Evolutionary variations in the biofilm-associated protein BslA from the genusBacillus

Abstract

BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly. IntroductionMany bacterial species live in multi-cellular communities known as biofilms. 1, 2 The Gram-positive bac-1 terium Bacillus subtilis forms biofilms with a characteristic wrinkled morphology and a surface that is 2 highly hydrophobic. A critical component in generating the hydrophobicity of the biofilm is the am-3 phiphilic protein BslA. 3 The structure of BslA contains a hydrophobic, surface-exposed 'cap' that sits 4 atop a hydrophilic immunoglobulin-like domain, and is reminiscent of the fungal hydrophobins. [3][4][5][6] We 5 recently characterised the mechanism by which BslA remains both stable and monomeric in aqueous 6 environments and how its adsorbs to an interface. 7 We showed that the hydrophobic cap of BslA, in 7 contrast to the hydrophobins, is structurally plastic; it undergoes an environmentally-responsive confor-8 mational change upon partitioning from an aqueous to a hydrophobic environment. Once at an interface, 9BslA self-assembles into an ordered rectangular 2D lattice that macroscopically forms an elastic protein 10 film. 3,7,8 Like the hydrophobins, BslA has been recognized as a 'natural' surface active agent that could 11 find a range of applications in the food and personal care industries e.g. as surface modifier, coating 12 agent, emulsifier, and/or foam stabiliser. 9-13 13 Although the gross structural changes that lead to the surface-stabilising function of BslA are under-14 stood, we wished to tease out the mechanism by which BslA interacts with a hydrophilic-hydrophobic 15 interface, i.e. what triggers the structural change between the conformational forms? Moreover, what 16 interactions are critical for the formation of the regular 2D lattice? To address these questions, we have 17 studied several BslA orthologues that are present in the genome of Bacillus species closely related to B. 18subtilis. In addition, we have investigated a paralogue of BslA called YweA. Using genetic modification 19 and biophysical techniques, we characterize the behaviour of these variants both in vivo and in vitro. We 20 find that key serine residues in the cap regions of the variants act as 'switches' that drive conformational 21 re-arrangement at an interface. We propose a classification system for BslA and its variants based upon 22 the behaviour of the protein film...

Bifunctionality of a biofilm matrix protein controlled by redox state

“…As previously observed, induction of wild-type yweA expression did not alter the biofilm morphology or wetting phenotype, compared with the parental bslA mutant (Fig. 4B) (14). In sharp contrast, appending the BslA C-terminal 11 amino acids to YweA allowed the chimeric YweA BslA171-181 protein to return hydrophobicity to the bslA mutant strain (114.8 ± 1.1°) (Fig.…”

“…S5 C-E). Together, these findings indicate that we can generate YweA oligomers in vitro by the addition of the BslA C-terminal 11 amino acids, but although they form an ordered 2D lattice, the chimeric proteins retain the fast film-relaxing properties of YweA (14).…”

“…The strict requirement for dimeric BslA to mediate biofilm hydrophobicity led us to hypothesize that if the monomeric paralogue of BslA, YweA, could be engineered to dimerize in vivo, it may become capable of reinstating the nonwetting phenotype to the bslA mutant. This outcome is not the case when the wild-type yweA coding region is expressed from a heterologous location in the bslA mutant (14). To test this prediction, we synthesized a chimeric YweA allele (Fig.…”

“…subtilis encodes a monomeric BslA paralogue called YweA (14). Deletion of yweA does not impact the overall morphology or hydrophobicity of the biofilm (8); however, deletion in combination with removal of bslA exacerbates the biofilm defect of the single bslA deletion (8,14).…”

“…subtilis encodes a monomeric BslA paralogue called YweA (14). Deletion of yweA does not impact the overall morphology or hydrophobicity of the biofilm (8); however, deletion in combination with removal of bslA exacerbates the biofilm defect of the single bslA deletion (8,14). Contrary to the marginal contribution of YweA to biofilm formation, but consistent with the high level of amino acid sequence similarity, in vitro recombinant YweA undergoes the partial structural rearrangement at an interface to reveal the hydrophobic cap and forms an elastic protein film, albeit with limited stability (14).…”