Open AccessA broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis
Author(s) -
Lotisha E. Garvin,
Rebecca Vande Voorde,
Mary S Dickinson,
Steven Carrell,
Kevin Hybiske,
Daniel D. Rockey
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0261088
Subject(s) - plasmid , chlamydiae , biology , transformation (genetics) , replicon , chlamydia trachomatis , genetics , cloning vector , t dna binary system , plasmid preparation , homologous recombination , microbiology and biotechnology , vector (molecular biology) , recombinant dna , gene , virology , pbr322
Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C . trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C . trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E . coli . Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.