High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells

Elspeth F. Garman,Kamel El Omari,Andriko von Kügelgen,Armin Wagner,Danguole Kureisaite-Ciziene,Ramona Duman,Dimitrios Kolokouris,Jan Löwe,Phillip J. Stansfeld,Matthew Herdman,Andreas Kjaer,Tanmay A.M. Bharat

doi:10.1016/j.str.2021.10.012

Abstract

SummarySurface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.

Highlights

Envelopes are the key platform for cellular interactions with the environment, critical for the regulation of import and export of materials, motility, and the cellular adherence to surfaces
While cell envelopes of prokaryotes are chemically and structurally diverse, many bacterial and almost all archaeal cells are encompassed by a paracrystalline, proteinaceous, macromolecular sheath known as a surface layer (S-layer) (Sara and Sleytr, 2000; Bharat et al, 2021; Sleytr and Beveridge, 1999; Beveridge, 1994; Messner and Sleytr, 1992; Fagan and Fairweather, 2014)
Using cryoelectron microscopy (cryo-EM) structure determination of a Holmium (Ho3+) and LPSbound RsaANTD, and long-wavelength X-ray diffraction studies on RsaACTD assembled with Ca2+ into stacked sheets in threedimensional crystals, we report the positions and identities of almost all metal ions in the S-layer, describing 108 experimentally confirmed Ca2+ ions bound to each RsaA hexamer in the S-layer

Summary

Introduction

Envelopes are the key platform for cellular interactions with the environment, critical for the regulation of import and export of materials, motility, and the cellular adherence to surfaces. Advances in structural biology and imaging techniques (Beck and Baumeister, 2016; Oikonomou and Jensen, 2017) have improved our understanding of S-layer biogenesis and assembly, revealing that S-layers have primarily a bipartite arrangement, often with separated lattice-forming and cell-anchoring domains in their constituent SLPs (Bharat et al, 2021; von Ku€gelgen et al, 2020; Gambelli et al, 2019; Phipps, 1990; Baumeister and Lembcke, 1992; Wildhaber and Baumeister, 1987; Pum and Sleytr, 2014; Veith et al, 2009) Another common feature of S-layers is lattice assembly mediated by the presence of divalent metal ions in the extracellular environment (Engelhardt, 2007a, 2007b), which has been observed in Archaea (Cohen et al, 1991; Kessel et al, 1988), Gram-positive (Baranova et al, 2012; Lupas et al, 1994), and Gram-negative bacteria (von Ku€gelgen et al, 2020; Bharat et al, 2017; Herrmann et al, 2020)

Methods

Results

Discussion

Conclusion