Fig 1.
Model system for studying recombination events involving the ICR.
1: DNA transfer is mediated by a conjugal mating between an HfrH donor and a F- recipient. Transfer begins at oriT and proceeds in the direction of the nearby ICR. Both strains are ΔrecA to prevent homologous recombination. The donor contains a tetracycline resistance regulon (tetRA) within the ICR. The recipient carries unselected scoreable drug markers within the ICR, and counter-selection markers (resistance to streptomycin, nalidixic acid, and/or chloramphenicol) elsewhere (AbR*). During the mating, leading-strand DNA synthesis starting at oriT in donor displaces the complementary single strand, which is transferred to the recipient, where lagging-strand synthesis copies it. Transfer begins with oriT and moves clockwise through the tetRA-ICR construct. 2: Cells are plated on Tet + Ab* media, selecting for recombinants that have received a stable copy of tetRA in a recipient background. 3: Recombinants are tested for the unselected marker, kanamycin sensitivity, to see if the incorporated tetRA replaced npt). 4: In some experiments, parents and recombinants were analyzed by sequencing: Whole genomes were assembled and annotated, then aligned for identification of 450 segregating variant positions.
Table 1.
Relevant features of the donor and recipient strains in the mating system.
Fig 2.
The ICR region in the standard donors (ER3276 & ER3435), standard recipients (ER3436 & ER3460) and ICR proximal border recipients (ER3472, ER3473, ER3480, & ER3481) (not to scale).
Fig 3.
The Effect of recA and repE on the mating system.
(A) The recombination efficiency per recipient per hour in mating crosses: 1) WT donor (ER3270) X WT recipient (ER1636), 2) ΔrecA donor (ER3276) X WT recipient (ER1636), 3) ΔrecA ΔrepE* donor (ER3435) X WT recipient (ER1636), 4) ΔrecA donor (ER3276) X recA recipient (ER3263), and 5) ΔrecA ΔrepE* donor (ER3435) X ΔrecA recipient (ER3436). See S2 Table for more details on individual crosses. The legend indicates status of recA and repE in the donor and recipient, mating duration, and which mating pair was used in each cross. Recombination efficiency is reduced by deleting recA from either the donor or recipient to eliminate homologous recombination, and with the ΔrepE* deletion in the donor to prevent F’-plasmid replication. All crosses were performed with a minimum of three biological replicates. Error bars represent standard error. (B) The proportion of recombinants that are kanamycin sensitive (KnS), kanamycin resistant (KnR), or UV resistant (UVR) from cross (4) ΔrecA donor X recA13 recipient and from cross (5) ΔrecA ΔrepE* donor X ΔrecA recipient. Most recombinants were KnR when the donor had repE in the leading F DNA because RepE allows for stabilization of donor DNA by plasmid replication (S3 Fig) [34]. In the absence of plasmid formation, crosses resulted primarily in KnS recombinants. In cross (4), UVR recombinants resulted from reversion of the ER3263 recipient’s recA13 point mutation allele [35,36]. To avoid this we deleted recA from recipient ER3436 for cross 5.
Fig 4.
YjiP promotes recA-independent recombination and reduces cell viability.
(A) The frequency of recombination during matings between the ΔrepE* ΔrecA donor and either the ΔrecA recipient (cross 6; ΔrecA) or a ΔrecA recipient with inducible overexpression of yjiP (cross 13; ΔrecA rhaBp-yjiPc), with and without 0.2% rhamnose. Recombination efficiency was calculated as the frequency of recombinant formation per viable recipient per hour in the mating mixture. Inducing yjiPc expression with rhamnose significantly increases recombination efficiency ~5 fold (P-value = 0.018), but rhamnose has no significant effect when the recipient lacks the rhaBp-yjiPc gene fusion. (B) Cell viability over time of the ΔrecA rhaBp-yjiPc recipient (ER3460) grown in 0.2% rhamnose. This experiment was performed three times with biological replicates, but only the results of a single representative trial are shown for clarity. Rhamnose was added at t = 0, and cells were mated after 3 hours of growth with ER3435. Untreated and unmated cells were also included as controls. Rhamnose-induced yjiPc expression reduces cell proliferation for the first three hours after treatment and begins to kill cells afterwards. Mating did not significantly affect cell viability. (C) Dose-response of cell killing: fraction starting titer for three strains at 18 hours as a function of inducer concentration. Strains were ΔrecA (ER3473), recA+ rhaBp-yjiPc (ER3480), and ΔrecA rhaBp-yjiPc (ER3460) grown in various concentrations of rhamnose for 18 hours relative to an untreated control. Higher concentrations of rhamnose are increasingly lethal to ER3460; 0.2% rhamnose kills ~90% of the normally viable cells. All experiments in panels A and C were performed with a minimum of three biological replicates with error bars representing standard error.
Fig 5.
Long Patch Replacements occur in RecA-independent crosses.
We compare (A) the entire genome and (B) the genomic region surrounding the ICR between the ΔrepE* ΔrecA donor (ER3435), the ΔrecA/ΔrecA rhaBp-yjiPc recipients (ER3440/ER3460), and six recombinants produced by mating these strains (Cross 6; uninduced recombinants, and Cross 13; yjiPc-induced recombinants). Each bar represents the genome for a single strain with the color indicating the origin of genomic DNA. See S5 Fig or an explanation of how the parent was identified for each position and the schematic created. Each recombinant exhibits a unique recombination patch that extends beyond the borders of the ICR. In four of the six cases, over 1.5 megabase of contiguous donor DNA replaced the corresponding recipient DNA. All markers are positioned to align with the common genomic backbone (i.e excluding indel variants such as F insertion). Coordinate 1 is placed at the translation start site of dnaA instead of the traditional coordinate #1 upstream of thrA.
Fig 6.
Identification of recombinants that replace the ICR only.
(A) Schematic of the mating configuration designed to allow rapid genetic screening of the proximal ICR border status. In the recipient, A KnR cassette replaced a gene inside the proximal border of the ICR (yjiT::npt) and a CmR cassette replaced a nearby gene outside the proximal border (yjiP::cat). We call these ICR proximal border recipients. With this arrangement, a recombinant with donor DNA confined to the ICR will lose KnR but keep CmR and thus can be quickly identified by antibiotic screening. The embedded table displays the distribution of recombinant phenotypes in matings with different recipient backgrounds. The ΔrecA ΔrepE* donor was crossed with recA+, ΔrecA or ΔrecA yjiPc-rhaBp proximal border recipients (Crosses 7, 8, and 10 respectively); at least 100 recombinants from at least three independent matings of each cross were tested. The proximal crossover yielding complete donor replacement (top configuration) dominated Rec+, with some recombinants showing proximal crossover within the ICR (bottom configuration). In recA, a larger fraction showed proximal crossover within the ICR, and a minority showed proximal crossover within the ICR border region (middle configuration). When YjiPc was overexpressed this distribution did not change greatly. (B) Detecting distal crossover events with PCR and Sanger sequencing. The schematic shows SNP configurations at the distal ICR border. Sanger sequencing of the closest two SNPs distal to the ICR in the ten KnS, CmR recombinants from Cross 8 showed that seven shared the SNPs of the donor, while three (ER3482, ER3483, and ER3510) shared the SNPs of the recipient. For these three, the distal-side limit of the replacement event lies within a ~13 Kb region that includes the downstream edge of the ICR.
Table 2.
Frequencies of recombination events in the conjugal system.