Periodic Reporting for period 2 - CryoET4Diversity (Cryo-electron tomography for microbial ecology and evolution)
Periodo di rendicontazione: 2023-07-01 al 2024-12-31
The cryo-electron microscopy (cryoEM) modalities of single particle analysis and cryo-electron tomography (cryoET) have revolutionized the fields of structural biology and cellular biochemistry, and they enabled groundbreaking insights into primarily hypothesis-driven, mechanistic problems, using well-established model systems. High-throughput sequencing technologies have revolutionized microbial community studies and changed our view of the diversity of life. In order to understand how microbes function and interact with other cells, however, sequencing- and cultivation-based techniques must be complemented with experiments that elucidate phenotypes in situ at the single-cell level.
In Aim 1, we will develop cryoET methods for their application to problems in microbial ecology. We will resolve technical challenges of cryoET application to complex environmental samples, including aspects of sample preparation, data collection, data analysis and data integration.
In Aim 2, we will apply the new methods to outstanding biological questions to advance our understanding of cell-cell interactions. We will study the role of unique bacterial tubulins in a bacterium-ciliate symbiosis, aspects of multicellularity in magnetotactic bacteria, and the diversity and roles of bacterial contractile injection systems.
This project resides at the interface of structural biology/biophysics and environmental/evolutionary biology. We will leverage the power of cryoET to generate three-dimensional images of cells in a frozen-hydrated life-like state, and at macromolecular resolution. The complementation with high-throughput and single-cell approaches from microbial ecology will allow us to make progress on specific biological questions. CryoET offers a new “sense” for the analysis of complex environmental samples. Our efforts will establish cryoET as a discovery tool that enables us to conceive how genetic variations manifest in structural and functional diversity.
In Aim 1, we will develop cryoET methods for their application to problems in microbial ecology. We will resolve technical challenges of cryoET application to complex environmental samples, including aspects of sample preparation, data collection, data analysis and data integration.
In Aim 2, we will apply the new methods to outstanding biological questions to advance our understanding of cell-cell interactions. We will study the role of unique bacterial tubulins in a bacterium-ciliate symbiosis, aspects of multicellularity in magnetotactic bacteria, and the diversity and roles of bacterial contractile injection systems.
This project resides at the interface of structural biology/biophysics and environmental/evolutionary biology. We will leverage the power of cryoET to generate three-dimensional images of cells in a frozen-hydrated life-like state, and at macromolecular resolution. The complementation with high-throughput and single-cell approaches from microbial ecology will allow us to make progress on specific biological questions. CryoET offers a new “sense” for the analysis of complex environmental samples. Our efforts will establish cryoET as a discovery tool that enables us to conceive how genetic variations manifest in structural and functional diversity.
Aim 1 deals with the development of cryo-electron tomography methods. We have made progress on different aspects. First, we have developed methods to using FACS sorting for sample preparation. Second, we have established a workflow to vitrify samples by high-pressure freezing. Third, we have implemented new software for the automation of focused-ion beam milling. Fourth, we have successfully applied for a new milling instrument that is in the process of installation. Advances have been made on cryoET data collection. For the identification of cells in cryotomograms, we are in the process of developing a new method involving laser microdissection technology. Furthermore, we have successfully developed and applied a method to use ribosome structures for the identification of cells (see publication Rodrigues-Oliveira et al, Nature).
Aim 2 applies the new methods to biological questions. We have made significant progress in characterizing novel tubulin genes from epixenosomes in vitro. We determined their structure by single particle cryoEM and showed that they form a heterodimer. We have also worked on imaging these organisms by cryoET. We have also collected initial datasets for magnetotactic bacteria. Regarding contractile injection systems, we have made progress on studying Chloroflexi in environmental samples and the role of ixotrophy in Bacteroidetes. As an unexpected event, we could apply the methods for the characterization of a new biological system, namely Asgard archaea (see publication Rodrigues-Oliveira et al, Nature). We are now investing more effort into the study of the Asgard cytoskeleton.
Aim 2 applies the new methods to biological questions. We have made significant progress in characterizing novel tubulin genes from epixenosomes in vitro. We determined their structure by single particle cryoEM and showed that they form a heterodimer. We have also worked on imaging these organisms by cryoET. We have also collected initial datasets for magnetotactic bacteria. Regarding contractile injection systems, we have made progress on studying Chloroflexi in environmental samples and the role of ixotrophy in Bacteroidetes. As an unexpected event, we could apply the methods for the characterization of a new biological system, namely Asgard archaea (see publication Rodrigues-Oliveira et al, Nature). We are now investing more effort into the study of the Asgard cytoskeleton.
Our advances to characterize an Asgard archaeal organism by cryo-electron tomography go beyond the state-of-the-art and were enabled by methods developments in the scope of the project.