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

Shen, Mengyu, Zhang, Huidong, Shen, Wei, Zou, Zhenyu, Lu, Shuguang, Li, Gang, He, Xuesong, Agnello, Melissa, Shi, Wenyuan, Hu, Fuquan, Le, Shuai·2018·Nucleic Acids Research
DOI 10.1093/nar/gky160PMC PMC5961081
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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 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.CC BY-NC · PubMed Central
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 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.CC BY-NC · PubMed Central
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 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.CC BY-NC · PubMed Central
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 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).CC BY-NC · PubMed Central
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.
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.CC BY-NC · PubMed Central

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Metadata

Authors
Shen, M., Zhang, H., Shen, W., Zou, Z., Lu, S., Li, G., He, X., Agnello, M., Shi, W., Hu, F., & Le, S.
Affiliations
  • Department of Microbiology, Third Military Medical University, Chongqing 400038, China
  • Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China
  • Department of Medical Laboratory, Chengdu Military General Hospital, Chengdu 610083, China
  • The Forsyth Institute, 245 First St, Cambridge, MA 02142, USA
  • School of Dentistry, University of California, Los Angeles, CA 90095, USA
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2018-03-05
Volume
46
Issue
9
Pages
4505-4514
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gky160
PMC
PMC5961081
Cited by
76
References
2
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • National Natural Science Foundation of China (31501004, 31570173)
  • National Key Research and Development Program (2017YFC1002002)
  • NIH (1R01DE023810, 1R01DE026186)

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