Abstract
Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.
Highlights
Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface
Using only stalked and predivisional cells oriented in the same direction, we found highly localized S-layer assembly in growing cells characterized largely by new protein enrichment at both cell poles and the division plane (Fig. 1f, i, j; Supplementary Fig. 3), in agreement with observations by electron microscopy[25]
We sought to determine the factors that contribute to S-layer assembly by examining how RsaA secretion, cell wall growth, and the presence of an existing S-layer structure affect the location of S-layer assembly in living cells
Summary
Imaging localized S-layer assembly in living cells. Covalently modifying CysRsaA with membrane-impermeable fluorophores via maleimide chemistry is a robust, highly specific labeling scheme for RsaA and enables live-cell STimulated Emission Depletion (STED) fluorescence microscopy showing a complete S-layer (Fig. 1c; Supplementary Fig. 2). To determine how these patches grew, we performed pulse-chase imaging of de novo S-layer assembly (Fig. 3d) This experiment revealed that initial S-layer patches expand from the perimeter of each patch (Fig. 3e), consistent with nucleation and growth characteristic of in vitro SLP crystallization observed by time-resolved atomic force microscopy[10,31]. To further evaluate this mode of assembly, very low concentrations (0.5 to 10 nM) of purified, STAR RED labeled CysRsaA were added to cultures of ΔRsaA cells (OD600nm = 0.2) and puncta were observed (Fig. 3f, g).
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