Sum of the Parts: Bacterial Biofilms by Solid-State NMR

Abstract

Bacteria harbor dedicated machinery to assemble amyloid fibers at their cell surface. Curli, produced by E. coli, are among the most well-studied bacterial amyloids and play a significant role in adhesion and in the assembly of bacterial biofilms. Curli interact with secreted polysaccharides to form a biofilm and play a crucial role in determining biofilm morphology, yet the exact composition of curli-integrated biofilms is poorly understood. The determination of biofilm composition by routine methods, such as amino acid hydrolysis and mass spectrometry, is complicated by incomplete dissolution, as well as the perturbative nature of degradative and enzymatic changes. Solid-state NMR can provide valuable information about intact biofilms and is uniquely suited to provide a quantitative perspective on biofilm composition and structure. To dissect the contributions and interactions of curli and polysaccharides in the biofilm framework, we have purified and obtained 13C CPMAS spectra of: (i) the wild-type UTI89 extracellular matrix (ECM); (ii) the curli-free ECM produced by the curli mutant strain UTI89ΔcsgA; and (iii) purified curli. These spectra indicate that the UTI89 ECM has two major components, curli and cellulose, each in a quantifiable amount. The curli and polysaccharide regions of the spectrum also harbor unique chemical-shift contributions that allow us to examine curli-polysaccharide interactions and the modes of action of biofilm inhibitors. Our data define quantitatively the composition of the intact extracellular matrix, including cellulose and amyloid fibers, which impacts bacterial physiology. We are also working to transform cartoon representations of the curli amyloid fiber into a molecular model using selective labeling and REDOR NMR.