mean diameter of motility (mm). (C) CdpA is dispensable for V. cholerae colonization of the infant mouse. The cdpA strain was competed against the fully virulent lacZ mutant strain AC66; the cdpA(pCdpA) complementation strain was competed against AC2365 containing empty vector. Each shape represents a CI from a single animal. The horizontal bars indicate the median CI.
- FIG. 4.
Expression of cdpA during infection of the mouse. Expression of cdpA during infection of the mouse was measured at 7 h and 21 h p.i. using RIVET. Expression in shaking broth culture (37°C) was included as a control. Expression of ctxA, a virulence gene known to be induced early during infection, was analyzed in parallel. Transcription of cdpA was not induced at 7 h p.i. compared to what was seen in vitro but was activated by 21 h p.i. (P = 0.002). In contrast, transcription of ctxA was induced by an early time point during infection (P = 0.001). In both figures, each symbol represents the percentage of CFU that were Km sensitive (resolved) under each condition at each time point in an individual sample. Horizontal lines indicate the median percent resolved CFU.
- FIG. 5.
Ectopic modulation of intracellular c-di-GMP by expression of vdcA. (A) To directly test the ability of vdcA expression to increase intracellular c-di-GMP, 2D-TLC was done to detect intracellular c-di-GMP in strains containing pVdcA (AC1902), pVdcAi (AC2389), or vector alone (AC1903). The c-di-GMP and GMP spots are indicated. (B) The pVdcA, pVdcAi, and vector-only strains were tested for the ability to activate biofilm formation. The mean intensities of crystal violet staining of the biofilms (A570) are shown. (C) The pVdcA, pVdcAi, and vector strains were tested for decreased motility. The mean diameters of motility in mm from three independent assays are shown. In panels B and C, asterisks indicate significant differences in biofilm and motility, respectively, compared to WT as determined by Student's t test.
- FIG. 6.
The effects of increased c-di-GMP on virulence of V. cholerae. (A) The pVdcA and pVdcAi strains were competed against WT containing vector alone in mice. In addition, the VdcA and VdcA(I) strains were competed directly to control for any effects of protein levels. Each symbol represents the CI obtained from an individual mouse. The horizontal bars indicate the geometric mean CI. Asterisks denote statistically significant differences in colonization (P < 1 × 10−4). (B) Transcription of toxT during infection of the mouse in the presence of WT (pVdcAi) and ectopically increased c-di-GMP (pVdcA) was measured using RIVET. The percentage of CFU that were Tc sensitive, and therefore the percentage of bacteria that have expressed toxT, at the indicated time points p.i. are shown. Each data point represents the mean from at least three mice. Asterisks indicate significant differences in resolution rate at that time point as determined by Student's t test.
Tables
- TABLE 1.
Strains used in this study
