Taqman qPCR assays of retrohoming in notable E. coli mutants.

The Table summarizes Taqman qPCR assays of Ll.LtrB intron retrohoming into a chromosomal rhlE target site in mutant strains. Keio deletion, non-temperature-sensitive mutants, and their parental wild-type strains containing donor plasmid pBL1-rhlE were grown to mid-log phase at 37°C and then intron expression was induced with 4 mM m-toluic acid for 1 h at 37°C. Five temperature-sensitive mutants (dnaEts, gyrBts, ligAts, rpoHts, and ssbts) that could not be grown at 37°C and their parental wild-type strains were grown at 30°C and then shifted to 37°C for 1 h prior to a 1-h induction with 4 mM m-toluic acid at 37°C. The priA deletion strain was grown and induced at 30°C and was confirmed by sequencing to lack second-site suppressor mutations in dnaC, which are known to accumulate in PriA mutants [72]. Taqman qPCR was carried out on extracted DNA to determine the number of 5′- and 3′-integration junctions relative to the number of rhlE genes (see Figure 2B and Materials and Methods). Values are the mean ± S.E.M. for three experimental replicates normalized to the retrohoming frequency of the parental wild-type strain assayed in parallel. Mutants that showed decreased retrohoming frequencies were assayed at least three times. Retrohoming frequencies of parental wild-type control strains (for the mutants indicated in parentheses) expressed as percent of available rhlE targets sites were: BW25113 (Keio deletion strains) 34%; AB1157 (dnaEts) 20%; N2603 (ligAts) 40%; BW30384 (polAexts) 25%; DG76 (dnaBts, dnaCts, and dnaGts) 56%; KL921 (ssbts) 40%; PR100 (pnpts) 31%; SS996 (ΔpriB, lexA) 60%; EJ1261(gyrBts) 30%; KY1445(rpoHts) 30%.tsTemperature sensitive.†Genes in which mutations decreased retrohoming frequencies in published genetic screens [12], [22], [30].#Genes also identified as contributing to retrohoming in the transposon-insertions screen using the TpR-RAM assay in this work. Retrohoming efficiencies in the TpR-RAM assay relative to the wild-type control were: gyrB, 1.1%; recJ, 18.9%: rpoH, 3.4%; and yjjB (upstream gene in operon with dnaC and dnaT), 0% (Table S1).aThe lexA mutant is lexA51::Tn5 in the SS996 strain background and has a constitutively induced SOS response.bThe sbcC mutant was not deficient in retrohoming in Taqman qPCR assays of retrohoming at 30°C.