ArticleApplied and environmental microbiology2009
Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei.
Article in Applied and environmental microbiology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 128 papers.
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Who cites it
128 citing papers in PubMed, 186 citations in OpenAlex.
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- A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.Applied biochemistry and biotechnology · 2024Article
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68 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
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.
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