Periodic Reporting for period 2 - BacNanoMachine (Reconstructing the coordinated self-assembly of a bacterial nanomachine)
Periodo di rendicontazione: 2022-04-01 al 2023-09-30
Life has evolved diverse protein machines and bacteria provide many fascinating examples. Despite being unicellular organisms of relatively small size, bacteria produce sophisticated nanomachines with a high degree of self-organization. The motility organelle of bacteria, the flagellum, is a prime example of complex bacterial nanomachines. Flagella are by far the most prominent extracellular structures known in bacteria and made through self-assembly of several dozen different kinds of proteins and thus represents an ideal model system to study sub-cellular compartmentalization and self-organization.
The flagellum can function as a macromolecular motility machine only if its many building blocks assemble in a coordinated manner. However, previous studies have focused on phenotypic and genetic analyses, or the characterization of isolated sub-components. Crucially, how bacteria orchestrate the many different cellular processes in time and space in order to construct a functional motility organelle remains enigmatic. Therefore, the major research aims of the project BacNanoMachine are to understand:
Aim 1: How is the hierarchical gene expression coupled to the assembly of multiple flagella?
Aim 2: What is the minimal genetic regulation required for assembly and function of the flagellum?
Aim 3: How are the flagellar building blocks targeted to the site of assembly?
Aim 4: How is robust protein secretion achieved in the presence of large cellular noise?
The flagellum can function as a macromolecular motility machine only if its many building blocks assemble in a coordinated manner. However, previous studies have focused on phenotypic and genetic analyses, or the characterization of isolated sub-components. Crucially, how bacteria orchestrate the many different cellular processes in time and space in order to construct a functional motility organelle remains enigmatic. Therefore, the major research aims of the project BacNanoMachine are to understand:
Aim 1: How is the hierarchical gene expression coupled to the assembly of multiple flagella?
Aim 2: What is the minimal genetic regulation required for assembly and function of the flagellum?
Aim 3: How are the flagellar building blocks targeted to the site of assembly?
Aim 4: How is robust protein secretion achieved in the presence of large cellular noise?
The bacterial flagellum and the evolutionarily related injectisome are capable of exporting proteins with a remarkable speed of several thousand amino acids per second using a conserved type-III secretion system (T3SS). Using extensive targeted and random mutagenesis approaches, we were able to show how bacteria control the integrity of the cytoplasmic membrane during high-speed protein translocation via the T3SS. We further developed an experimental setup that allows us to synchronize flagella assembly and to monitor early and late substrate secretion of T3SS. Our results demonstrate that the T3SS features a remarkable specificity for only the substrates required at the respective flagellar assembly stage. The developed secretion substrate reporter system further provides a platform to investigate the molecular mechanism of substrate recognition by the T3SS.
In this project, we combine the visualization of the dynamic self-assembly of individual flagella with quantitative single-cell gene expression analyses, re-engineering of the genetic network and biophysical modeling in order to develop a biophysical model of flagella self-assembly. This novel, integrative approach will allow us to move beyond the classical, descriptive characterization of protein complexes towards an engineering-type understanding of the extraordinarily robust and coordinated assembly of a multi-component molecular machine.