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

Abstract

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.

Keywords: Acinetobacter; Conditional suicide plasmid; Escherichia coli; IPTG; Pseudomonas; Transposon mutagenesis.

Fig. 1

IPTG-controlled conditional suicide plasmids. a Plasmid pMMB208 and its conditional-suicide derivatives. pMMB208 contains an RSF1010 oriV for replication and an oriT for conjugation. Genes repA, mobA/repB and repC encode proteins required for plasmid replication, and repF encodes a transcription repressor that binds promoter P4. pMMB208 also has Cam^R and lacI^Q genes and a Ptac promoter. Plasmid pMMB-repF is a derivative of pMMB208 that has a second copy of the repF gene inserted downstream of Ptac. Plasmids pMMB-repAK42A and pMMB-repAD139A have a dominant-negative repA mutant gene, either K42A or D139A, inserted downstream of Ptac. b Amount of E. coli DH10B cells retaining the indicated plasmids after 24 h growth in the absence of antibiotics, either with or without IPTG induction. Results were average of three independent experiments, and bars represent mean ± SD (standard deviation). *p < 0.0001, **p < 0.0001, and ***p < 0.0001 by unpaired Student’s t-test for IPTG induced cultures. c pMMB208 and its derivatives are digested with HindIII (H) and PstI (P). Comparing to pMMB208 and pMMB-repF, the repA K42A and D139A mutants showed reduced yields in plasmid minipreps (no IPTG induction; 3.0% and 4.7% of that of pMMB208, respectively). HindIII and PstI digestion generates two fragments for each plasmid. The ~ 9 kb fragment is seen on the gel, while the shorter ones, ranging from 18 bp for pMMB208 to 861 bp for the repA mutants, are not visible. Another large band (~ 9 kb) is also seen in restriction digestion of pMMB208 and its derivatives, even after complete digestion, and the cause is unknown

Fig. 2

mTn5 transposon mutagenesis using an IPTG-controlled conditional suicide plasmid. a Diagram of plasmid pSNC-mTn5. pSNC-mTn5 is a derivative of pMMB-repF that contains a Kan^R-tagged mTn5, a lac promoter-controlled hyperactive transposase gene (tnp^H), and a sacB counter selection marker (with its own promoter). OE and IE are outside and inside ends of the mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. A “+” symbol for IPTG indicates that the inducer was added to the liquid culture, and a “+” symbol for Suc, Cam, and Kan indicates that the chemicals were added to the plates. Black columns represent plasmid retention frequencies, and the blue column represents Tn5 retention frequency. Results were average of three independent experiments, and bars represent mean ± SD (*p < 0.0001 and **p = 0.0054 by unpaired t-test). (see Methods for details) (c) Colony restreaking. 150/150 Suc^RKan^R colonies of DH10B were found to be Kan^RCam^S and 50 are shown here. d Colony PCR of 10 restreaked clones in (c). Primer sets P1&P2 and P3&P4 detect Kan^R and repF, respectively. All were mTn5-positive and plasmid-negative. Primers P3 and P4 are a functional pair for PCR-amplification of the plasmid sequence (data not shown). e Plasmid and transposon retention frequencies in A. baylyi. Results were average of three independent experiments, and bars represent mean ± SD (*p = 0.024 and **p < 0.0001 by unpaired t-test). f Plasmid and transposon retention frequencies in P. aeruginosa. Results were average of three independent experiments, and bars represent mean ± SD (*p = 0.0013 and **p = 0.0038 by unpaired t-test). Colony restreaking and PCR analysis are shown in Additional file 2: Figure S2

Fig. 3

mTn5 insertion sites in different bacteria. a mTn5 insertion sites in E. coli DH10B. b mTn5 insertion sites in A. baylyi 33,305. c mTn5 insertion sites in P. aeruginosa PAO1. Only the chromosomal sequences next to the OE are shown. The 9 bp duplicated sequences are shown in capital letters. Identical clones are shown only once, with numbers indicated in parenthesis. Either gene names or locus tags are given as genetic locations

Fig. 4

Generation of a P. aeruginosa insertion library with pSNC-mTn5ME. a Diagram of pSNC-mTn5ME, a derivative of pSNC-mTn5 that has MEs instead of OE and IE at the termini of mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. Results were average of three independent experiments, and bars represent mean ± SD (*p < 0.0001 and **p = 0.0004 by unpaired t-test). Colony restreaking and PCR assays are shown in Additional file 4: Figure S4. c Plasmid and transposon retention frequencies in A. baylyi. Results were average of three independent experiments, and bars represent mean ± SD (*p = 0.0029 and **p = 0.0006 by unpaired t-test). Colony restreaking and PCR assays are shown in Additional file 5: Figure S5. d Plasmid and transposon retention frequencies in P. aeruginosa PAO1. Results were average of three independent experiments, and bars represent mean ± SD (*p < 0.0001 and **p = 0.0065 by unpaired t-test). Colony restreaking and PCR assays are shown in Additional file 6: Figure S6. e Plasmid and transposon retention frequencies in the P. aeruginosa PAO1 mutant library generated with pSNC-mTn5ME. f Colony restreaking. 100/100 Suc^RKan^R colonies of the mTn5ME library of P. aeruginosa were found to be Kan^RCam^S. 50 are shown here. g Colony PCR of ten restreaked clones in (f) with the indicated primers. All were mTn5-positive and plasmid-negative. h Transposon insertion sites of 46 mutant clones from the mTn5ME insertion library of P. aeruginosa. Identical clones are shown only once, with their duplication numbers indicated in parenthesis