Knock-out or knock-in? Converting a SacB-based gene disruption system for site-specific chromosomal integration in Pseudomonas syringae pv. tomato DC3000.

Recent advances in next generation sequencing technology allow us to retrieve the whole genome sequence of a requested bacterium in less than a day. Thus, development of quick, easy and efficient means to systemically analyze the functions of all genes is required in the post-genome era. Here, a procedure of finding a suitable chromosome integration site and developing a gene disruption system into a knock-in system in Gram-negative bacteria is proposed. As a proof of concept, we successfully modified a sacB-based gene knock-out strategy into a site-specific gene integration system to deliver a DNA fragment into the genome site between 313,520bp and 313,521bp of the model phytopathogenic bacterium, Pseudomonas syringae pv. tomato (Pst) DC3000. The expression levels of avrPtoB and hcp2 integrated using this method exhibited steady and similar expression levels as those in the wild type. In the future, this concept could allow us to easily develop gene replacement and delivery systems at the same time using a counter-selectable suicide vector-based allelic exchange strategy, and facilitate functional genomics studies of any bacterium whose genome has been sequenced.