Abstract
Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.
Highlights
The predominant state in which bacteria and archaea live on Earth is in the form of biofilms (Flemming et al, 2016): aggregates of microorganisms embedded in a self-made extracellular matrix
We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture
Through this analysis we have expanded our knowledge of the protein TapA which is needed for biofilm formation by B. subtilis
Summary
The predominant state in which bacteria and archaea live on Earth is in the form of biofilms (Flemming et al, 2016): aggregates of microorganisms embedded in a self-made extracellular matrix. As the level of TasA (calculated molecular mass 25.7 kDa) in the biofilm is substantially reduced in the absence of functional TapA (Romero et al, 2014) (Figure S1d), the bioactivity of the tapA truncations was supported by immunoblot analysis which showed a recovery of TasA levels back to those seen for NCIB3610 (Figure S1d) The identification of this region of tapA as sufficient for TapA activity is consistent with, but significantly extends, the previous identification of amino acids 50-57 as being needed for TapA function in the context of the full-length protein (Romero et al, 2014). The impact of removing the extracellular proteases on the size of TapA in vivo was assessed by immunoblot analysis of proteins extracted from the WT and KO8 strain after growth for 12, 18, 24 or 48 hr under colony biofilm formation conditions. We noted that the ~16 kDa TapA band was not detected in the protein samples extracted from colony biofilms once the coding region for vpr was deleted (Figure 4d). This hypothesis is strengthened if you take into account that this is the conserved and stable core of the protein
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