Isolation of bacterial type IV machine subassemblies
Mayukh K Sarkar1, Seyyed I Husnain, Simon J Jakubowski
1Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|January 10, 2013
Summary
This study details a new protocol for isolating bacterial type IV secretion systems (T4SSs) and their protein complexes. The method, adaptable for various bacteria, aids in studying DNA transfer mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacterial type IV secretion systems (T4SSs) are crucial for transferring DNA and proteins to target cells, often requiring direct contact.
- Understanding T4SS architecture is key to deciphering their function in inter-cellular communication and pathogenesis.
Purpose of the Study:
- To describe a protocol for isolating and characterizing VirB protein complexes from the Agrobacterium tumefaciens VirB/VirD4 T4SS.
- To provide a method adaptable for isolating T4SS subassemblies from diverse bacterial species, including Gram-negative and Gram-positive bacteria.
Main Methods:
- Isolation and biochemical/ultrastructural analysis of VirB protein complexes from Agrobacterium tumefaciens.
- Adaptation of the protocol for isolating T4SS subassemblies from other bacterial types.
- Utilizing a modified chromatin immunoprecipitation assay, termed transfer DNA immunoprecipitation (TrIP), to assess substrate copurification.
Main Results:
- A detailed protocol for isolating and characterizing T4SS protein complexes is presented.
- The protocol's adaptability to various bacterial systems is highlighted.
- Demonstration of TrIP for recovering cross-linked DNA substrate-channel subunit complexes during conjugative DNA transfer.
Conclusions:
- The described protocol facilitates the study of bacterial T4SS architecture and function.
- The TrIP assay enables the investigation of substrate engagement within T4SS channel complexes.
- This work provides a valuable tool for advancing research on bacterial secretion systems and DNA transfer.
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