Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery

Abstract

Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

FIG. 1.

History of construction of S17-1 λpir(pSC189) and SM10 λpir(pSC189) donor strains for transposon-based random mutagenesis using the R6K suicide plasmid pSC189. The RP4-2-Tc::Mu plasmid carries loci coding for RP4 conjugation machinery (Tra1, Tra2, and Tra3), a tetracycline resistance marker interrupted by Mu (Tc::Mu), and genetic elements coding for kanamycin resistance (Km^r). RP4-2-Tc::Mu integration into the chromosome of E. coli was isolated after cotransformation with the incompatible mini-RP4 plasmid pSR120 and selection for clones displaying both kanamycin and tetracycline resistance (step 1). The clones obtained were cured from pSR120, leading to SM10 (parental strain, S49-20 Km^r) or S17-1 (parental strain, E. coli 294 Sm^r Tp^r, following inactivation of the RP4-associated kanamycin resistance gene by Tn7 insertion), which contain the integrated RP4-2-Tc::Mu plasmid (ChRP4), as described previously (step 2) (40). For simplicity, only a single depiction is presented for strains SM10 and S17-1. Finally, the Π-encoding gene pir was introduced at the λ site (step 3), and the strains were transformed with the suicide vector pSC189, which carries a mariner-based transposon (Tn) and its C9 transposase (transposase) and harbors the RP4-dependent origin of conjugation, oriT, for mobilization and the Π-dependent origin of replication, oriR6K (step 4) (11, 14, 28).

FIG. 2.

Mu particles are released into the supernatant of S17-1 and S17-1 λpir cultures. Confluent lysis was observed when a drop of filter-sterilized supernatant of S17-1 or S17-1 λpir culture was spotted onto a lawn of growing MG1655 Δlac cells (Mu⁻). In contrast, no (S17-1 supernatant) or very few (S17-1 λpir supernatant) plaques appeared when the same supernatants were spotted on a lawn of a Mu lysogenic derivative (Mu⁺), confirming that the lysis is Mu dependent and that Mu particles are present in the supernatant. Mu-independent lysis observed using S17-1 λpir supernatant probably results from release of λ phage particles into the medium.

FIG. 3.

Genetic map of Mu insertions in E. coli S17-1λpir. (A) Structure of the RP4-2-Tc::Mu insertion region in E. coli S17-1 λpir showing the lacZ region and the oriT-carrying RP4 core plasmid flanked by the two copies of bacteriophage Mu, Mu1 and Mu2, in the same orientation. (B) PCR analysis of the junction between Mu and lacZ in S17-1 λpir, MG1655 Δlac, and a random Lac⁺ transconjugant obtained by conjugation between these strains, using primers lacZatg+100-3 and MuL.100-3. The region amplified using these primers is shown in panel A.

FIG. 4.

De novo infection by Mu during conjugation results in a second mutation event in the recipient strain. (Step 1) During conjugation, pSC189 is transferred to the recipient cell using the mobilization and conjugation functions of the RP4 plasmid integrated into the chromosomes of donor strains S17-1 λpir and SM10 λpir (ChRP4). (Step 2) Once in the cell, the pSC189-carried transposon (gray box) integrates into the chromosome, altering the expression of a gene and its associated function. (Step 3) In parallel, the ChRP4-associated Mu prophage (triangle) is induced spontaneously into a subpopulation of donor cells. Mu particles (tailed hexagons) are then released into the environment and infect the recipient cells, leading, in some cases, to a second mutagenic event by lysogenization (2).

FIG. 5.

Construction of a new donor strain, MFDpir (see the text for details). The introduction of RP4-2-Tc::Mu into the chromosome of SM10 and that of its derivative, ω7249, occurred in glvB. This resulted in duplication of Mu, with each copy (Mu1 and Mu2) flanking the core RP4 and displaying the same orientation. The two Mu1 and Mu2 copies, along with the remains of the interrupted tetracycline resistance region (′tetA-R and tetA"), were removed and replaced by cassettes providing resistance to apramycin (apra) and zeocin (zeo), respectively. After transduction of the Mu-free RP4 region into MG1655, the kanamycin resistance gene aphA was replaced by a cat-FRT cassette; the chloramphenicol resistance gene, cat, was subsequently excised using Flp recombinase. Then, the Π-encoding dapA::pir116-erm locus was introduced into the strain. The recA gene was finally replaced by a kan-FRT cassette, and the kanamycin resistance gene, kan, was removed with Flp, giving the new donor strain, MFDpir.