Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants

WANG Chuhan, MA Peng-jiao, LUO Tao, BAO Lang

Abstract

To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53.   Methods   The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants.  Results   The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully.   Conclusion   pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

 

Keywords: Plasmid optimization, Mycobacterium smegmatis, Mutant screening, Plasmid shedding, Rifampin resistance

 

 

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COURT D L, SAWITZKE J A, THOMASON L C. Genetic engineering using homologous recombination. Annu Rev Genet .2002, 36:361-388[2018-11-27]. https;//doi. org/10. 1146/annurev. genet. 36. 061102. 093104.

VAN KESSEL J C, HATFULL G F. Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering; characterization of antimycobacterial drug targets. Mol Microbiol,2008,67(5); 1094-1107.

HATFULL G F, PEDULLA M L, JACOBS-SERA D, et al. Exploring the mycobacteriophage metaproteome; phage genomics as an educational platform. PLoS Genet, 2006, 2 (6): e92[2018-12-04]. https://doi.org/10.1371/journal. pgen. 0020092.

VAN KESSEL J C, HATFULL G F. Recombineering in Mycobacterium tuberculosis. Nat Methods, 2007, 4 ( 2 ) : 147- 152.

VAN KESSEL J C, HATFULL G F. Mycobacterial recombineering. Methods Mol Biol, 2008, 435; 203-215 [ 2018-12-04 ]. https://link. springer, com/protocol/10.1007%2F978-1-59745-232-8_ 15. doi: 10.1007/978-1-59745- 232-8_ 15.

VAN KESSEL J C, MARINELLI L J, HATFULL G F. Recombineering mycobacteria and their phages. Nat Rev Microbiol, 2008,6(11); 851 -857.

HUANG L, YUAN Z, LIU P. et al. Effects of promoter leakage on dynamics of gene expression. BMC Syst Biol, 2015,9:16[2018-12-04]. https://doi. org/10. 1186/sl2918- 015-0157-z.

RAJU A, KULKARNI S, RAY M K. et al. E84G mutation in dihydrofolate reductase from drug resistant strains of Mycobacterium tuberculosis ( Mumbai, India ) leads to increased interaction with Trimethoprim. Int J Mycobacteriol,2015,4(2);97-103.

NEBENZAHL-GUIMARAES H, JACOBSON К R. FARHAT M R. el at. Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis. J Antimicrob Chemother, 2014,69(2):331-342.

KHAN M Z, BHASKAR A, UPADHYAY S, et al. Protein kinase G confers survival advantage to Mycobacterium tuberculosis during latency-like conditions. J Biol Chem, 2017,292(39):16093-16108.

HATFULL G F. Mycobacteriophages. Microbiol Spectr, 2018,6(5) [2018-12-04]. http;//www. asmscience. org/ content/journal microbiolspec, 10. 1128/microbiolspec. GPP 3-0026-2018. doi; 10. 1128/microbiolspec. GPP3-0026-2018.

PELICIC V, REYRAT J M, GICQUEL B. Expression of the Bacillus subtil is sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol, 1996,178(4) ; 1197-1199.

JACKSON M, REINALDO CAMACHO L, GICQUEL B, et al. Gene replacement and transposon delivery using the negative selection marker sacB. Methods Mol Med,2001,54 ; 59-75 [2018-12-05]. https://doi.org/10. 1385/1-59259-147- 7:059.

MENDEZ-LORENZO L, PORRAS-DOM1NGUEZ J R, RAGA-CARBAJAL E, et al. Intrinsic Levanase activity of Bacillus subtilis 168 levansucrase (SacB). PLoS One,2015, 10( 11):е0143394[2018-12-05]. https://doi.org/10. 1371/ journal, pone. 0143394.

SATYAGAL V N, AGRAWAL P. Modeling the behavior of plasmid-harboring cells in nonselective media. Biotechnol Bloeng,1989.34(2):265-272.

MILLER L P. CRAWFORD J T, SHINNICK T M. The rpoB gene of Mycobacterium tuberculosis. Antimicrob Agents Chemother , 1 994,38(4) : 805-811.

HORNG Y T, JENG W Y, CHEN Y Y, et al. Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin. Antimicrob Agents Chemother,2015,59(3); 1542-1548.

SRIVASTAVA G, TRIPATHI S, KUMAR A, et at. Molecular insight into multiple RpoB clinical mutants of Mycobacterium tuberculosis, an attempt to probe structural variations in rifampicin binding site underlying drug resistance. Int J Biol Macromol,2018,120(Pt B) :2200-2214.

JAMIESON F B, GUTHRIE J L, NEEMUCHWALA A, et al. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis. J Clin Microbiol. 2014,52(6):2157-2162.

MACIAG A, DAINESE E. RODRIGUEZ G M, et al. Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J Bacteriol,2007,189(3):730-740.

SERAFINI A, BOLDRIN F. PALU G, et al. Characterization of a Mycobacterium tuberculosis ESX-3 conditional mutant; essentiality and rescue by iron and zinc. J Bacteriol,2009,191(20): 6340-6344.


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