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

Abstract

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.

Keywords: Caulobacter; RsaA; biomaterial; engineered living materials; quantum dots.

Figure 1.

RsaA forms a 2D hexameric lattice on the surface of C. crescentus. (a) Structure of the RsaA lattice. (b) High resolution AFM images of the wild-type RsaA lattice (strain MFm111), (c) RsaA485:SpyTag (strain MFm 118), (d) RsaA690:SpyTag (strain MFm 120) on the surface of C. crescentus cells. In all three cases, a well-ordered, hexagonal protein lattice is observed. The unit cell length (center-to-center distance between adjacent hexagons) is 22 ± 1 nm, which is the same as reported in literature. Scale bar is 40 nm. See Methods for experimental details of AFM.

Figure 2.

Design and expression of RsaA-SpyTag in C. crescentus. (a) Ribbon diagram of the RsaA monomer structure indicating SpyTag insertion sites (orange). Inset shows a space-filling model of the RsaA hexamer. (b) Design of engineered C. crescentus strains expressing RsaA-SpyTag. SpyTag flanked by upstream and downstream (GGSG)₄ spacers was directly inserted into the genomic copy of rsaA. (c) Immunoblot with anti-RsaA antibodies of C. crescentus strains whole cell lysate. The band corresponding to RsaA increases in molecular weight from wild-type RsaA (lane 2) to RsaA-SpyTag at each insertion site (lanes 3–10).

Figure 3.

SpyCatcher protein fusions ligate specifically to the surface of C. crescentus expressing RsaA-SpyTag. (a−d) Confocal fluorescence images of C. crescentus cells visualized in DAPI and RFP channels. Cells expressing wild-type RsaA incubated with (a) mRFP1 or (b) SpyCatcher-mRFP1. Cells expressing RsaA690-SpyTag with (c) mRFP1 or (d) SpyCatcher-mRFP1. Only when the SpyCatcher-mRFP1 probe is introduced to cells displaying SpyTag (d) is RFP fluorescence tightly associated with the cell membrane observed, including the stalk region. Scale bar = 3 μm.

Figure 4.

SpyCatcher protein fusions covalently bind to RsaA-SpyTag with variable occupancy according to the SpyTag location. (a) Flow cytometry histograms of RFP fluorescence per cell for strains expressing wild-type RsaA (black) and RsaA-SpyTag (colored lines) incubated with SpyCatcher-mRFP1 for 1 h. Baselines are offset for clarity. All eight strains displaying RsaA-SpyTag show an increase in the intensity of RFP fluorescence over the negative control with their intensity varying based on where SpyTag is inserted within RsaA. (b) SDS-PAGE of whole cell lysates from the rsaA467:SpyTag strain incubated for 24 h without (lane 2) and with (lane 3) SpyCatcher-mRFP1 protein. Appearance of a higher molecular weight band only in the reaction containing SpyCatcher-mRFP1 indicates covalent binding to RsaA-SpyTag.

Figure 5.

Engineered RsaA assembles biopolymers on the C. crescentus cell surface. (a–c) Confocal fluorescence images of C. crescentus cells incubated with ELP-mCherry fusion proteins visualized in DAPI and mCherry channels. Cells expressing (a) wild-type RsaA incubated with SpyCatcher-ELP-mCherry and (b) expressing RsaA690:SpyTag incubated with ELP-mCherry. Only the rsaA690:SpyTag strain incubated with SpyCatcher-ELP-mCherry (c) shows signal along the cell membrane in the mCherry channel, indicating specific assembly on the cell surface. Scale bar = 5 μm.

Figure 6.

Engineered RsaA assembles inorganic nanocrystals on the C. crescentus cell surface. (a–c) Interference reflection microscopy (IRM) and confocal fluorescence images of C. crescentus cells incubated with QDs. Cells expressing (a) wild-type RsaA incubated with SpyCatcher-QDs and (b) expressing RsaA690:SpyTag incubated with PEG-QDs. (c) Cells expressing RsaA690:SpyTag incubated with SpyCatcher-QDs show QD fluorescence along the cell surface, indicating specific assembly of SpyCatcher-QDs by the engineered strain. Scale bar = 5 μm.

Figure 7.

Engineered C. crescentus with ligated SpyCatcher-QDs remain viable over 2 weeks. (a) Viability of CB15NΔsapA (wild-type) and CB15NΔsapA rsaA467:SpyTag strains incubated without or with SpyCatcher-QDs (+ QD) was assessed by quantifying colony forming units/mL (CFU/mL) as described in the Methods section. Data shown represent mean ± standard deviation of three replicates per condition. The CFU/mL of cells with SpyCatcher-QDs is very similar to that of cells grown without SpyCatcher-QDs. (b) Confocal images of rsaA467:Spytag + SpyCatcherQD show QD fluorescence over the two week duration indicating sustained attachment of SpyCatcher- QDs to the engineered strain. Scale bar = 3 μm.