X - R A Y N A N O P R O B E

S C I E N T I F I C H I G H L I G H T S

7 6 H I G H L I G H T S 2 0 2 2 I

PRINCIPAL PUBLICATION AND AUTHORS

Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity, D.N. Azulay (a,b), O. Spaeker (c), M. Ghrayeb (a,b), M. Wilsch-Bräuninger (d), E. Scoppola (c), M. Burghammer (e), I. Zizak (f), L. Bertinetti (f), Y. Politi (f), L. Chai (a,b), Proc. Natl. Acad. Sci. 119(4), e2118107119 (2022); https:/doi.org/10.1073/pnas.2118107119 (a) Institute of Chemistry, The Hebrew University of Jerusalem (Israel) (b) The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem (Israel) (c) Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam (Germany) (d) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden (Germany) (e) ESRF (f) Department Structure and Dynamics of Energy Materials, Helmholtz-Zentrum Berlin (Germany)

REFERENCES

[1] H. Vlamakis et al., Genes. Dev 22, 945-953 (2008). [2] J.N. Wilking et al., Proc. Natl. Acad. Sci. 110, 848 (2013).

Amorphous-to-crystal transition in biominerals

State-of-the-art optical and X-ray microscopy methods were combined to investigate early- mineralised calcareous units from two bivalve species, revealing chemical and crystallographic structural insights.

Biomineralisation integrates complex physical and chemical processes bio-controlled by living organisms through ionic regulation and the production of organic molecules, allowing for the tuning of structural, optical and mechanical properties of hard tissues during ambient-condition crystallisation. The investigation

of the developing biomineralised units allows one to capture some of the transient states involved in the biomineralisation process. For many marine calcareous biomineralising organisms, the production of single- crystal-like elements escapes classical nucleation and crystallisation theory. The repeated observation of an amorphous precursor, an organo-granular structure and a layer-by-layer growth mode are generic features, which point towards an amorphous-to-crystalline transition [1].

Mollusc bivalve shells are composed of calcium carbonate and form an integral part of the global CO2 cycle. The mollusc bivalve species chosen for this study, Pinctada margaritifera and Pinna nobilis oyster shells, present a rather simple morphology at the edge of their shell,

hump (Figure 64b). In non-sporulating younger colonies (24h) [1], the doublet is absent, exposing a broad hump (Figure 64b). In even younger colonies, where matrix production has just begun, a high spatial inhomogeneity with respect to the presence of this scattering signal suggests that it originates from the presence of matrix biopolymers. This signal is altered in matrix mutants lacking the most abundant ECM protein, TasA (ΔtasA), and polysaccharide, EPS (Δeps) (Figure 64b).

The matrix-related scattering signal from biofilms, as well as from different polymorphs of TasA fibres formed in different conditions, is reminiscent of cross-β signal found in amyloid fibres. However, the broad XRD peaks point at short cross-β-sheet domains (Figure 64c). XRF analysis revealed that all the protein fibres bind calcium ions, whereas other metal ions such as iron, zinc and manganese are only bound by some fibre polymorphs.

The time-dependent expression and organisation of matrix components give rise to spatial heterogeneity in matrix organisation. This is demonstrated by scanning mm-scale areas of an intact, naturally hydrated WT

biofilm. Mapping the spatial distribution of the spore- related doublet reflections in the biofilm revealed increased abundance along large wrinkles relative to areas between them (Figure 64d). Along the wrinkles, increased contributions of free water were observed, in agreement with previous demonstrations of the function of biofilm wrinkles as water channels for nutrient transport [2]. XRF mapping revealed that calcium, the most abundant metal ion, is uniformly distributed across the biofilm, whereas zinc, iron and manganese ions are concentrated along the biofilm wrinkles (Figure 64e).

The results suggest a mechanism for the preferential accumulation of water, metal ions and spores in biofilm wrinkles (Figure 65). Water and metal ions are drawn into the biofilm via evaporation-driven water flow [2]. While calcium is bound to the ECM, other metal ions accumulate along water channels, where increased surface area enhances evaporation [2]. Metal ion accumulation leads to local biological stress, eventually resulting in sporulation. Thus, this work links molecular and elemental organisation to architecture and biological differentiation across whole biofilms.