RT Journal Article
SR Electronic
T1 Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 419473
DO 10.1101/419473
A1 Santa S. Naorem
A1 Jin Han
A1 Stephanie Y. Zhang
A1 Junyi Zhang
A1 Lindsey B. Graham
A1 Angelou Song
A1 Cameron V. Smith
A1 Fariha Rashid
A1 Huatao Guo
YR 2018
UL http://biorxiv.org/content/early/2018/09/17/419473.abstract
AB Background Transposon 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.Results We 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.Conclusion We 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.IPTGIsopropyl β-D-1-thiogalactopyranoside;tsTemperature-Sensitive;bpbase pair;OEOutside End;IEInside End;MEMosaic End;ODOptical Density;LBLuria Broth;LBNSLB no salt;CamChloramphenicol;Kankanamycin.