Mobilization of tet (X4) by IS 1 family elements in porcine Esc herichia coli isolates

The dissemination mechanism of the high-level tigecycline resistance gene tet (X4) in porcine Escherichia coli was investigated . tet (X4) and other antimicrobial resistance genes were located on the plasmids p1919D3-1 and p1919D62-1 and flanked by two or three copies of IS 1 family elements, which can form one to three translocatable units (TUs). Using a reduced transposition model, IS 1A was experimentally demonstrated to mediate the transposition of tet (X4) from a suicide plasmid into the E. coli chromosome.

A transposon-associated CRISPR/Cas9 system specifically eliminates both chromosomal and plasmid-borne mcr-1 in Escherichia coli

The global spread of antimicrobial-resistant bacteria has been one of the most severe threat to public health. The emergence of mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by IS Apl1- formed transposon Tn 6330 and mediated rapid dispersion among Enterobacteriaceae species. Here we developed a CRISPR-Cas9 system flanked by IS Apl1 in a suicide plasmid capable of exerting the sequence-specific curing against mcr-1 bearing plasmid and killing the strain with chromosomal-borne mcr-1 . The constructed IS Apl1 -carried CRISPR-Cas9 system either restored the sensitivity to colistin of strains with plasmid-borne mcr-1 or directly eradicated the bacteria harbored the chromosomal-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting mcr-1 gene. This method is highly efficient in removing mcr-1 gene from Escherichia coli and thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with the immunity against the acquisition of the exogenous mcr-1- containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.ResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~108, which is ~2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.ConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.