The Tn-Based Genomic Integration Is Dependent on an Tn Box in the Gene and Is Site-Specific With Monocopy in Species Complex.

The Tn-based genomic integration system enables direct insertion of foreign gene elements into the chromosome downstream of in many bacteria species. The gene is greatly conserved in species complex (RSSC), while its downstream regions are mostly different in the RSSC. Here, we provided genetic evidence to validate that this Tn integration is dependent on a conserved 30-bp motif in the , called an Tn box, and artificial Tn boxes elsewhere are competent for the Tn integration, which is further confirmed to be simultaneous downstream of both original and artificial Tn boxes, using PCR. With the whole-genome resequencing on 500 Tn-colonies, the Tn integration was confirmed to be site- specific at 25 bp downstream of with monocopy as a chromosome of the RSSC. Characteristic of a monocopy in a chromosome enables the Tn-based complementation to fully restore phenotypes of mutants to those of parent strains that are advantageous rather than those based on plasmids with low-copy numbers. The Tn-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.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.