The propensity of the bacterial rodlin protein RdlB to form amyloid fibrils determines its function in Streptomyces coelicolor

Abstract

Streptomyces bacteria form reproductive aerial hyphae that are covered with a pattern of pairwise aligned fibrils called rodlets. The presence of the rodlet layer requires two homologous rodlin proteins, RdlA and RdlB, and the functional amyloid chaplin proteins, ChpA-H. In contrast to the redundancy shared among the eight chaplins, both RdlA and RdlB are indispensable for the establishment of this rodlet structure. By using a comprehensive biophysical approach combined with in vivo characterization we found that RdlB, but not RdlA, readily assembles into amyloid fibrils. The marked difference in amyloid propensity between these highly similar proteins could be largely attributed to a difference in amino acid sequence at just three sites. Further, an engineered RdlA protein in which these three key amino acids were replaced with the corresponding residues from RdlB could compensate for loss of RdlB and restore formation of the surface-exposed amyloid layer in bacteria. Our data reveal that RdlB is a new functional amyloid and provide a biophysical basis for the functional differences between the two rodlin proteins. This study enhances our understanding of how rodlin proteins contribute to formation of an outer fibrillar layer during spore morphogenesis in streptomycetes.

Figure 1

(A) The ESPript output of the mature rodlin sequences aligned by ClustalW. Conserved residues have a black background: 71 out of 105 of residues in RdlB are identical to RdlA. (B) The hydropathy patterns of RdlA (black) and RdlB (red) appear to be highly similar.

Figure 2. RdlA and RdlB have a different propensity to assemble into amyloid-like fibrils.

(A) Vortexing induces RdlB to adopt β-sheet rich secondary structure (red solid line) from random coil (red dash line), while RdlA (black solid line) retains the random coil conformation observed before vortexing (black dash line). The protein concentration used was 4 μM in 100 mM sodium phosphate buffer (pH 7.0) measured in a 1 mm cuvette. (B,C) Increase in ThT fluorescence intensity by RdlA (B) and RdlB (C) at different concentrations. Readings were taken every 3 min for over 15 hours in a microplate reader while shaking at 700 rpm. The data shown are the average of at least 4 replicates and the error bars represent the standard error of the mean. (D) No well-defined structure was found in negatively-stained electron micrographs of RdlA (40 μM) after samples were incubated for 24 hours. (E) Negatively stained electron micrograph of mature RdlB fibrils (40 μM) incubated for 24 hours. (F) Negatively-stained electron micrograph of RdlB fibrils after 24 hours proteinase K digestion; the thin, needle-like fibrils tend to align into bundles. Scale bars represent 100 nm in images (D–F).

Figure 3. X-ray diffraction pattern of a dried stalk of RdlB fibrils.

(A) 2-D X-ray diffraction profile with significant reflections marked. (B) The reflections in axial and equatorial directions are indicated by the solid and dotted lines, respectively, in the 1-D profile of the X-ray diffraction pattern. The decrease in intensity observed near 5.50 Å (0.18 A⁻¹) in both axial and equatorial directions results from the loss of pixels on the image plate shown by the crossed pattern.

Figure 4. The in vitro characterization of RdlB mutant aggregation.

(A) Aggregation of RdlB and its mutants monitored by ThT fluorescence intensity. All conditions were identical and all protein concentrations 10 μM. (B) In vitro self-polymerization of three synthetic peptides based on the N-terminal sequence of RdlB monitored by ThT fluorescence. The peptide concentration was 40 μM. (A,B) Readings were taken every 3 min in a FLUOstar Omega microplate reader with 700 rpm orbital shaking. (C,D) Negatively stained electron micrograph of aggregates formed by RdlB peptide fragments after incubation for 4 days. Scale bars are 100 nm. (C) Peptide corresponding to residues 11–27 of RdlB (NGNGASQYFGNSMTTGN). Fibrils appear as short, rigid rods. (D) Peptide corresponding to residues 28–42 of RdlB (MSPQMALIQGSFNKP). Fibrils appear significantly shorter and thinner, the morphology of these fibrils appears more fragile as shreds of fibrils were often observed indicating fragmentation.

Figure 5. Mutants of RdlA and RdlB exhibited reversed propensity for amyloid formation.

(A) Sequence alignment of the N-terminal regions of rodlins and the designed mutants. (B) The aggregation of wild-type rodlins and the mutants RdlB- and RdlA* monitored by ThT fluorescence over 30 h. The protein concentration for each protein was 10 μM. Readings were taken every 3 min in a FLUOstar Omega microplate reader with 700 rpm orbital shaking. (C) Negatively stained electron micrograph of RdlA* (80 μM) after incubation for 6 days with gentle shaking. The scale bar is 100 nm in length.

Figure 6. The propensity of rodlins to form amyloid fibrils correlates with the presence of rodlets.

The outer surface of spores of the wild-type strain (A) is characterized by the rodlet layer. In contrast, no rodlets are detected on the surface of spores of the ∆rdlB strain (B). Introduction of the wild-type rdlB gene (contained on plasmid pIJ8630-rdlB) in the ∆rdlB strain restored rodlet formation (C). No complementation is observed when pIJ8630-∆17-42rdlB (D) or pIJ8630-rdlB− (E) are introduced in the ∆rdlB mutant strain. (F) Rodlets are also formed by the introduction of plasmid pIJ8630-rdlA*2 in the ∆rdlAB mutant. (G) Spore chains of the S. coelicolor wild-type strain as observed with scanning electron microscopy without platinum plasma coating. A transparent sheath-like structure is visible surrounding the separating spores. (H) Proposed model for the developmental transition of S. coelicolor aerial hyphae into chains of spores, which are enveloped by two amyloidal layers containing assembled chaplins (blue/purple) and rodlins (orange/green). Note that the rodlins are part of the outermost layer. The scale bar represents 100 nm (A–F) or 1 μm (G).