Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity

David N. Azulay, Oliver Spaeker, Mnar Ghrayeb, Michaela Wilsch-Bräuninger, Ernesto Scoppola, Manfred Burghammer, Ivo Zizak, Luca Bertinetti, Yael Politi*, Liraz Chai*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Original languageAmerican English
Article numbere2118107119
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number4
StatePublished - 25 Jan 2022

Bibliographical note

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  • Biofilms
  • Extracellular matrix
  • Functional amyloid proteins
  • Phenotypic heterogeneity
  • Small angle X-ray scattering (SAXS)/wide angle X-ray scattering (WAXS)


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