Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms
- University of California, Santa Cruz, United States
- Columbia University, United States
- Max Planck Institute for Terrestrial Microbiology, Germany
- National Research Council, Canada
Figures
Figure 1 with 2 supplements
RbmA binary switch regulates dimerization.
(A) Two views of the RbmA dimer structure (PDB: 4KKP) with the FnIII-1 domain binary switch depicted in its two states (D-loop, O-loop) via surface representation (olive). Dashed line, backbone …
Figure 1—figure supplement 1
Larger view of βc switch region.
Two views of the RbmA dimer structure (PDB: 4KKP) with the binary switch depicted in its two states (D-loop, O-loop) in olive; FnIII-1 domain (gray), FnIII-2 domain (white). Dashed line, backbone …
Figure 1—figure supplement 2
Mutations at D97 and R234 influence populations of the binary switch in RbmA.
Full 1H-15N TROSY-HSQC spectra of various full-length 15N RbmA proteins as indicated: (A) R234A, (B) wild-type (WT), (C) D97A, (D) D97K, (E) comparison of spectra for full-length WT RbmA and the …
Figure 2 with 1 supplement
Switch mutations at D97 and R234 exert changes in the global structure of RbmA.
(A) Limited proteolysis of full-length wild-type (WT), R234A and D97A mutants in the presence of 1:150 (w/w) ratio of trypsin for the indicated times at room temperature. Tryptic fragments were …
Figure 2—figure supplement 1
Western blot analysis of RbmA stability in wild-type smooth strain (low-VPS producing strain) at various growth phases.
Anti-BSA was used as an additional loading control. Culture supernatant (CS) fraction fractions were spiked with BSA. WC, whole-cell fraction.
Figure 3 with 2 supplements
RbmA loop conformational transitions impacts biofilm architecture.
(A) Colony corrugation phenotypes and (B) darkfield images of thin-sectioned paraffin-embedded spot colonies after 5 and 2 days of growth, respectively, at 25°C. Scale bars, 0.5 mm (in A), 1 mm (in B…
Figure 3—figure supplement 1
Analysis of RbmA production and degradation.
Western blot analysis of RbmA in culture supernatant (CS) and whole-cell (WC) fractions of rugose, various RbmA strains harboring chromosomal rbmA deletion or point mutations (D97A, D97K and R234A), …
Figure 3—figure supplement 2
Quantitative analysis of (A) biofilm biomass, (B) average biofilm thickness, (C) maximum biofilm thickness, and (D) colony counts (CFU/mL) in the effluent of the biofilms formed by the rugose strain, ΔrbmA and various mutant strains at 24 hr post-inoculation as determined by COMSTAT and dilution plating analysis.
***p<0.0003; **p<0.002; *p<0.04; n.s., not significant. (E) Approximate colony height measurements at 48 hr of rugose parental strain and various mutants. (F) Top-views of spot biofilms of rugose …
Figure 4 with 1 supplement
Biofilm development at single-cell resolution.
(A) Average nearest-neighbor distance between individual cells, measured as a function of time and height of a biofilm colony. At each time point in this heatmap, the spatial variation of the …
Figure 4—figure supplement 1
Nearest-neighbor distance, vertical orientation, radial orientation and volume of individual cell in the biofilms formed by rugose parental strain and various mutants, analyzed at single-cell resolution.
In each row, the same biofilm of a particular strain, indicated on the left, is shown as an x-z-projection (see Figure 4B for a definition of the coordinate system). In each column of the figure, …
Figure 5 with 1 supplement
VPS binding induces formation of higher order structures in full-length RbmA dimers.
(A) Selected regions of overlaid 1H-15N TROSY-HSQC spectra of full-length 15N RbmA WT (top) and R234A (bottom). Dashed line represents horizontal trace for peak intensity analysis shown above. Blue, …
Figure 5—figure supplement 1
VPS preferentially induces peak broadening in closed, dimeric forms of RbmA.
Stacked 1D 1H spectra of various full-length 15N RbmA proteins as indicated: (A) Wild-type (WT). VPS concentration increases from bottom to top as indicated; asterisk indicates new peaks that …
Figure 6 with 3 supplements
RbmA FnIII-2 domain binds VPS.
