Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

  • Gomaa, Ahmed E. (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Deng, Zhiping (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Zhimin (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Shang, Liguo (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Zhan, Yuhua (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Lu, Wei (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Lin, Min (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yan, Yongliang (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
  • Received : 2016.08.09
  • Accepted : 2016.10.28
  • Published : 2017.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

Keywords

References

  1. Aagot N, Nybroe O, Nielsen P, Johnsen K. 2001. An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media. Appl. Environ. Microbiol. 67: 5233-5239. https://doi.org/10.1128/AEM.67.11.5233-5239.2001
  2. Garrido-Sanz D, Meier-Kolthoff JP, Göker M, Martín M, Rivilla R, Redondo-Nieto M. 2016 Genomic and genetic diversity within the Pseudomonas fluorescens complex. PLoS One 11: e0150183. https://doi.org/10.1371/journal.pone.0150183
  3. Liu P, Jenkins NA, Copeland NG. 2003. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13: 476-484. https://doi.org/10.1101/gr.749203
  4. Lalucat J, Bennasar A, Bosch R, Garcia-Valdes E, Palleroni NJ. 2006. Biology of Pseudomonas stutzeri. Microbiol Mol. Biol. Rev. 702: 510-547.
  5. Christie-Oleza JA, Nogales B, Martin-Cardona C, Lanfranconi MP, Alberti S, Lalucat J, Bosch R. 2008. ISPst9, an ISL3-like insertion sequence from Pseudomonas stutzeri AN10 involved in catabolic gene inactivation. Int. Microbiol. 11: 101-110.
  6. White AK, Metcalf WW. 2004. The htx and ptx operons of Pseudomonas stutzeri WM88 are new members of the Pho regulon. J. Bacteriol. 186: 5876-5882. https://doi.org/10.1128/JB.186.17.5876-5882.2004
  7. Heurlier K, Denervaud V, Pessi G, Reimmann C, Haas D. 2003. Negative control of quorum sensing by RpoN (${\sigma}^{54}$) in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185: 2227-2235. https://doi.org/10.1128/JB.185.7.2227-2235.2003
  8. Mulet M, David Z, Nogales B, Bosch R, Lalucat J, Garcia-Valdes E. 2011. Pseudomonas diversity in crude-oilcontaminated intertidal sand samples obtained after the prestige oil spill. Appl. Environ. Microbiol. 77: 1076-1085. https://doi.org/10.1128/AEM.01741-10
  9. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. 2013. Repurposing CRISPR as an RNAguided platform for sequence-specific control of gene expression. Cell 152: 1173-1183. https://doi.org/10.1016/j.cell.2013.02.022
  10. Clark ST, Diaz Caballero J, Cheang M, Coburn B, Wang PW, Donaldson SL, et al. 2015. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis. Sci. Rep. 5: 10932. https://doi.org/10.1038/srep10932
  11. Hauberg-Lotte L, Klingenberg H, Scharf C, Böhm M, Plessl J, Friedrich F, et al. 2012. Environmental factors affecting the expression of pilAB as well as the proteome and transcriptome of the grass endophyte Azoarcus sp. strain BH72. PLoS One 7: e30421. https://doi.org/10.1371/journal.pone.0030421
  12. Hayrapetyan H, Tempelaars M, Nierop Groot M, Abee T. 2015. Bacillus cereus ATCC 14579 RpoN (Sigma 54) is a pleiotropic regulator of growth, carbohydrate metabolism, motility, biofilm formation and toxin production. PLoS One 10: e0134872. https://doi.org/10.1371/journal.pone.0134872
  13. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821. https://doi.org/10.1126/science.1225829
  14. Mauchline TH, Chedom-Fotso D, Chandra G, Samuels T, Greenaway N, Backhaus A, et al. 2015. An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota. Environ. Microbiol. 17: 4764-4778. https://doi.org/10.1111/1462-2920.13038
  15. Poole K. 2012. Bacterial stress responses as determinants of antimicrobial resistance. J. Antimicrob. Chemother. 67: 2069-2089. https://doi.org/10.1093/jac/dks196
  16. Wang P, Yu Z, Li B, Cai X, Zeng Z, Chen X, Wang X. 2015. Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas. Microb. Cell Fact. 14: 11. https://doi.org/10.1186/s12934-015-0194-8
  17. Wang Y, Zhang Y, Jiang T, Meng J, Sheng B, Yang C, et al. 2015. A novel biocatalyst for efficient production of 2-oxocarboxylates using glycerol as the cost-effective carbon source. Biotechnol. Biofuels 8: 186. https://doi.org/10.1186/s13068-015-0368-y
