Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice

Abstract

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a "dissect and build" structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca²⁺ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several "repeats-in-toxin" motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Fig. 1. Overall structure of MpIBP.

(A) Linear domain map of MpIBP drawn to scale. The MpIBP amino acid (aa) sequence is shown in fig. S1. RII and RIV (colored light blue and orange, respectively) are known from two structures solved previously (10, 11, 22). RI, RIII, and RV in white are new three-dimensional structures determined in this study. (B) Expanded view of the RI and RIII to RV linear domain maps colored as in (C). Sequence identities (%) to a 104–amino acid RII repeat are shown for the RIC and RIII_1 domains. (C) NMR and x-ray crystal structures of linked MpIBP domains from N to C termini are shown in cartoon representation: RIN (blue), RIC (red), RII repeats (cyan), RIII_1–4 (dark blue), RIII_5 (dark green), RIV (orange), and RV (magenta). Small green spheres indicate calcium ions. OM is indicated by horizontal lines on either side of RIM. The solution structure of RIM determined by SAXS is illustrated as a gray cylinder. Hatched lines indicate the ~108 RII repeats that are not shown in the figure. The linker regions between RIII_5/RIV (94 residues) and RIV/RV (112 residues) are indicated by wavy lines.

Fig. 2. Detailed structural features of the OM anchoring RI.

(A) The NMR structure of RIN (bottom) and the 2 Å crystal structure of RIC (top) are colored red and fitted into the gray solution structure of the whole RI construct determined by SAXS. The RI solution envelope is fitted through the purple TolC pore homology model embedded in the OM. (B) Close-up view of the cylindrical RIM (gray) determined by SAXS without showing the TolC pore. Dimensions of RIM are indicated. (C) Top-down view of the TolC OM pore model. The internal diameter is indicated. (D) The 20-member NMR structural ensemble of RIN is colored red and shown in ribbon representation. The N and C termini and the height of the protein are marked. (E and F) SAXS data were collected from MpIBP_RI at a concentration of 7 mg/ml. (E) Experimental scattering data of MpIBP_RI (magenta symbols) and fit result of ab initio modeling (DAMMIF, black line). (F) Radial distribution function obtained after Indirect Fourier Transform (IFT) analysis of the scattering data, with data points starting from the first Guinier regime at low q up to the Porod regime at high q values (0.013 Å⁻¹ ≤ q ≤ 0.12 Å⁻¹). a.u., arbitrary units.

Fig. 3. CD spectra of RINM, RIII_5, and RV measured in EDTA and different concentrations of CaCl₂ or MgCl₂.

(A) The far-UV CD spectra of RINM were plotted as molar ellipticity versus wavelength. The spectra in the presence of 1 mM EDTA (green line), 1 mM CaCl₂ (red line), and both 1 mM CaCl₂ and MgCl₂ (broken black line) are coincident. (B) The far-UV CD spectra of RIII_5. Spectra in the presence of 0.5 mM EDTA, 1 mM CaCl₂, and both 1 mM CaCl₂ and MgCl₂ are indicated by black, blue, and red lines, respectively. (C) The far-UV CD spectra of RV. Spectra in the presence of 1 mM EDTA or 2, 3, and 5 mM CaCl₂ are indicated by black, green, magenta, and broken black lines, respectively.

Fig. 4. Detailed structural features of the MpIBP_RIII ligand-binding domains.

(A) RIII_1–4 is colored in rainbow representation, whereas the RIII_5 construct is colored yellow. Calcium ions in the ligand-binding sites are shown as magenta spheres, whereas the other Ca²⁺ are shown as green spheres. (B) Enlarged view of the sugar-binding site of the RIII_5 structure, showing the 1 Å 2F_o − F_c map and the carbon atoms of the glucose molecule colored magenta. Oxygen atoms are red, and nitrogen atoms are blue. (C) Enlarged view of the ligand-binding cavity of RIII_3 is shown with the 2.1 Å resolution 2F_o − F_c map contoured at 1 σ [as in (B)]. Ca²⁺ coordination by the C-terminal Pro and Asp residues from a symmetry-related molecule are shown in stick representations. (D) Enlarged view of the Ca²⁺-stiffened linker region between RIII_2 and RIII_4. Ca²⁺ coordinating residues are shown in stick representation.

Fig. 5. M. primoryensis selectively binds the diatom C. neogracile.

SEM images of (A) single or (B) multiple C. neogracile (indicated by white arrows) bound by M. primoryensis (indicated by yellow arrows). Representative bright-field microscopy images of C. neogracile (marked by white arrows) in the presence of (C) M. primoryensis (yellow arrows) or (D) ice. Bright-field microscopy images of M. primoryensis + C. neogracile microcolonies with (F, G, and H) or without (E) ice. Light (I, L, and O) and fluorescence microscopy images (J, M, and P) of a mixture of diatoms, C. neogracile (white arrows), and F. cylindrus (green arrows), incubated with TRITC-labeled RIII_1–5. Slight shifts in the merged images (K, N, and Q) between the fluorescence and bright-field images are due to drifting of the cells between image capture.

Fig. 6. RIII_5 sugar-binding and RIII_3 peptide-binding domains are responsible for binding to C. neogracile.

Light (A, D, G, J, M, and P), fluorescence (B, E, H, K, N, and Q), and merged (C, F, I, L, O, and R) images of diatom C. neogracile (white arrows) incubated with green fluorescent protein–tagged MpIBP domains: RII (A to F), RIII_5 (G to L), and RIII_3 (M to R). All images were captured with the same length of exposure.

Fig. 7. Structural comparison between the RIV and RV of MpIBP.

(A) Cross section of the RTX repeats of RIV (gray). (B) Cross section of the RTX repeats of RV (cyan). (C) The 1.45 Å structure of RV. The N-terminal moiety is colored green, whereas the C-terminal moiety is colored magenta.

Fig. 8. Model of M. primoryensis collectively binding with diatoms to ice.

(A) Ice/snow that covers the surface of Ace Lake to a depth of 1 to 2 m is represented by a gray rectangle with three internal brine channels of irregular shape. Lake water is colored blue with a light to dark gradient from top to bottom signifying the increased availability of light and oxygen toward the top of the water column as indicated by the gray arrow. Bacteria and photosynthetic microorganisms such as diatoms within the brine pits and underneath the ice are drawn as small white ovals and large green ovals, respectively. The phototrophic and anoxic zones are indicated on the right. (B) Expanded view of (A) showing two linked bacterial cells bound to ice and a diatom. Cell-surface proteins and carbohydrates are drawn as fuzzy black hairs, and the polar flagella are drawn as squiggles. MpIBPs protrude from cell surfaces. RII, RIII_1–4, RIII_5, RIV, and RV are drawn as cyan rods, blue ovals, dark green hexagons, orange rectangles, and magenta triangles, respectively. (C) Expanded view of the cell surface–anchoring domains of MpIBP near the OM. OM is drawn the same way as in Fig. 2A. Surface glycans are drawn as connected brown hexagons. RIN, RIM, and RIC are drawn as a blue triangle, a gray cylinder, and red ovals, respectively. The hollow TolC OM pore is outlined in black. The arrow with a broken line indicates the protein continues to RII to RV. (D) Enlarged view of MpIBP_RIII interacting with the peptide and sugar molecules on the cell surface of a diatom. Ligand-binding Ca²⁺ are drawn as magenta spheres. Surface protein is indicated by a wavy line from the cell surface.