(A) Schematic representation of RbmA structure. (B) Zoomed-in regions of 1H-15N HSQC spectra of 15N RbmA proteins in the absence (RbmA*, magenta; FnIII-2 domain, blue) and presence (gray) of 4.5 …
Figure 6—figure supplement 1
Domain mapping and characterization of RbmA FnIII domains.
(A) Schematic representation of RbmA structure with constructs indicated. Color-coding applies throughout the figure. (B) Full-length and spontaneously cleaved RbmA proteins resolved on a 18% SDS …
Figure 6—figure supplement 2
NMR analysis of the FnIII-2 domain R234A mutation.
(A) Schematic representation of RbmA structure with constructs and mutation indicated. Color-coding applies throughout the figure. (B) Full 1H-15N HSQC spectrum of 15N FnIII-2 domain with R234A …
Figure 6—figure supplement 3
Complementation by FnIII domains and RbmA*.
Colony morphology of rugose and ΔrbmA strains carrying vector only, and ΔrbmA harboring various expression plasmids grown in the absence (top) and presence (bottom) of IPTG inducer after 3 days of …
Figure 7
Models of various RbmA variants and their involvement in biofilm formation.
(A) Cartoon models representing the various RbmA oligomerization and switch states. The VPS binding is indicated in blue on the FnIII-2 domain (white); dashed line indicates flexible or unfolded …
Videos
Video 1
Time lapse movie of colony biofilm development of rugose strain and various rbmA mutants.
https://doi.org/10.7554/eLife.26163.011
Video 2
Reconstructions of 3D biofilm containing approximately 1500 cells of rugose strain and various rbmA mutants.
Color code depicts average distance to the nearest neighbor.
Tables
Table 1
Size exclusion chromatography in-line with multi-angle light scattering (SEC-MALS) analysis.
https://doi.org/10.7554/eLife.26163.005| Construct | Calculated MW (kDa) (monomer: dimer) | Observed MW (kDa) |
|---|---|---|
| WT | 26.9: 53.8 | 1 peak; 51.8 ± 0.5% |
| R234A | 26.9: 53.8 | 1 peak; 55.2 ± 0.8% |
| D97A | 26.9: 53.8 | 1 peaks; 33.8 ± 1.2%, 46.6 ± 8.4% |
| D97K | 26.9: 53.8 | 1 peak; 30.6 ± 7.9% |
| RbmA* | 22.0: 44.0 | 2 peaks; 25.7 ± 7.9%, 59.8 ± 20.8% |
Table 2
Bacterial strains and plasmids used in this study.
https://doi.org/10.7554/eLife.26163.022| Strain or plasmid | Relevant genotype | Source |
|---|---|---|
| E. coli strains | ||
| CC118λpir | Δ(ara-leu) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1 λpir | (Herrero et al., 1990) |
| S17-1λpir | Tpr Smr recA thi pro rK- mK+ RP4::2-Tc::MuKm Tn7λpir | (de Lorenzo et al., 1994) |
| TOP10 | F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strepr) endA1 nupG | Invitrogen |
| BL21(DE3) | F- ompT hsdSB (rB-mB-) gal dcm (DE3) | Invitrogen |
| V. cholerae strains | ||
| FY_VC_1 | Smooth wild-type, V. cholerae O1 El Tor A1552, Rifr | (Yildiz and Schoolnik, 1999) |
| FY_VC_2 | Rugose variant, V. cholerae O1 El Tor A1552, Rifr | (Yildiz and Schoolnik, 1999) |
| FY_VC_6431 | Rugose variant rbmA-Myc, Rifr | (Berk et al., 2012) |
| FY_VC_4327 | Δvps-IΔvps-II, deletion of vpsA-K and vpsL-Q in rugose variant, Rifr | (Fong et al., 2010) |
| FY_VC_105 | ΔrbmA, in-frame chromosomal deletion in rugose variant, Rifr | (Fong et al., 2006) |
| FY_VC_10035 | rbmA-D97A, chromosomal point mutant in rugose variant, Rifr | This study |
| FY_VC_11995 | rbmA-D97A-Myc, chromosomal point mutant in rugose variant, Rifr | This study |
