Monday 27 March 2023

Synchrotron X-ray diffraction and fluorescence reveals more about the nature of biofilms


Image by Kaspar Kallip - Own work, CC BY-SA 4.0,

Biofilms are multicellular microbial communities made of bacterial cells surrounded by a network of secreted biopolymers. Biofilms enable bacteria to attach on surfaces and the extracellular matrix barrier structurally isolates bacterial cells from external environmental fluctuations. For instance, biofilms can confer bacterial cell protection against mechanical stress and toxins like antimicrobials and disinfectants.


While the bacterial cells are genetically identical, their different phenotypes within biofilms allow the communities to respond differently to nutrients and oxygen levels, as well as threats.


Spatiotemporal gene expression analyses are the most common methods used to understand biofilm-associated phenotypic heterogeneity at the molecular level. Spatiotemporal gene expression is the activation of genes within specific tissues of an organism at specific times during development. Gene activation patterns vary widely in complexity.


Despite the method, it remains unknown how the elemental and structural properties of biofilms differ across space and time because most studies digest biofilms before analyzing the isolated molecules for their quantities.


New methods can reveal the importance of the physicochemical properties of the molecules secreted by the cells in a biofilm. This leads to the differential distribution of nutrients that propagate through macroscopic length scales and hence different biofilm properties.


An alternative technique is X-ray diffraction. This method can help to determine the structures of molecules present in a sample using electron densities of diffracted X-rays. A second method is X-ray fluorescence, which can be used to detect emissions of characteristic fluorescent X-rays from a material that is bombarded with high energy X-rays for elemental analysis.


Such technologies have shown how TasA fibers can greatly influence how stable the biofilms are and explain for differences in structural stability between old and young biofilms. TasA proteins can form fibers into three different morphologies: aggregate in acidic conditions, form thin and long fibrils at high salt concentration, and establish fiber bundles at high protein and salt concentrations. Thin and long fibrils were most common in young biofilms (1-2-day-old), showing that as biofilm ages, the proportion of TasA fibers could change a biofilm’s mechanical integrity.


Researchers have made use of both methods to scan intact Bacillus subtilis biofilm samples of different ages. This revealed spatiotemporal division of four main components including water, spores, extracellular matrix, and metal ion. The data shows how these components interact to affect processes like sporulation and polymer fibril formation.


One observation made is where water is bound in biofilm wrinkles and the role of wrinkles acting as water channels for nutrient transport and waste removal. Biofilm wrinkles could serve as channels filled with water to transfer nutrients across biofilms, and the water in wrinkles is not freely floating.


Importantly, bacterial spores can survive long periods of dehydration without detectable damage, and the presence of water can trigger germination. In terms of signalling when germination may be possible, spore coat protein swelling in biofilms may serve as humidity sensors for spore germination.


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (

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