Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

Abstract

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.

Keywords: AddAB; DNA repair; PrimPol; Thermus thermophilus; bacterial transformation; comet assay.

FIGURE 1

Schematic overview of all strains tested in this work. Outline of the genomic context of ppol and addAB in Thermus thermophilus HB27. Individual genes are indicated as well as the genomic coordinates of the ends. Integrity of the topA gene is also shown. Mutations generated are designated in white with dashed arrows and major resultant compensatory mutations are in black with dashed squares. The order of construction among strains is indicated with solid arrows. Strains presented are HB27 (Wild type), ppol:kat [knockout of ppol by insertion of a kanamycin-resistant marker (kat) through homologous recombination], ppol_cat [removal of primase-polymerases (Ppol) catalytical activity by the generation of D70A and D72A residues substitution with CaldoCas9 system], ppol:lox72 (markerless deletion of ppol through Cre–lox deletion system), ppol_comp (restoration of ppol in ppol:lox72 strain with CaldoCas9 system), addAB:kat (addAB knockout by kat insertion through homologous recombination). The gene denominations as well as genomic coordinates for HB27 chromosome are shown.

FIGURE 2

Transformation efficiency of Thermus thermophilus mutants. (A) Hundred nanogram of a replicative plasmid (pMotH1103A) or an homologous-recombination integrative linear DNA fragment conferring resistance to Hyg (pyrEH), were used to transform Tth HB27pyrEK control strain and ppol:kat and addAB:kat insertion mutant. (B) Hundred nanogram of a replicative plasmid (pMotK1103A) or an homologous-recombination integrative linear DNA fragment (pyrEK) conferring resistance to Kn, were used to transform Tth HB27 cells and its derivatives ppol:lox72, ppol_cat, ppol_comp, addAB:lox72, and addAB_ppol mutants. (C) Viable cells per mL of the kat-marked transformed strains in Kn plates. (D) Viable cells per mL of the markerless transformed strains in TB plates. (E) Serial dilutions of transformed cells of the indicated strains plated on Kn (upper panel) or non-selective plates (lower panel) and incubated for 48 h at 65°C. Transformation is represented as the number of colonies on selection plates at 65°C, relative to the number of viable colonies on non-selective plates. Transformation frequencies were calculated as an average of at least five biological replicates. Error bars correspond to the standard deviation of the means. Asterisks indicate statistically different values observed in mutant strains compared to those in the corresponding control strains (*P-value <0.005 and **P-value <0.0001).

FIGURE 3

Resistance to DNA damaging agents in the Thermus thermophilus mutants. (A) The HB27pyrEK control strain and the ppol:kat and addAB:kat insertion mutants were subjected to different DNA damaging agents. The cell cultures were incubated at 65°C until OD₆₀₀ of 0.3 (3 × 10⁸ cell/ml) was reached. Then, cells were further incubated for 2 h with the indicated concentrations of the different DNA damaging agents: peroxide hydrogen (H₂O₂), 4-Nitroquinoline-N-oxide (4-NQO) and Bleomycin (Bleo). Then, 10 μl of serial decimal dilutions were drop-inoculated on non-selective plates that were incubated at 65°C for 48 h. Fraction of surviving cells following DNA damage (4-NQO, left. Bleo, medium. H₂O₂, right) is calculated relative to an untreated control. (B) The HB27 control strain and its derivatives ppol:lox72, ppol_cat, ppol_comp, addAB:lox72, and addAB_ppol mutants were subjected to the same DNA damaging agents used in panel (A). Each data point is an average of at least five biological replicates. Error bars correspond to the standard deviation of the means.

FIGURE 4

DNA integrity analysis in Thermus thermophilus mutants. Representative images of control and mutant strains analyzed by neutral comet assay and stained with GelRed^®. The whole extent of the comet tail relative to the head provides qualitative information about the amount of DNA lesions (A) HB27. (B) ppol::kat. (C) addAB::kat. (D) ppol_cat. (E) ppol::lox72. (F) ppol_comp. In the HB27 cultures, the majority of the cells were intact showing little or no DNA migration (fluorescence limited to nucleoids). On the other hand, in the mutants DNA leaked out from the nucleoid indicating the presence of strand fragmentation. The different halos and migration patterns are indicative of the amount of DNA damage. Cells with extensive lesions had almost all DNA in the tail due to migration from the nucleoid.

FIGURE 5

Plasmid stability in the Thermus. thermophilus mutants. (A) Comparison of plasmid loss frequencies using a replicative plasmid conferring Hyg resistance (pMotH1103A) in HB27pyrEK control strain and in ppol:kat and addAB:kat insertion mutant strains. Transformed cells were re-streaked twice on selective plates and grown on selective media liquid for 24 h. After that, cells were further incubated for another 48 h in the absence of antibiotic to allow plasmid loss to occur. We finally plated cells on both non-selective and selective plates and the plasmid loss frequency was measured as the CFUs on selective media divided by CFUs on non-selective plates (total cells). (B) Comparison of plasmid loss frequencies using a replicative plasmid conferring Kn resistance (pMotK1103A) in HB27 control strain and its derivatives ppol:lox72, ppol_cat, ppol_comp, addAB:lox72 and addAB_ppol mutants. Each data point is an average of 3 independent experiments. Error bars correspond to the standard deviation of the means. Asterisks indicate significant statistical differences (**P-value <0.0002 when comparing the mean of addAB:lox72 with the mean of addAB_ppol).