Utilization of the existing mutant libraries to construct specific mutants in Escherichia coli strains

Onderzoeksoutput: Editorial

Samenvatting

Since the importance of mutants in determining the cellular role of specific gene products, numerous mutant libraries have been established in E. coli. It would be very useful if interesting mutations in one strain could be conveniently moved to other strains, such as from a cystitis isolate to a pyelonephritis isolate of uropathogenic E. coli (UPEC). The traditional method to transfer mutation between bacteria is transduction mediated by phages, in particular P1 [Masters (1996) in Ecosal: Escherichia coli and Salmonella, 2nd Edition, available online http://ecosal.org/generalized-transduction.html]. However, transducing phages are not available for all strains. In addition, the length of the transduced region can be much larger than the intended mutation. In contrast to transduction, the Red recombination system of phage Lambda provides an efficient and precise way to introduce specific mutations into the bacterial genome, especially in E. coli K-12 strains.
In this study, we extended the method established by Datsenko and Wanner [Datsenko & Wanner (2000) Proc Natl Acad Sci USA 97: 6640-6645] by amplifying the linear fragments containing an antibiotic resistance gene from existing mutants. Then we compared the efficiency of P1 transduction and Red-mediated recombination from transferring an existing Delta-phoA::km mutation from the E. coli K-12 strain MG1655 to the UPEC strain UTI89. In addition, the Delta-nadC::km mutation from Keio Collection [Baba et al. (2006) Mol Syst Biol 2: 2006.0008] was successfully transferred from E. coli MG1655 Delta-nadC::km to UTI89Nad+. Also, mutations in the degP and yohJ genes, in a home-made transposon insertion mutant library derived from UTI89 Delta-phoA, were transferred to the acute pyelonephritis isolate CFT073 and the acute cystitis isolate UTI89. Comparing the bladder and kidney colonization data from UTI89degP and CFT073degP mutants [Redford & Welch (2006) Infect Immun 74: 4030-4038] in two mouse lines, we confirmed that DegP has a similar function in both bacteria, while the mouse strain has a strong influence on the colonization ability of UPEC strains. From the morphology of the colonies of yohJ mutants in different backgrounds, we concluded that the mucoid morphology resulted from an unknown spontaneous mutation, that was not transferred by the Red method, and not from the yohJ mutation.
From the above application, the Lambda Red recombination system can be used widely in moving of alleles between strains. The short and controlled length of the transferred DNA segment minimizes the risk of simultaneously transferring unknown additional mutations or polymorphisms between strains.
Originele taal-2English
Pagina's (van-tot)51-51
Aantal pagina's1
TijdschriftAbstract book, Belgian Society for Microbiology, Annual Meeting 2011, Life, Death and Survival of Micro-organisms, Brussels, Nov. 16, 2011
StatusPublished - 16 nov 2011

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