Isolation of bacterial type IV machine subassemblies

The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through isolation of machine sub-assemblies for further biochemical and ultrastructural analysis. Here, we describe a protocol for isolation and characterization of VirB protein complexes from the paradigmatic VirB/VirD4 T4SS of Agrobacterium tumefaciens. This protocol can be adapted for isolation of T4SS subassemblies from other gram-negative bacteria as well as gram-positive bacteria. The biological importance of isolated T4SS subcomplexes can be assessed by assaying for copurification of trapped or cross-linked substrates. This can be achieved with a modified form of the chromatin immunoprecipitation (ChIP) assay termed transfer DNA immunoprecipitation (TrIP). Here, a TrIP protocol is described for recovery of formaldehyde-cross-linked DNA substrate–channel subunit complexes from cells employing T4SSs for conjugative DNA transfer.