Curli amyloid fibers inEscherichia colibiofilms: the influence of water availability on their structure and functional properties

Abstract

Microbial biofilms are multicellular communities where bacteria produce an extracellular matrix mainly consisting of proteins and polysaccharides. These biofilms not only confer resistance against external stresses (e.g. antibiotics), but their physical and chemical properties can also adapt to environmental conditions (e.g. temperature, humidity, etc.). Gaining insight on how such cues affect the molecular structure of biofilms would greatly benefit to the emerging field of engineered living materials. In this work, we studied the physical-chemical properties of curli amyloid fibers extracted from biofilms grown on substrates with different water contents. Electron microscopy, FT-IR and fluorescence spectroscopy were used to characterize the conformation and physical-chemical properties of these fibers as a function of the biofilm growth conditions. The results show that varying the water content of the substrate leads to differences in the yield of curli fibers purified from the biofilms and to differences in the packing, hydrophobic character and chemical stability of the fibers. Fibers from biofilms grown on lower water content substrates presented a higher hydrophobicity and chemical stability than those from biofilms grown on higher water content substrates. Such fundamental knowledge on the biophysical features of amyloid fibrils sheds light on the influence of water on biofilm behavior and how the molecular structure of biofilm matrix defines biofilm macroscopic properties. Understanding the molecular behavior of the biofilm matrix formed in various growth conditions may inspire future strategies to engineer biofilm-based materials.

Nutrient availability influencesE. colibiofilm properties and the structure of purified curli amyloid fibers

“…The differences observed here suggest differences in the packing or in the β-sheet content of the fibers. 13…”

“…We used a band fitting method to decompose the spectra into the secondary structure components. 13,19 Purified curli fibers obtained from biofilm grown at low nutrient availability (0.75 and 1.5% w/v) had the highest content of β-sheet and the lowest content of β-turn structures ( Figure 3d and Table S.3 ). In contrast, purified curli fibers obtained from biofilm grown at higher nutrient availability (3.0 – 12.0 % w/v) had the lowest content of β-sheet and the highest content of β-turn structures ( Figure 3d and Table S3 ).…”

“…Curli fibers are made of CsgA monomers, each of them containing a single tryptophan (Trp). Trp is an amino acid behaving as a solvatochromic fluorophore ( Figure S9b ), 13 which position of maximum fluorescence emission gives information on the hydrophobicity of its environment ( Figure 4b, inset ). 22 Curli fibers grown at a low nutrient concentration (0.75 % and 1.5 % w/v) presented a Trp population in a less hydrophobic environment than the other fibers.…”

“…Fiber denaturation into CsgA monomers was monitored by the fluorescence intensity emitted by ThioT. 13 Curli fibers obtained from biofilms grown on salt-free LB agar containing 1.5 % w/v nutrients had the highest stability, while fibers grown at lower nutrient availability (0.75 % w/v) or higher than 3.0 % w/v presented low chemical stability ( Figure 4c ). Interestingly, fibers with a high packing degree and a high β-sheet content, presented high chemical stability ( Figure 4e-f ).…”

“…Considering the mode of action of urea, this result is consistent with the idea that the access of urea to the peptide is easier in loosely packed fibers, thus facilitating amyloid denaturation. 13,38…”