Strain Description Reference or source V. cholerae strains AC51 El Tor C6709, Smr 39 AC66 C6709 lacZ::res-tet-res 7 AC1901 C6709 pMMB67EH::vieA-His6 This work AC1902 C6709 pMMB67EH::VCA0956(pVdcA) This work AC1903 C6709 lacZ::res-tet-res pMMB67EH This work AC2131 C6709 cdpA::pGP704 This work AC2280 C6709 ΔvpsR This work AC2284 C6709 ΔvpsR pMMB67EH::vieA-His6 This work AC2286 C6709 ΔvpsR pVdcA This work AC2365 C6709 lacZ::res-tet-res pMMBneo This work AC2377 C6709 cdpA::pGP704 pMMBneo::cdpA-His6 This work AC2380 C6709 pMMBneo::cdpA-His6 This work AC2389 C6709 pMMB67EH::VCA0956(E258A) pVdcA(I) This work AC2390 C6709 pMMB67EH::vieA(E170A) This work AC2395 C6709 lacZ pVdcA This work AC2434 C6709 cdpA::pGP704 pMMBneo This work AC2435 C6709 ΔvpsR pMMBneo This work AC2483 C6709 toxT::tnpR lacZ::res-tet-res pVdcA This work AC2484 C6709 toxT::tnpR lacZ::res-tet-res pVdcA(I) This work AC2595 C6709 lacZ::res-neo-sacB-res This work AC2606 C6709 cdpA::tnpR lacZ::res-sacB-neo-res This work AC2655 C6709 pMMBneo::cdpA(AVGAW)-His6 This work AC2658 C6709 cdpA::pGP704 pMMBneo::cdpAΔEAL-His6 This work AC2659 C6709 cdpA::pGP704 pMMBneo::cdpA(AVGAW)-His6 This work E. coli strains DH5α F− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1 Laboratory strain DH5αλPir F− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1 λ::pir Laboratory strain SM10λPir thi recA thr leu tonA lacY supE RP4-2-Tc::Mu λ::pir Laboratory strain AC575 DH5α pMMB67EH 33 AC751 DH5αλPir pCVD442lac 11 AC2364 DH5α pMMBneo 35 AC1817 DH5α pMMB67EH::vieA-His6 48 AC1835 DH5α pMMB67EH::vieA(E170A)-His6 48 AC1887 DH5α pVdcA This work AC2129 SM10λPir pGP704::′cdpA′a This work AC2142 SM10λPir pCVD442lac::ΔvpsR This work AC2376 DH5α pMMBneo::cdpA-His6 This work AC2386 DH5α pVdcA This work AC2528 SM10λPir pRes 35, 44 AC2599 SM10λPir pIVET5ncdpA This work; 44 AC2641 DH5α pMMBneo::cdpAΔEAL-His6 This work AC2642 DH5α pMMBneo::cdpAAVGAW-His6 This work ↵a An internal fragment of cdpA was cloned in order to make the plasmid insertion.
- TABLE 2.
Primers used in this study
Primer name Sequencea (5′ to 3′) (reference) oriR CAGCAGTTCAACCTGTTG 67EHF CGACATCATAACGGTTCTGG 67EHR TTCACTTCTGAGTTCGGCAT 0130FX GGTCTAGAAGAGCGGTTTGTATGCGATT 0130RX GGTCTAGAGCCGGCTCAAACGAGTATAG 0130PF TAATCGCCCTGAAAGTGACC C0130F TTCTAGATTTAGGATACATTTTTATGTTTACGGTCTCGC C0130R TTGCATGCCTAATGGTGATGGTGATGGTGACCCAAGCGTGAAGGT 0130EAL TTCTAGATTTAGGATACATTTTTATGGCGGTGGTACGTGATGAT vpsRF1 TGCATGCCTACAACCCAAATCACGC vpsRR1 AATCAGCAAAACTTACATGAACCTATATTCCTT vpsRF2 GAATATAGGTTCATGTAAGTTTTGCTGATTTAC vpsRR2 TTTCTAGAGGTAAACTCAAGCCGATT vpsRF0 CTCTGTGGCGTTAGAAG vpsRR0 CCTGTCCTTAGTGATGTG A0956F1 CCGAGCTCTTTAGGATACATTTTTGTGATGACAACTGAAGAT A0956R1 GGGCATGCAGTTTAGAGCGGCATGAC A0956EAF GGCGGTGAAGCGTTTGCACTG A0956EAR CAGTGCAAACGCTTCACCGCC 0130GEF TTCTAGATTTAGGATACATTTTTATGACGGACAATTTGGCAC 0130GAF GCTGTCGGTGCTTGGGCAACGGTTTTT 0130GAR CCAAGCACCGACAGCAATCGCATACAAACC 0130EAF TACGCTTGTTTAGTGCGGATTGAA 0130EAR CACTAAACAAGCGTAGGAAGCCAC 0130CGR TTGCATGCCTAATGGTGATGGTGATGGTGCTCCTGACGAACACTTTC 0130GAS GCGATTGCTGTCGGTGCT 0130EAS ATTGTGGCTTCCTACGCT 3287qF TTCTCTCCTTTGAGTCGCGAGCAT 3287qR ATTTGCAGCAACGTCACCTGATGG vpsRqF TGGATTCAGTACCTGGCTCT vpsRqR CGCAATCCCGTTAAGGCTAA RPB2FD CTGTCTCAAGCCGGTTACAA (38) RPB2RV TTTCTACCAGTGCAGAGATGC (38) ↵a Restriction sites are underlined; sequence encoding the His6 tag is both underlined and in boldface; boldface text without underlining indicates codons changed to obtain desired amino acid mutations.