  18. Yan Y, Ping S, Peng J, Han Y, Li L, Yang J, et al. 2010. Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501. BMC Genomics 11: 11. https://doi.org/10.1186/1471-2164-11-11
  19. Damron FH, Owings JP, Okkotsu Y, Varga JJ, Schurr JR, Goldberg JB, et al. 2012. Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN. J. Bacteriol. 194: 1317-1330. https://doi.org/10.1128/JB.06105-11
  20. Lapage SP, Hill LR, Reeve JD. 1968. Pseudomonas stutzeri in pathological material. J. Med. Microbiol. 1: 195-202. https://doi.org/10.1099/00222615-1-2-195
  21. You CB, Lin M, Fang XJ, Song W. 1995. Attachment of Alcaligenes to rice roots. Soil Biol. Biochem. 27: 463-466. https://doi.org/10.1016/0038-0717(95)98620-4
  22. Nicolay T, Devleeschouwer K, Vanderleyden J, Spaepen S. 2012. Characterization of esterase A, a Pseudomonas stutzeri A15 autotransporter. Appl. Environ. Microbiol. 78: 2533-2542. https://doi.org/10.1128/AEM.07690-11
  23. Aranda J, Poza M, Pardo BG, Rumbo S, Rumbo C, Parreira JR, et al. 2010. A rapid and simple method for constructing stable mutants of Acinetobacter baumannii. BMC Microbiol. 10: 279. https://doi.org/10.1186/1471-2180-10-279
  24. Boyle NR, Reynolds TS, Evans R, Lynch M, Gill RT. 2013. Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing. Biotechnol. J. 5: 515-522.
  25. Gao C, Wang Y, Zhang Y, Lv M, Dou P, Xu P, Ma C. 2015. NAD-independent L-lactate dehydrogenase required for L-lactate utilization in Pseudomonas stutzeri A1501. J. Bacteriol. 197: 2239-2247. https://doi.org/10.1128/JB.00017-15
  26. Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP. 2012. Integration of DNA into bacterial chromosomes from plasmids without a counterselection marker. Nucleic Acids Res. 408: e59.
  27. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 2013. RNA-guided editing of bacterial genomes using CRISPRCas systems. Nat. Biotechnol. 31: 233-239. https://doi.org/10.1038/nbt.2508
  28. Katzen F, Becker A, Ielmini MV, Oddo CG, Ielpi L. 1999. New mobilizable vectors suitable for gene replacement in gram-negative bacteria and their use in mapping of the 3' end of the Xanthomonas campestris pv. campestris gum operon. Appl. Environ. Microbiol. 65: 278-282.
  29. Newman JR, Fuqua C. 1999. Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227: 197-203. https://doi.org/10.1016/S0378-1119(98)00601-5
  30. Yan Y, Yang J, Dou Y, Chen M, Ping S, Peng J, et al. 2008. Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc. Natl. Acad. Sci. USA 105: 7564-7569. https://doi.org/10.1073/pnas.0801093105
  31. Zhang T, Yan Y, He S, Ping S, Alam KM, Han Y, et al. 2012. Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501. Res. Microbiol. 163: 332-339. https://doi.org/10.1016/j.resmic.2012.05.002
  32. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, et al. 2014. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33: 73-80. https://doi.org/10.1038/nbt.3081
  33. Wang Y, Lv M, Zhang Y, Xiao X, Jiang T, Zhang W, et al. 2014. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production. Sci. Rep. 4: 6939.
  34. Ohba H, Satoh K, Yanagisawa T, Narumi I. 2005. The radiation responsive promoter of the Deinococcus radiodurans pprA gene. Gene 363: 133-141. https://doi.org/10.1016/j.gene.2005.07.035
  35. Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, et al. 2014. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9: e98186. https://doi.org/10.1371/journal.pone.0098186
  36. Kojic M, Venturi V. 2001. Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator. J. Bacteriol. 183: 3712-3720. https://doi.org/10.1128/JB.183.12.3712-3720.2001
  37. Tremblay J, Richardson A-P, Lepine F, Deziel E. 2007. Selfproduced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour. Environ. Microbiol. 9: 2622-2630. https://doi.org/10.1111/j.1462-2920.2007.01396.x
  38. Faulds-Pain A, Wren BW 2013. Improved bacterial mutagenesis by high-frequency allele exchange, demonstrated in Clostridium difficile and Streptococcus suis. Appl. Environ. Microbiol. 79: 4768-4771. https://doi.org/10.1128/AEM.01195-13
  39. Zhan Y, Yan Y, Deng Z, Chen M, Lu W, Lu C, et al. 2016. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501. Proc. Natl. Acad. Sci. USA 113: E4348-E4356. https://doi.org/10.1073/pnas.1604514113

Cited by

  1. An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species vol.7, pp.1, 2017, https://doi.org/10.1186/s13568-017-0425-y
  2. A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number a vol.84, pp.18, 2017, https://doi.org/10.1128/aem.01167-18