Abstract:

【Objective】To construct broad host range replication type fluorescent protein plasmid and to evaluate the applicability of fluorescent protein plasmid by labeling four strains of Gram-negative bacteria. 【Method】gfp, mCherry and the constitutive promoter PpsbA were cloned into the plasmid pBBR1MCS2' in order to construct the expression vector pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry, and the expression vectors were introduced into Ralstonia solanacearum EP1, Pectobactererium carotovorum subsp. carotovorum WB, Klebsiella Pneumoniae JF and Burkholderia cepacia 1-2 by conjugative transfer. Their fluorescence performance of colonies and cells was observed, growth curves and plasmid retention rates were detected, and relevant functions were verified. 【Result】The fluorescent protein expression vectors pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry were successfully constructed, and the four Gram-negative bacteria were successfully labeled with green and red fluorescent proteins. The colonies and individual cells of the fluorescent protein-labeled bacteria emitted corresponding fluorescence, and the growth rate was consistent with that of the wild-type strains. However, the stability of the fluorescent protein plasmids varied greatly among the four strains. The green and red fluorescent protein plasmids were the most stable in the strain JF, with plasmid retention rates of 90.33% and 94.67%, respectively after 40 h of incubation without selective pressure; followed by strain WB and EP1, and the worst in strain 1-2, with fluorescence not detected after 5 and 25 h of incubation, respectively. In addition, there was no significant difference in the pathogenicity between fluorescently labeled WB and wild-type strain WB. 【Conclusion】 Two fluorescent protein expression vectors are successfully constructed and introduced into four different Gram-negative bacterial strains, including R. solanacearum EP1. In the plasmid retention rate, green and red fluorescent proteins are less stable in strain 1-2, and are stably expressed in the remaining three strains. These vectors provide important research materials for the subsequent study of labelling Gram-negative bacteria and investigating their related functions.

Key words: Gram-negative bacteria, broad host range replication type, fluorescent protein labeling, conjugative transfer, plasmid retention rate