Pseudomonas aeruginosa MutL promotes large chromosomal deletions through non-homologous end joining to prevent bacteriophage predation

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen with a relatively large genome, and has been shown to routinely lose genomic fragments during environmental selection. However, the underlying molecular mechanisms that promote chromosomal deletion are still poorly understood. In a recent study, we showed that by deleting a large chromosomal fragment containing two closely situated genes, hmgA and galU, P. aeruginosa was able to form 'brown mutants', bacteriophage (phage) resistant mutants with a brown color phenotype. In this study, we show that the brown mutants occur at a frequency of 227 ± 87 × 10-8 and contain a deletion ranging from ∼200 to ∼620 kb. By screening P. aeruginosa transposon mutants, we identified mutL gene whose mutation constrained the emergence of phage-resistant brown mutants. Moreover, the P. aeruginosa MutL (PaMutL) nicking activity can result in DNA double strand break (DSB), which is then repaired by non-homologous end joining (NHEJ), leading to chromosomal deletions. Thus, we reported a noncanonical function of PaMutL that promotes chromosomal deletions through NHEJ to prevent phage predation.

Figure 1.

Characteristics of deletion mutants selected by phage infection. (A) Brown mutants are selected after phage PaoP5 infection. (B) Phage-resistant mutant frequencies in total occur at 1083 ± 524 × 10⁻⁸. The brown mutants were detected with a frequency of 227 ± 87 ×10⁻⁸. The data was calculated from five biological replicates. (C) Both the hmgA and galU genes were deleted in the brown mutants, as was detected by PCR. (D) Deletion sites and deleted genes in all the brown mutants. (E) Locations of deletions in the chromosomes of nine brown mutants compared with wild-type PAO1. (F) Close-up of E, indicating the deleted chromosomal regions of the nine brown mutants. The locations of the hmgA and galU genes are indicated.

Figure 2.

MutL is required for large chromosomal deletions in P. aeruginosa. (A) Representative pictures of the phage-resistant mutants of PAO1, ΔmutL, ΔmutL::mutL and PAO1::mutL. Many more white mutants were generated in the ΔmutL background, and most mutants generated from the mutL overexpression strain were brown mutants. (B) The frequency of brown mutants and white mutants for each strain selected by phage. No brown mutants were detected from ΔmutL, while overexpression of mutL significantly increased the frequency of brown mutants to 1468 ± 427 × 10⁻⁸. (P < 0.05, calculated by one way ANOVA). (C) Genomic organization of PAO1::sacB. Two copies of sacB were inserted into the PAO1 genome immediately downstream from galU. Thus, PAO1::sacB is sensitive to both phage and 5% sucrose selection. (D) The frequency of brown mutants and white mutants for each strain selected by phage and 5% sucrose. Inserting two copies of the sacB gene significantly decreased the rate of white mutants after selection with both phage and 5% sucrose. More than 10⁸ΔmutL::sacB cells were subjected to selection by phage and sucrose without any brown mutants being detected. Each experiment was repeated five times. The asterisks mark P-value of < 0.05 as calculated by Student's t-test.

Figure 3.

Nicking activity of PaMutL results in DSB and is essential for chromosomal deletions. (A) Confirmation that the nicking function of PaMutL can results in DSB in vitro. Linear DNA was observed from PaMutL (500nM) digested plasmid DNA, but NTD (500 nM) did not cleave dsDNA. SC, supercoiled; L, linearized; N, nicked. (B) DSB made by PaMutL is not site-specific. After incubation of plasmid pUCP24 with PaMutL (200 nM) or NTD (200 nM), the DNA was linearized with EcoRI. The products were analyzed by gel electrophoresis after ethidium bromide staining. (C) Key residues for PaMutL nicking function. (D) A20V,G97S, N37H, R474A and NTD mutants cannot restore the brown mutants, nor could decrease the frequency of white mutants in mutL::sacB.

Figure 4.

NHEJ promotes chromosomal deletions while HR inhibits it. (A) Knockout of LigD and Ku significantly decreased the frequency of brown mutants, while complementation of LigD or Ku increased the deletion frequency to 1947 ± 420 × 10⁻⁸ and 558 ± 205 × 10⁻⁸, respectively (P < 0.05, calculated by one way ANOVA). (B) Knockout of recA and recB significantly increased the frequency of brown mutants to 11192 ± 9569 × 10⁻⁸ and 3823 ± 2898 × 10⁻⁸, respectively (P < 0.05, calculated by one way ANOVA).

Figure 5.

Model of the mechanism by which MutL makes DSB and results in a DNA fragment deletion through NHEJ that protects P. aeruginosa from phage infection. PaMutL generates DSB occasionally. The end points of DSB are then joined by Ku and LigD. Brown mutants with a deleted a galU/hmgA region are identified after phage infection.