Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei.
Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZalpha gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P(BAD)-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Deltaasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.
López
Carolina M
CM
Department of Microbiology, Immunology and Pathology, Colorado State University, IDRC at Foothills Campus, Campus Delivery 2025, Fort Collins, CO 80523-2025, USA.
Rholl
Drew A
DA
Trunck
Lily A
LA
Schweizer
Herbert P
HP
D000483:Alleles; D001483:Base Sequence; D016957:Burkholderia pseudomallei / Q000235:genetics* / Q000472:pathogenicity; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005821:Genetic Techniques; D006801:Humans; D008554:Melioidosis / Q000382:microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial
J Bacteriol. 2007 Dec;189(23):8746-9
17905994
Appl Environ Microbiol. 2009 Oct;75(19):6062-75
19648360
Infect Immun. 2006 Mar;74(3):1949-53
16495572
Trans R Soc Trop Med Hyg. 2008 Dec;102 Suppl 1:S1-4
19121666
J Microbiol Methods. 2009 Mar;76(3):320-3
19150470
Appl Environ Microbiol. 2008 Feb;74(4):1064-75
18156318
J Bacteriol. 1997 Jan;179(2):538-40
8990308
Clin Microbiol Rev. 2005 Apr;18(2):383-416
15831829
Antimicrob Agents Chemother. 2006 Oct;50(10):3460-3
17005832
Antimicrob Agents Chemother. 1999 Mar;43(3):465-70
10049252
Int J Syst Bacteriol. 1998 Jan;48 Pt 1:317-20
9542103
Appl Environ Microbiol. 2008 Jul;74(14):4498-508
18502918
Appl Environ Microbiol. 2008 May;74(10):2985-9
18310423
J Bacteriol. 1997 Apr;179(7):2116-25
9079894
Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14240-5
15377794
Environ Microbiol. 2008 Jun;10(6):1652-60
18341581
FEBS Lett. 1994 Jul 18;348(3):311-6
8034061
J Bacteriol. 1999 Sep;181(17):5498-504
10464226
Biotechniques. 2006 Feb;40(2):191-7
16526409
Gene. 2009 Feb 1;430(1-2):123-31
19010402
Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10191-6
10963681
Nucleic Acids Res. 1990 Mar 25;18(6):1407-13
2183191
Appl Environ Microbiol. 2008 Dec;74(24):7529-35
18952878
J Bacteriol. 1988 Jun;170(6):2575-83
2836362
Nat Rev Microbiol. 2006 Apr;4(4):272-82
16541135
Methods Enzymol. 1994;235:458-65
8057916
Nucleic Acids Res. 1999 Nov 15;27(22):4409-15
10536150
Appl Microbiol Biotechnol. 2005 Nov;69(2):151-61
15843930
Gene. 1995 May 26;158(1):15-22
7789804
Emerg Infect Dis. 2008 Nov;14(11):1689-92
18976550