We’ve been developing methods to identify cells carrying TIDs (“Tandem Inverted Duplications”). Sophie Maisnier-Patin uses P22 transduction to introduce various tools which will introduce a drug resistance cassette only if they are able to recombine into such inverted structures.
Sophie asks whether a kanR MudJ element can nucleate TIDs by virtue of its terminal inverted repeat? This entity is 104 bp long and can form a perfectly paired 48 bp stem with an 8 base loop. The hypothesis is something like the following:
The tool Sophie used in this experiment contained two oppositely facing MudJ elements:
She transduced this into a recipient containing MudJ and a deletion which should prevent recombination with cob and his. :
The resulting strain, SMP1666, should have a lac+ camR kanR trp- phenotype. Unfortunately, no TIDs was found. Instead, Illumina and subsequent PCR analyses showed that one end of the tool inserted by a strand annealing mechanism, as the his:cob deletion was not quite large enough. The strain was unstable for chloramphenicol resistance, expected from the flanking trpB::MudJ direct repeats. Sophie showed by transduction to trp+ that the SMP1666 parent, as expected, contained a MudJ-bisected trpB locus, and that trp+transductants, whether camR or camS, were invariably lac-, another prediction of the hypothesis. The corrected model became:
A large fraction of the trp+ lac- transductants retained kanamycin resistance! How could this be, when elimination of the last MudJ in the chromosome should, by definition, also remove the remaining kanR and lac loci? Clearly, something was wrong. There had to be another MudJ with an impaired promoter incapable of driving lacZ. We wrestled for weeks with elaborate models. All of them suffered from the need for at least two concerted events, implying a low rearrangement frequency, when the opposite had been observed.
We remembered some interesting results from two other Sophian strains, SMP1750 & TT26263, in which the donor DNA had recombined with the recipient through the MudJ inverted repeat locus itself, even though the element orientations were divergent. Although the inverted-repeat region is short, when folded it is prone to attack by SbcCD. This would provide many 3′ ends which could then anneal and rescue the cell. A corollary is that the MudJ stem-loop structure is recombinationally potent.
In light of this, we have refined our model:
The head-to-head Muds in the middle would have no promoter at all for lac, but the internal constitutive kanR promoter would still be active. The trpB gene would, of course, have been restored exactly as expected in earlier models.-- Eric Kofoid