Multi-scale imaging of macromolecular complexes in environmental bacteria
EMBARGOED UNTIL 2028-10-03
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Date
2025
Publication Type
Doctoral Thesis
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yes
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EMBARGOED UNTIL 2028-10-03
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Abstract
Bacteria inhabit a wide range of ecosystems from hot springs to the ocean. Their ability to survive and flourish in such diverse environments is reflected in their biological diversity and complexity. Nevertheless, the diversity of bacteria that most studies focus on is not representative of environmental diversity. Hence, bringing environmental bacteria to the forefront of scientific studies will enable us to discover even more of the existing bacteria diversity and understand novel bacterial biologies.
One contributing factor of bacterial complexity is their interactions with neighbouring microorganisms. In nature, bacteria are not isolated and in fact, their fitness is dependent on their interactions with their neighbours. These interactions can range from cooperative to antagonistic, but they are often mediated by macromolecular complexes. As a result, to fully capture and comprehend these microbial communities, environmental samples should be studied at a community, cellular and protein level. This can be achieved via a multi-scale imaging approach. Cryo-single particle analysis can provide structural and mechanistic insights into macromolecular complexes mediating cell-cell interactions. Complemented by cryo-electron tomography (cryoET), which allows cells to be imaged at a near-native state, cellular and functional context for these macromolecular complexes can be obtained.
Combined with expansion microscopy and phylogenetic analyses, the aforementioned methodologies were applied in chapter 2 to investigate the defensive function of epixenosomes, a bacterial epibiont of a ciliate. We uncovered the presence of several macromolecular complexes including tubulin polymers, a refractile (R) body and two contractile injection systems (CISs). Based on our findings, we posited that epixenosomes undergo R-body-mediated cell lysis, releasing CISs into the environment and ultimately repel predators of the host. Cell lysis is achieved via the extension of the R body, which is likely facilitated by the tubulin polymers. Intriguingly, apart from eukaryote-like tubulins, we discovered the presence of other eukaryotic signature proteins like actin and we discuss their evolutionary implications.
To explore how bacteria interact in stratified macroscopic microbial communities, we studied the use of CISs in hot spring mats. Because of the microbial complexity of hot spring mats, in chapter 3, we applied cryo-focused ion beam milling and cryoET, guided by metagenomics and proteomics. We found that the most abundant Roseiflexus bacteria utilise CISs to either compete with cyanobacteria or mediate their own cell death. We then discovered CISs in other thermophiles and showed that they all belong to a new lineage of CISs from thermophilic bacteria.
Overall, this thesis will hopefully encourage the study of more environmental samples as well as the incorporation of multi-scale imaging approaches to conventional environmental microbiology workflows.
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Examiner : Pilhofer, Martin
Examiner : Ishikawa, Takashi
Examiner : Horn, Matthias
Examiner: Wieczorek, Michal
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ETH Zurich
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Subject
cryo-electron tomography,; microbial mats; PVC bacteria; epixenosomes; Roseiflexus; contractile injection system; focused ion beam milling; refractile bodies
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09463 - Pilhofer, Martin / Pilhofer, Martin