Understanding the role of TapA in the formation of B. subtilis biofilms

: a biochemical and genetic analysis

  • Christopher Earl

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Once thought of as unicellular organisms, it is now appreciated that bacteria can exist as multicellular collectives with emergent properties that distinguish them from their free living planktonic forms. Biofilm formation is an example of such a multicellular behaviour which is widespread in the prokaryotic world. Biofilms can take diverse forms but this work focusses on the surface-attached biofilm formed by the organism Bacillus subtilis. This study explores the properties of the extracellular matrix which gives structure to the biofilm and protects the encased cells from a variety of environmental threats.

The B. subtilis biofilm matrix is composed of exopolysaccharide (EPS) and the protein components BslA, TasA and TapA. TasA is a fibre forming protein which has been described in the literature as an amyloid-forming fibre, however, recent work contradicts this finding. The current work focusses on TapA (TasA anchoring/assembly protein; formerly YqxM) which is essential for the development of complex, structured biofilms. TapA has previously been described as a cell-wall associated protein needed for the formation of TasA fibres. TapA secretion has since been demonstrated to involve the action of the signal peptidase SipW with the tapA-sipW-tasA genes found together on the B. subtilis genome.

Recent work from Erskine et al has demonstrated that TasA fibres (fTasA) form spontaneously in vitro (Erskine et al., 2018). The findings of this thesis determine that TapA is not needed for the ability of TasA fibres to restore structure when added ex vivo to a ΔtasA sinR mutant biofilm. Biochemical and genetic analysis of B. subtilis biofilms has led to the discovery that TapA is processed to a low molecular weight form in vivo and that this process is dependent on the proteolytic activity of secreted proteases. Subsequently, it was found that a limited part of the N-terminus of the TapA protein is sufficient to restore rugosity to a ΔtapA mutant biofilm. Genetic analysis of the minimal functional unit identified key amino acids needed for the function of TapA, highlighting the importance of a potential β-strand secondary structure in the Nterminus of TapA. Evidence is provided that SipW is not required for the ability of exoproteases to process TapA, suggestive that TapA secretion is not SipWdependent. Experimental evidence indicates that TapA could act as a chaperone facilitating the stability of TasA or as a peptide aiding the formation of TasA fibres in vivo.
Date of Award2018
Original languageEnglish
Awarding Institution
  • University of Dundee
SupervisorNicola Stanley-Wall (Supervisor) & Sarah Coulthurst (Supervisor)

Cite this

Understanding the role of TapA in the formation of B. subtilis biofilms: a biochemical and genetic analysis
Earl, C. (Author). 2018

Student thesis: Doctoral ThesisDoctor of Philosophy