Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Abstract

19Bacteria assemble the cell envelope using localized enzymes to account for growth and 20 division of a topologically complicated surface 1-3 . However, a regulatory pathway has not been 21 identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein 22 coat surrounding the curved 3D surface of a variety of bacteria 4,5 . By specifically labeling, 23 performed time-resolved, super-resolution fluorescence imaging and single-molecule tracking 47 (SMT) of S-layer assembly on living C. crescentus cells. In C. crescentus, the S-layer is made of 48 a single 98 kDa SLP, RsaA, which accounts for around 30% of the cell's total protein synthesis 7 . 49RsaA, like other SLPs, self-assembles into crystalline sheets upon the addition of divalent 50 calcium (Ca 2+ ) in vitro [8][9][10][11][12] . Given the many fitness-related functions ascribed to crystalline 51 bacterial S-layers, we hypothesized that SLP self-assembly may play a role in generating the S-52 layer coat in vivo 4,6 . 53RsaA covers the cellular surface of C. crescentus by forming a 22 nm-repeat hexameric 54 crystal lattice and is non-covalently anchored to an ~18 nm thick LPS outer membrane 13-17 55 (Figure 1a,b). The surface topology of stalked and predivisional C. crescentus includes a 56 cylindrical stalk measuring roughly 100 nm in diameter while the crescentoid cell body 57 approaches 800 nm in width 18,19 (Figure 1d). This large variety of curved topologies guarantees 58 that crystal distortion and defects within the S-layer lattice structure are present, defects which 59 enable complete coverage of the bacterial surface 4,13,20,21 . We can use Gaussian curvature, the 60 product of the maximum and minimum curvatures at a given point, to quantify the cellular 61 topology 22 . Crystalline defects cluster at regions with high absolute values of Gaussian curvature 62 such as the cell poles and division plane, while grain boundaries occur where Gaussian curvature 63 approaches zero such as the cell body (Figure 1d) 20,21 . 64Specifically labeling the S-layer in vivo has proven difficult due to the SLP's life cycle 65 and functions, which include secretion, refolding, anchoring, and crystallization 11,16,17,23,24 . 66

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

“…RsaA self-assembles quickly and efficiently in vivo at extremely low concentrations, (9) suggesting an evolutionary basis for non-canonical crystallization kinetics if the same or similar assembly pathway occurs on the cellular surface. SLPs continuously crystallize on the curved and growing LPS outer membrane and the topology of the surface has been shown to affect S-layer crystal growth in vivo (9). Flexibility between domains might allow the crystallization domain to position itself for lattice formation while relieving torque originating from anchoring a 2D lattice on a variably curved surface.…”

“…RsaA, the 98 kDa SLP from Caulobacter crescentus , undergoes environmentally triggered self-assembly, forming sheets of crystalline protein with hexameric 22 nm repeats when exposed to calcium (7, 8). Recent work from our lab demonstrated that fast, efficient protein crystallization drives S-layer assembly in vivo (9). Therefore, we examined RsaA self-assembly in vitro using time-resolved small angle x-ray scattering (SAXS), circular dichroism spectroscopy (CD), and time-resolved Cryo-EM to determine the structural basis for S-layer assembly.…”

“…Using super-resolution microscopy and single-molecule tracking, we have recently shown that during native S-layer assembly, RsaA monomers diffuse while non-covalently attached to the cell surface until incorporated into a growing crystal lattice (9). When no previous S-layer lattice exists, RsaA nucleates 2D crystals at remarkably low concentrations to form stable crystalline rafts with as few as ~50 molecules (9).…”

“…Using super-resolution microscopy and single-molecule tracking, we have recently shown that during native S-layer assembly, RsaA monomers diffuse while non-covalently attached to the cell surface until incorporated into a growing crystal lattice (9). When no previous S-layer lattice exists, RsaA nucleates 2D crystals at remarkably low concentrations to form stable crystalline rafts with as few as ~50 molecules (9). Advances in imaging modalities such as atomic force microscopy (AFM) and Cryo-EM have allowed for the direct observation of macromolecular nucleation and crystallization for a select few systems at the nanometer scale (15–18).…”

Lipid Anchoring of Archaeosortase Substrates and Mid-Cell Growth in Haloarchaea

“…In spite of the bacteria that use the localization of specialized proteins to promote mid-cell elongation, it is important to point out that these mechanisms are likely not evolutionary related to the proposed haloarchaeal S-layer lipidation and cell elongation. First, even though there are examples of bacterial species with S-layer that carry out peptidoglycan cellwall synthesis at mid cell (6), their new S-layer material is inserted as patches distributed all around the cell (35,36). Second, lack of conservation in the protein architecture between archaeal and bacterial S-layers argue that they may have emerged independently of each other (8,37).…”