Fig 1.
Deletion frequency between repeated sequences depends on the genetic context.
A. Schematic representation of the genetic recombination assay. The 5’ and 3’ parts of the tetA gene, containing 438 bp repeats (black boxes) separated by an intervening sequence of 1,479 bp encoding a spc gene conferring resistance to spectinomycin, were introduced into the dispensable amyE gene locus. Recombination between the repeats leads to the tetA gene reconstitution conferring tetracycline resistance to bacteria. B. Medians of [TetR] frequencies in WT (GY12949), ΔrecA (GY15184), ΔradA (GY12956), ΔrecAΔradA (GY12971), ΔrecO (GY12963), ΔrecF (GY12955), ΔuvrD (GY12953), ΔddrB (GY16016), ΔddrB ΔrecA (GY16628), and ΔddrB ΔrecO (GY16638) mutant strains are represented by Tukey box plots. Outliers are represented by open circles. Medians were calculated from 12 to 53 independent values (except for medians calculated from at least 4 independent values in ΔradA and ΔrecA ΔradA bacteria). Statistically significant differences in the medians of recombination frequencies of the mutants, compared to those observed in strain GY12949, were calculated using the non-parametric Dunn's multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; ns if P > 0.05.
Fig 2.
Recombination between chromosomal and plasmid DNA is RecA- and RecF-dependent.
A. Schematic representation of the recombination assay between chromosomal and plasmid DNA. The 5’tetA and the 3’tetA regions of the tetA gene, containing 438 bp repeats, were introduced into the chromosomal dispensable amyE gene and into the p11554 plasmid giving rise to plasmid p15002, respectively. One crossing over between the two 438 bp repeated sequences (black boxes) leads to the reconstitution of a functional tetA gene and the integration of the plasmid into chromosomal DNA. B. Medians of [TetR] frequencies in WT (GY15147), ΔrecA (GY15158), ΔrecF (GY15160), ΔradA (GY15149), and ΔuvrD (GY15156) bacteria, all containing the p15002 plasmid, are calculated from at least 3 independent values and represented by Tukey boxplots. Outliers are represented by open circles. The small arrows attached to the horizontal line representing the upper limit of detectable [TetR] frequencies indicate that [TetR] frequencies were < 5 10−7 for ΔrecA and ΔrecF bacteria.
Fig 3.
Effect of base pair changes in the repeats on their recombination frequencies.
A. Schematic representation of the recombination substrates containing 1 or 3 bp changes. The positions of the base pair changes calculated from the initiation codon in the 5’tetA region are indicated. B. Medians of [TetR] frequencies calculated from 14 to 43 independent values in WT (GY12949), ΔrecA (GY15184), ΔmutS (GY12978), ΔmutS ΔrecA (GY16620) bacteria containing identical repeated sequences, WT (GY12950), ΔrecA (GY16624), ΔmutS (GY12980), ΔmutS ΔrecA (GY16618) containing one base difference in the repeated sequences, and WT (GY12948), ΔrecA (GY16622), ΔmutS (GY12979), ΔmutS ΔrecA (GY16616) containing 3 base differences in the repeated sequences, are represented by Tukey boxplots. Outliers are represented by open circles. Statistically significant differences in the medians of recombination frequencies of the mutants and WT containing sequence differences in the repeated sequence compared to GY12949 were calculated using the non-parametric Dunn's multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; ns if P > 0.05.
Fig 4.
Impaired growth and stationary-phase lethality of recombination-deficient mutant cells.
GY9613 (WT) (black diamonds), GY13915 (ΔddrB) (grey squares), GY15125 (ΔrecO) (green triangles), GY12968 (ΔrecA) (blue squares), GY16626 (ΔddrB ΔrecA) (red squares and interrupted lines), GY16636 (ΔddrB ΔrecO) (orange circles) were grown from independent colonies at 30°C to an OD650nm = 0.1 (time 0 of the growth curves). A. OD650nm as a function of time. B. Colony forming units as a function of time. C. Colony forming units as a function of OD650nm.
Fig 5.
Deletion frequencies between repeated sequences separated by 3,500, 6,500, and 10,500 bp.
A. Schematic representation of the constructions used. B., C., D. Bacteria contain 3,500 bp (panel B), 6,500 bp (panel C), and 10,500 bp (panel D) intervening sequences between the tetA repeats. The medians of [TetR] frequencies calculated from 10 to 35 independent values in the tested strains are represented by Tukey boxplots. Outliers are represented by open circles. Statistically significant differences in the medians of recombination frequencies of the mutants compared to the WT GY16209, GY16227, and GY16235 in panel B, C, and D, respectively, were calculated using the non-parametric Dunn's multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; (ns) if P > 0.05. B. WT (GY16209), ΔrecA (GY16238), ΔrecO (GY16262), ΔrecF (GY16264), ΔuvrD (GY16608), ΔddrB (GY16268), ΔddrB ΔrecA (GY16630), ΔddrB ΔrecO (GY16640) C. WT (GY16227), ΔrecA (GY16244), ΔrecO (GY16278), ΔrecF (GY16276), ΔuvrD (GY16612), ΔddrB (GY16282), ΔddrB ΔrecA (GY16632), ΔddrB ΔrecO (GY16642) D. WT (GY16235), ΔrecA (GY16252), ΔrecO (GY16290), ΔrecF (GY16292), ΔuvrD (GY16614), ΔddrB (GY16296), ΔddrB ΔrecA (GY16634), ΔddrB ΔrecO (GY16644).
Fig 6.
γ-irradiation induced recombination between repeated sequences.
A. Induction of recombination between repeated sequences separated by 1,479 bp as a function of the radiation dose. [TetR] frequencies were measured in at least 5 independent cultures after 20 hours of post irradiation incubation of GY12949 in TGY medium after exposure to different γ-irradiation doses. B. Induction by exposure to γ-irradiation of recombination between repeats separated by intervening sequences of increasing length: 1,479 bp (GY12949), 3,500 bp (GY16209), 6,500 bp (GY16227) and 10,500 bp (GY16235). C. γ-promoted induction of recombination between overlapping sequences separated by 1,479 bp in WT (GY12949), ΔddrB (GY16016), and ΔuvrD (GY12953) bacteria. D. γ-promoted induction of recombination between overlapping sequences separated by 10,500 bp in WT (GY16235), ΔddrB(GY16296), ΔuvrD(GY16614). B., C., D. Medians of the [TetR] frequencies calculated from 5 to 30 independent values are represented by Tukey boxplots. Outliers were represented by open circles. Statistically significant differences in the medians of recombination frequencies between irradiated and the corresponding non-irradiated bacteria were calculated using the non-parametric Mann-Withney test: * P < 0.05; ** P < 0.01; *** P < 0.001; (ns) if P> 0.05. NI: non-irradiated bacteria. IR: irradiated bacteria.
Table 1.
Bacterial strains and plasmids.