Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

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.

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.

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.

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.