Investigation of the biofilm contributing eps gene operon of Bacillus subtilis

Abstract

Many bacteria secrete an extracellular matrix to form sessile multicellular communities known as biofilms, which provide a multitude of survival benefits. The bacterial species Bacillus subtilis produces an extracellular matrix composed of proteins, extracellular DNA, and polysaccharides. The 15 gene eps operon in B. subtilis encodes proteins which synthesise an exopolysaccharide which is key for biofilm structure and adhesion to surfaces. Through morphology imaging and footprint quantification of eps single gene deletion strains we show that genes contribute at varying degrees to biofilm formation and therefore exopolysaccharide synthesis. Six of the fifteen genes of the eps operon encode for glycosyltransferases (GT), which are enzymes that catalyse the formation of glycosidic linkages between sugar residues. In the formation of the exopolysaccharide these GTs catalyse the extension of the protoexopolysaccharide. The colony biofilm imaging carried out shows that most of these GTs are vital for biofilm formation. Investigation into potential active site residues showed an amino acid DxD motif found in the GT-A folding enzymes which when disrupted by mutation causes a loss in biofilm morphology. To show the DxD mutation causes a loss in active site function and not a protein instability antibody blotting was deployed, however, as the antibodies were new and uncharacterised, they had to be tested which showed large amounts of nonspecific binding. Further bioinformatic investigation led us to the E. coli protein WbnH which had sequence similarities to that of the Eps GT-B folding enzymes. The coding region for wbnH was introduced into the eps GT-B deletion strains and then subsequently shown via biofilm morphology and footprint assays that it could not recover wild-type biofilm morphology. Overall this work has enhanced our working understanding of the eps gene cluster.

Date of Award	2022
Original language	English
Awarding Institution	University of Dundee
Supervisor	Nicola Stanley-Wall (Supervisor) & Megan Bergkessel (Supervisor)