Table 1. Taqman qPCR assays of retrohoming in notable E. coli mutants.
Gene | Function | Retrohoming frequency (% WT) | |
5′ Junction | 3′ Junction | ||
dinB † | DNA polymerase IV | 125±4% | 127±3% |
dnaB ts | Replicative DNA helicase | 41±13% | 77±3% |
dnaC ts , # | Replication/restart initiation | 21±1% | 10±1% |
dnaE ts , † | DNA polymerase III, α-subunit | 27±4% | 47±9% |
dnaG ts | DNA primase | 36±5% | 26±7% |
dnaQ † | DNA polymerase III, ε-subunit | 135±5% | 128±5% |
dnaT # | Primosomal protein I | 39±3% | 67±3% |
gyrB ts , # | DNA gyrase, subunit B | 14±8% | 26±10% |
hns † | Histone-like nucleoid structuring protein | 20±5% | 16±5% |
lexA a | Transcriptional repressor of SOS | 87±13% | 81±13% |
ligA ts , † | DNA ligase | 113±1% | 101±3% |
ligB | DNA ligase | 122±4% | 102±4% |
mnmE † | tRNA modification | 128±12% | 109±11% |
pnp † | Polynucleotide phosphorylase | 88±4% | 102±1% |
polAex ts , † | DNA polymerase I | 38±5% | 50±6% |
polB † | DNA polymerase II | 98±9% | 84±7% |
priA | Primosomal protein N′ | 52±2% | 54±1% |
priB | Primosomal protein N | 92±1% | 79±2% |
priC | Primosomal protein N″ | 35±3% | 23±3% |
recF † | ss/dsDNA binding protein | 125±6% | 111±4% |
recJ † | 5′→3′ exonuclease | 112±2% | 104±5% |
recQ † | ATP-dependent DNA helicase | 115±7% | 90±2% |
rep † | ATP-dependent DNA helicase | 167±5% | 99±5% |
rnhA † | RNase HI | 11±1% | 15±2% |
rnhB † | RNase HII | 106±3% | 79±2% |
rpoH # | RNA polymerase σ32 (σH) factor | 14±4% | 11±2% |
sbcC † , b | ATP-dependent dsDNA exonuclease | 36±3% | 31±1% |
sbcD † | ATP-dependent dsDNA exonuclease | 101±7% | 103±8% |
seqA † | Inhibitor of replication initiation | 51±8% | 44±7% |
ssb ts | ssDNA binding protein | 37±3% | 41±1% |
stpA † | H-NS-like DNA/RNA-binding protein | 146±3% | 123±5% |
tus † | Inhibition of replication at Ter sites | 39±8% | 31±9% |
umuC † | DNA polymerase V | 119±3% | 117±7% |
umuD † | DNA polymerase V | 142±5% | 121±4% |
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%.
Temperature 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).
The lexA mutant is lexA51::Tn5 in the SS996 strain background and has a constitutively induced SOS response.
The sbcC mutant was not deficient in retrohoming in Taqman qPCR assays of retrohoming at 30°C.