Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly

Abstract

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.

FIG 1

TapA accelerates the polymerization of TasA. Polymerization of purified matrix protein assayed in vitro by ThT fluorescence (filled dots; No addition) was the TasA-TapA (100:1) mix purified from wild-type cells. The other curves are for mixtures of the same protein preparation to which TapA was added (+TapA) at the following molar ratios: 1:100 (triangles), 1:20 (circles), or 1:3 (diamonds). TapA alone, TapA with no TasA used as a negative control (squares). The x axis is time after ThT addition, and the y axis is fluorescence, in arbitrary units (AU).

FIG 2

Sequence analysis of TapA. (A) Schematic of the TapA protein sequence. Red, signal sequence; green, five TapA cysteine residues (C1 to C5); yellow, the two imperfect repeats identified by TRUST analysis (33); black lines above the diagram, sequences deleted or replaced in the TapA^Δ194-230, TapA^Δ50-57, or TapA^replace mutant. (B) Alignment of TapA sequences from closely related Bacillus species generated using the Clustal W2 program. Identical residues are marked with asterisks, conserved substitutions are marked with colons, and semiconserved substitutions are marked with periods. The color coding is the same as that described in the legend to panel A.

FIG 3

Cysteines contribute to the robustness of biofilms. Top-down views show pellicles of the wild type, a tapA-null mutant, and mutant strains DR10 (tapA^C1,C3,C4/A), DR11 (tapA^C2-C5/A), and DR12 (tapA^C1-C5/A) (upper row) or the corresponding strains that also harbored a deletion of the epsA-O genes (bottom row). Pellicles were grown in MSgg medium for 24 h prior to imaging.

FIG 4

Deletion of TapA residues 50 to 57 inhibits biofilm formation. (A) (Top row) Pellicle formation of tapA strains harboring no tapA (tapA), alleles of wild-type tapA from B. subtilis (tapA^WT), B. amyloliquefaciens (tapA^amylo), or B. subtilis mutants (tapA^Δ194-230, tapA^Δ50-57), or a replacement of amino acids 50 to 57 (tapA^replace). (Bottom row) Colony morphology of the same strains from the top row. Pellicles were grown in MSgg broth for 24 h prior to imaging, and colonies were grown in MSgg agar for 72 h prior to imaging. (B) Cells were grown in MSgg medium for 24 h prior to harvesting and separation into medium (Med), cell (C), and extracellular matrix (Mat) fractions. Samples were concentrated and immunoblotted with either anti-TapA (top) or anti-TasA (bottom) antibodies. Numbers on the left are molecular masses (in kilodaltons). (C) Immunocytochemistry with anti-TapA antibody and FITC-conjugated secondary antibody performed on intact cells of wild-type (WT) tapA or the tapA^Δ50-57 mutant harvested from pellicles grown in MSgg broth for 24 h. The fluorescence image (green) is overlaid over transmitted light (gray). Bars, 2 μm.

FIG 5

Effect of different TapA proteins on polymerization of TasA. In vitro polymerization of TasA-TapA (100:1) (assayed using ThT fluorescence) was performed with no addition or the addition of different TapA proteins to TasA at a 1:3 molar ratio, as indicated. Samples were prepared as described in the legend to Fig. 1.

FIG 6

TapA^Δ50-57 inhibits the functionality of native TapA. A top-down view shows the pellicle formation of the WT strain or merodiploid strains containing the wild-type tapA allele (WT), the wild-type tapA allele and another copy of wild-type tapA (WT + tapA^WT), or the wild-type allele and the tapA^Δ50-57 allele (WT + tapA^Δ50-57). Pellicles were grown in MSgg medium for the times indicated on the left prior to imaging.