Coiled coils are a robust motif for exploring amino acid interactions, generating unique supramolecular structures, and expanding the functional properties of biological materials. A recently discovered antiparallel coiled-coil hexamer (ACC-Hex, peptide 1) exhibits a unique interaction in which Phe and Ile residues from adjacent α-helices interact to form a Phe-Ile zipper within the hydrophobic core. Analysis of the X-ray crystallographic structure of ACC-Hex suggests that the stability of the six-helix bundle relies on specific interactions between the Phe and Ile residues. The Phe-Ile zipper is unprecedented and represents a powerful tool for utilizing the Phe-Ile interactions to direct supramolecular assembly. To further probe and understand the limits of the Phe-Ile zipper, we designed peptide sequences with natural and unnatural amino acids placed at the Phe and Ile residue positions. Using size exclusion chromatography and small-angle X-ray scattering, we found that the proper assembly of ACC-Hex from monomers is sensitive to subtle changes in side chain steric bulk and hydrophobicity introduced by mutations at the Phe and Ile residue positions. Of the sequence variants that formed ACC-Hex, mutations in the hydrophobic core significantly affected the stability of the hexamer, from a ΔGuw of 2–8 kcal mol–1. Additional sequences were designed to further probe and enhance the stability of the ACC-Hex system by maximizing salt bridging between the solvent-exposed residues. Finally, we expanded on the generality of the Phe-Ile zipper, creating a unique sequence that forms an antiparallel hexamer that is topologically similar to ACC-Hex but atomistically unique.

Bacteria form interface-associated communities called biofilms, often comprising multiple species. Biofilms can be detrimental or beneficial in medical, industrial, and technological settings, and their stability and function are determined by interspecies communication via specific chemical signaling or metabolite exchange. The deterministic control of biofilm development, behavior, and properties remains an unmet challenge, limiting our ability to inhibit the formation of detrimental biofilms in biomedical settings and promote the growth of beneficial biofilms in biotechnology applications. Here, we describe the development of growth surfaces that promote the growth of commensal Escherichia coli instead of the opportunistic pathogen Pseudomonas aeruginosa. Periodically patterned growth surfaces induced robust morphological changes in surface-associated E. coli biofilms and influenced the antibiotic susceptibilities of E. coli and P. aeruginosa biofilms. Changes in the biofilm architecture resulted in the accumulation of a metabolite, indole, which controls the competition dynamics between the two species. Our results show that the surface on which a biofilm grows has important implications for species colonization, growth, and persistence when exposed to antibiotics.Keywords: antibiotic susceptibility; bacterial signaling; biofilms; microfabricated surfaces; probiotic surfaces;

The bacterium Geobacter sulfurreducens is a model biological catalyst in microbial electrochemical devices. G. sulfurreducens forms electrically conductive, electrode-associated biofilms, but the biological structures mediating electrical conduction from cells to the electrodes are a matter of debate. Bacteria in these communities produce a network of fiber-like Type IV pili, which have been proposed to act either as inherent, protein-based electronic conductors, or as electronically inert scaffolds for cytochromes mediating long-range charge transport. Previous studies have examined pilus conduction mechanisms under vacuum and in dry conditions, but their conduction mechanism under physiologically relevant conditions has yet to be characterized. In this work, we isolate G. sulfurreducens pili, and compare the electronic conduction mechanism of both live biofilms and purified pili networks under dry and aqueous conditions. Solid-state I–V characteristics indicate that electronic transport in films of purified pili is representative of conduction in a fiber percolation network. Electrochemical gating measurements in a bipotentiostat device configuration confirm previous results suggesting redox currents dominate live biofilm conduction. Purified pili films, however, exhibit non-redox electronic conduction under aqueous, buffered conditions, and their conductivity increases with decreasing temperature. These findings show that isolated pili possess inherent, non-redox-mediated conductivity consistent with a metallic-like model of charge carrier transport. The results demonstrate an experimental platform for studying electronic transport in biomaterials and suggest that pili serve as an exemplary model for designing bioelectronic interfaces.

Bacterial biofilms are problematic in natural and anthropogenic environments, and they confer protective properties on their constituent cells, making them difficult to treat with conventional antibiotics. Antibiofilm strategies, therefore, represent a promising direction of research for treating biofilm infections. Natural autodispersal and interspecies dispersal signaling pathways provide insight into cell–cell communication mechanisms, species dynamics in mixed communities, and potential targets for infection therapies. Here, we describe a novel interspecies dispersal signaling pathway between Pseudomonas aeruginosa and Escherichia coli. E. coli biofilms disperse in response to compounds in P. aeruginosa culture supernatant. Two components of the P. aeruginosa Las and Rhl quorum sensing systems, N-(3-oxo-dodecanoyl) homoserine lactone (3oxoC12HSL) and rhamnolipids, are found to act cooperatively to disperse E. coli biofilms. Our results indicate that rhamnolipids do not affect growth, biofilm development, or dispersal in E. coli but instead complement 3oxoC12HSL signaling by inducing selective permeability of the E. coli membrane. The increased target cell permeability is consistent with rhamnolipid-mediated removal of lipopolysaccharide from E. coli membranes and appears to selectively increase the permeability of lipophilic acyl homoserine lactones. This work suggests that rhamnolipids play a critical role in P. aeruginosa–E. coli interspecies signaling. Rhamnolipids and other biosurfactants may have similar effects in other intra- and interspecies chemical signaling pathways.

The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.