S C

IE N

T IF

IC H

IG H

LI G

H T

S X

-R A

Y N

A N

O P

R O

B E

7 5 I H I G H L I G H T S 2 0 2 2

X-ray techniques reveal links between structural hierarchy and elemental heterogeneity in Bacillus subtilis biofilms

Cells in biofilms benefit from increased resistance to antibiotic treatment relative to planktonic cells, partly due to phenotypic heterogeneity, where they differentiate into functionally distinct subgroups. Spatial and temporal analysis of whole biofilms using X-ray diffraction and fluorescence exposes heterogeneous elemental distribution and molecular structure relationships to biofilm architecture.

Biofilms (Figure 64a) are surface-associated aggregates of bacterial cells held together by an extracellular matrix (ECM). The ECM, composed mainly of secreted polysaccharides and proteins, provides biofilms with mechanical support and resistance to toxins. Cells in biofilms differentiate into subgroups expressing specific sets of genes, which, in the example of the model organism Bacillus subtilis, are motile cells, matrix-forming cells and spores [1].

The molecular order and the compositional heterogeneity in B. subtilis biofilms were studied in situ using simultaneous X-ray diffraction (XRD) and X-ray fluorescence (XRF) measurements at beamline ID13. Mature (72h) wild-type (WT) biofilms show scattering signals composed of a sharp doublet, originating from spore coat proteins, and a broad

Fig. 65: Schematic representation of the relationship between biofilm structures and metal ion distribution. a) Cross-section of B. subtilis biofilm with a water-filled (blue) wrinkle. Bacterial cells represented by brown ovals; spores represented by circles. Water and nutrients are taken up by the biofilm (blue arrows) driven by surface evaporation (curled arrows). b) The biofilm is composed of cells and ECM (brown background and darker lines) in which

c) TasA fibrils contain short cross-β-sheet domains. d) Zoom into a column across the biofilm wrinkle; water and

metal ions (Ca: purple and Fe/Zn/K/Mn: red and green) are free inside water channels (bottom). The metals are adsorbed by the ECM (middle), with preference to Ca that remains mostly

bound to the ECM whereas Zn, Mn, and Fe are free to diffuse in the matrix, concentrated in the biofilm wrinkles (top). Metal ion accumulation at biofilm wrinkles possibly leads to sporulation.

Image for illustration purposes, not to scale.

Fig 64: Heterogeneity in biofilm extracellular matrix. a) B. subtilis WT biofilm (72h). Scale bar: 5 mm. b) XRD pattern of WT biofilms of different ages (12-72h) and matrix-mutant biofilms. c) XRD pattern of polymorphs of TasA protein fibres prepared under different conditions (aggregates are formed in acidic solutions, fibrils in high-salt or high-protein solution, bundles at high-salt and -protein

concentration). d) Mapping the intensity of one of the spore-related doublet reflections. Arrows point to wrinkles in the biofilm. e) XRF overlay map of Mn (blue), Zn (red) and Ca (green). Magenta: overlap of red and blue. Look out tables (LUTs) stand for XRF counts.

Fe co-localises with Mn and Zn (data not shown).