A Practical Guide to Recombineering in Photorhabdus and Xenorhabdus.

Fluent genetic manipulation of prokaryote genomes is still limited to only a few commonly used hosts. Ideally the advanced technologies available for cloning into recombinant Escherichia coli should also be applicable in other prokaryotes. In particular, 'recombineering' is mediated by the lambda Red operon that permits fluent and precise engineering of the E. coli genome and associated recombinant DNA. The major limitation is that host-specific phage-derived recombination systems are also required in more distant species. Recently, an endogenous Red-like operon Pluγβα has been reported to be effective in both Photorhabdus and Xenorhabdus bacteria. The Pluγβα recombineering system is based on three host-specific phage proteins from Photorhabdus luminescens, Plu2935, Plu2936, and Plu2934, which are functional analogs of Redβ, Redα, and Redγ, respectively. In this chapter, we provide a comprehensive and up-to-date method for P. luminescens and Xenorhabdus stockiae genome engineering via the Pluγβα recombineering system. In order to facilitate the rapid construction of knock-in vectors, recET-mediated recombineering is incorporated in the pipeline. Concerted recET system in E. coli with Pluγβα system in Photorhabdus and Xenorhabdus could promote reverse genetics, functional genomics, and bioprospecting research for these two genera.