Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials.

Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogues of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundation for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled on the basis of the insertion location within the S-layer protein, RsaA. Moreover, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm2. Finally, we show that ligation of quantum dots to the cell surface does not impair cell viability, and this composite material remains intact over a period of 2 weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step toward building hierarchically ordered engineered living materials with emergent properties.

Visualizing Chemoattraction of Planktonic Cells to a Biofilm.

Bacterial chemotaxis in response to continuous chemical gradients has been extensively studied at the individual cell and population levels using a variety of well-established in vitro methods (Englert et al., Microfluidic techniques for the analysis of bacterial chemotaxis. Methods Mol Biol 571:1-23, 2009). In nature, bacteria are surrounded by heterogeneous chemical gradients; hence, it is essential to understand chemotaxis behavior under such conditions. Here, we describe a setup that allows visualization of the chemotaxis response of motile cells to the complex microenvironment of a biofilm maintained under static conditions. The biofilm is separated from the motile cells by a semi-permeable membrane. Cells swimming toward the biofilm are captured on the membrane and imaged using confocal laser scanning microscopy (CLSM). Chemotaxis toward specific molecules produced by the biofilm, such as autoinducer-2 (AI-2), can be studied using this setup. This system can be adapted to study chemotaxis toward poly-species biofilms, or even mammalian cells.

Chemotaxis to self-generated AI-2 promotes biofilm formation in Escherichia coli.

Responses to the interspecies quorum-sensing signal autoinducer-2 (AI-2) regulate the patterns of gene expression that promote biofilm development. Escherichia coli also senses AI-2 as a chemoattractant, a response that requires the periplasmic AI-2-binding protein LsrB and the chemoreceptor Tsr. Here, we confirm, as previously observed, that under static conditions highly motile E. coli cells self-aggregate and form surface-adherent structures more readily than cells lacking LsrB and Tsr, or than ΔluxS cells unable to produce AI-2. This difference is observed both at 37 and 30 °C. Cells deleted for the genes encoding the lsrACDBFG operon repressor (ΔlsrR), or the AI-2 kinase (ΔlsrK), or an AI-2 uptake channel protein (ΔlsrC), or an AI-2 metabolism enzyme (ΔlsrG) are also defective in biofilm formation. The Δtsr and ΔlsrB cells are totally defective in AI-2 chemotaxis, whereas the other mutants show normal or near-normal chemotaxis to external gradients of AI-2. These data demonstrate that chemotaxis to external AI-2 is necessary but not sufficient to induce the full range of density-dependent behaviours that are required for optimal biofilm formation. We also demonstrate that, compared to other binding-protein-dependent chemotaxis systems in E. coli, low levels (on the order of ~250 molecules of periplasmic LsrB per wild-type cell and as low as ~50 molecules per cell in some mutants) are adequate for a strong chemotaxis response to external gradients of AI-2.

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.

The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.