Two-step PCR mediated Red recombination technique for rapid deletion of Yersinia pestis sRNA and large chromosomal fragment
(2.State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China 100071)
【Abstract】[Objective] Based on the λ Red recombination system, a two-step PCR method was developed to delete small non-coding RNA (sRNA) and large chromosomal fragment in Yersinia pestis. [Methods] Two PCR procedures were done to amplify product formed of a kanamycin resistance gene flanked by long (600–1 000 bp) homology arms. The PCR fragment carrying a kanamycin resistance gene flanked by regions homologous to the target locus was electroporated into a recipient 201 strain of Yersinia pestis expressing the homologous recombination system encoded by plasmid pKD46, which promoted the replacement of the target gene with kanamycin resistance fragment. Finally, the recombinant clones were identified by PCR. [Results] The homologous extensions of 600–1 000 bp were constructed by two PCR method, which increased the efficiency of homologous recombination, the sRNA RyhB1 (108 bp) and RyhB2 (106 bp) and the large chromosomal fragments 47-2 (10.4 kb), 47-3 (21.6 kb), 47-3a (9.2 kb) and 47-3b (6.1 kb) were successfully deleted. [Conclusion] The two-step PCR mutation technique was a simple and efficient method for the precise modification of sRNA and large fragment chromosome of Yersinia pestis. This method was suitable for gene knockout of the whole genome of Yersinia pestis, which provided a powerful tool for gene expression and regulation, pathogenicity and virulence study of Yersinia pestis.
【Keywords】 Yersinia pestis; Red recombination system; sRNA; large chromosomal fragments; gene knockout;
(Translated by SONG GJ)
 Waters LS, Storz G. Regulatory RNAs in bacteria. Cell, 2009, 136 (4): 615–628.
 Babski J, Maier LK, Heyer R, Jaschinski K, Prasse D, Jäger D, Randau L, Schmitz RA, Marchfelder A, Soppa J. Small regulatory RNAs in Archaea. RNA Biology, 2014, 11 (5): 484–493.
 Papenfort K, Vanderpool CK. Target activation by regulatory RNAs in bacteria. FEMS Microbiology Reviews, 2015, 39 (3): 362–378.
 Deng ZL, Meng XR, Su SC, Liu ZZ, Ji XL, Zhang YQ, Zhao XN, Wang XY, Yang RF, Han YP. Two sRNA RyhB homologs from Yersinia pestis biovar microtus expressed in vivo have differential Hfq-dependent stability. Research in Microbiology, 2012, 163 (6/7): 413–418.
 Deng ZL, Liu ZZ, Bi YJ, Wang XY, Zhou DS, Yang RF, Han YP. Rapid degradation of Hfq-free RyhB in Yersinia pestis by PNPase independent of putative ribonucleolytic complexes. BioMed Research International, 2014 (2): 798918.
 Yan YF, Su SC, Meng XR, Ji XL, Qu Y, Liu ZZ, Wang XY, Cui YJ, Deng ZL, Zhou DS, Jiang WC, Yang RF, Han YP. Determination of sRNA expressions by RNA-seq in Yersinia pestis grown in vitro and during infection. PLoS One, 2013, 8 (9): e74495.
 Liu ZZ, Wang HL, Wang HD, Wang J, Bi YJ, Wang XY, Yang RF, Han YP. Intrinsic plasmids influence MicF-mediated translational repression of ompF in Yersinia pestis. Frontiers in Microbiology, 2015, 6: 862.
 Liu ZZ, Gao XF, Wang HD, Fang HH, Yan YF, Liu L, Chen R, Zhou DS, Yang RF, Han YP. Plasmid pPCP1-derived sRNA HmsA promotes biofilm formation of Yersinia pestis. BMCMicrobiology, 2016, 16: 176.
 Jiang WY, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 2013, 31 (3): 233–239.
 Mali P, Yang LH, Esvelt KM, Aach J, Guell M, Di Carlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science, 2013, 339 (6121): 823–826.
 Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157 (6): 1262–1278.
 Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nature Methods, 2013, 10 (10): 957–963.
 Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97 (12): 6640–6645.
 Husseiny MI, Hensel M. Rapid method for the construction of Salmonella enterica serovar Typhimurium vaccine carrier strains. Infection and Immunity, 2005, 73 (3): 1598–1605.
 Janes BK, Pomposiello PJ, Perez-Matos A, Najarian DJ, Goss TJ, Bender RA. Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes. Journal of Bacteriology, 2001, 183 (8): 2709–2714.
 Lesic B, Rahme LG. Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa. BMC Molecular Biology, 2008, 9: 20.
 Yamamoto S, Izumiya H, Morita M, Arakawa E, Watanabe H. Application of λ Red recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes. Gene, 2009, 438 (1/2): 57–64.
 Derbise A, Lesic B, Dacheux D, Ghigo JM, Carniel E. A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunology & Medical Microbiology, 2003, 38 (2): 113–116.
 Ge TD, Feng EL, Yan BJ, Wang HL, Huang LY. Construction of deletion mutant of Shigella flexneri acid resistance genes. Letters in Biotechnology, 2005, 16 (5): 488–491 (in Chinese).
 Deng ZL, Liu ZZ, He JM, Wang J, Yan YF, Wang XY, Cui YJ, Bi YJ, Du ZM, Song YJ, Yang RF, Han YP. TyrR, the regulator of aromatic amino acid metabolism, is required for mice infection of Yersinia pestis. Frontiers in Microbiology, 2015, 6: 110.