| FY_VC_10039 | rbmA-D97K, chromosomal point mutant in rugose variant, Rifr | This study |
| FY_VC_11998 | rbmA-D97K-Myc, chromosomal point mutant in rugose variant, Rifr | This study |
| FY_VC_8795 | rbmA-R234A-Myc, chromosomal point mutant in rugose variant, Rifr | (Giglio et al., 2013) |
| FY_VC_10283 | ΔhapAΔprtVΔivaP, in-frame chromosomal deletion in rugose variant, Rifr | This study |
| FY_VC_240 | Rugose-gfp, V. cholerae O1 El Tor A1552, rugose variant, Rifr Gmr | (Beyhan and Yildiz, 2007) |
| FY_VC_224 | ΔrbmA-gfp, Rifr Gmr | (Fong et al., 2006) |
| FY_VC_10084 | rbmA-D97A-gfp, Rifr Gmr | This study |
| FY_VC_10086 | rbmA-D97K-gfp, Rifr Gmr | This study |
| FY_VC_8832 | rbmA-R234A-gfp, Rifr Gmr | This study |
| Plasmids | ||
| pGP704sacB28 | pGP704 derivative, mob/oriT sacB, Apr | G. Schoolnik |
| pFY-4183 | pGP-rbmA-D97A, for chromosomal point mutation, Apr | This study |
| pFY-4523 | pGP-rbmA-D97A-Myc, for chromosomal point mutation, Apr | This study |
| pFY-4185 | pGP-rbmA-D97K, for chromosomal point mutation, Apr | This study |
| pFY-4524 | pGP-rbmA-D97K-Myc, for chromosomal point mutation, Apr | This study |
| pFY-3509 | pGP-ΔhapA, Apr | This study |
| pFY-3512 | pGP-ΔprtV, Apr | This study |
| pFY-4277 | pGP-ΔivaP, Apr | This study |
| pMMB67EH | Low copy number IPTG inducible vector, Apr | (Fürste et al., 1986) |
| pFY-4260 | prbmA, pMMB67EH containing wild-type rbmA in-frame with rtx type I secretion signal, no native type II secretion signal, Apr | This study |
| pFY-4261 | pFnIII-1, pMMB67EH containing FnIII-1 in-frame with rtx type I secretion signal, Apr | This study |
| pFY-4262 | pFnIII-2, pMMB67EH containing FnIII-2 in-frame with rtx type I secretion signal, Apr | This study |
| pFY-4263 | prbmA* pMMB67EH containing rbmA* in-frame with rtx type I secretion signal, Apr | This study |
| pGEX-6P-2 | IPTG-inducible vector for expression of recombinant proteins with N-terminal GST tag, cleavable by PreScission protease, Apr | GE |
| pHisGST | IPTG-inducible vector for expression of recombinant proteins with N-terminal His6-GST tags, cleavable by TEV protease, Apr, | (Xu et al., 2015) |
| pFY-1429 | pGEX-rbmA, no native type II secretion signal Apr | This study |
| pFY-4194 | pHisGST-noSP-rbmA-D97A, no native type II secretion signal, Apr | This study |
| pFY-4196 | pHisGST-noSP-rbmA-D97K, no native type II secretion signal, Apr | This study |
| pFY-3491 | pGEX-noSP-rbmA-R234A, no native type II secretion signal, Apr | This study |
| pFY-4219 | pHisGST-noSP-rbmA* no native type II secretion signal, Apr | This study |
| pFY-3551 | pHisGST-FnIII-1, Apr | This study |
| pFY-3066 | pHisGST-FnIII-2, Apr | This study |
| pFY-3435 | pHisGST-FnIII-2-R234A, Apr | This study |
| pUX-BF13 | oriR6K helper plasmid, mob/oriT, provides Tn7 transposition function in trans, Apr | (Bao et al., 1991) |
| pMCM11 | pGP704::mTn7-gfp, Gmr Apr | M. Miller and G. Schoolnik |
| pNUT542 | Ptac_sfgfp expression plasmid, Gmr | This study |
| pNUT1029 | Ptac_mRuby expression plasmid, Gmr | This study |
Additional files
-
Supplementary file 1
List of primers used in this study for generating various strains and plasmids.
- https://doi.org/10.7554/eLife.26163.023
-
Transparent reporting form
- https://doi.org/10.7554/eLife.26163.024
