The Tn7-based genomic integration is dependent on an attTn7 box in the glms gene and is site-specific with monocopy in Ralstonia solanacearum species complex.

Abstract

The Tn7-based genomic integration system enables direct insertion of foreign gene elements into chromosome downstream of glms in many bacteria species. The glms gene is greatly conserved in Ralstonia solanacearum species complex (RSSC), while its downstream regions are mostly different in the RSSC. Here, we provided genetic evidence to validate that this Tn7-integration is dependent on a conserved 30-bp motif in the glms, called attTn7 box, and artificial attTn7 boxes elsewhere are competent for the Tn7-integration, which is further confirmed to be simultaneous at downstream of both original and artificial attTn7 boxes using the PCR. With the whole genome re-sequencing on 500 Tn7-colonies, the Tn7-integration was confirmed to be site- specific at 25-bp downstream of glms with monocopy as chromosome of the RSSC. Characteristic of the monocopy in chromosome enables the Tn7-based complementation to fully restore phenotypes of mutants to those of parent strains that is advantageous than those based on plasmids with low-copy numbers. The Tn7-based genomic integration system provides a generally applicable and versatile genetic tool for studies of complementation, pathogenesis, overexpression, and in-vivo promoter activity assays with monocopy in the RSSC.