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 (dnaE^ts, gyrB^ts, ligA^ts, rpoH^ts, and ssb^ts) 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 (dnaE^ts) 20%; N2603 (ligA^ts) 40%; BW30384 (polAex^ts) 25%; DG76 (dnaB^ts, dnaC^ts, and dnaG^ts) 56%; KL921 (ssb^ts) 40%; PR100 (pnp^ts) 31%; SS996 (ΔpriB, lexA) 60%; EJ1261(gyrB^ts) 30%; KY1445(rpoH^ts) 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 Tp^R-RAM assay in this work. Retrohoming efficiencies in the Tp^R-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.