Elsevier

Gene

Volume 470, Issues 1–2, 1 January 2011, Pages 31-36
Gene

An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose

https://doi.org/10.1016/j.gene.2010.09.005Get rights and content

Abstract

Counterselectable markers are powerful tools in genetics because they allow selection for loss of a genetic marker rather than its presence. In mycobacteria, a widely used counterselectable marker is the gene encoding levan sucrase (sacB), which confers sensitivity to sucrose, but frequent spontaneous inactivation complicates its use. Here we show that the Escherichia coli galactokinase gene (galK) can be used as a counterselectable marker in both Mycobacterium smegmatis and Mycobacterium tuberculosis. Expression of E. coli galK, but not the putative M. tuberculosis galK, conferred sensitivity to 2-deoxy-galactose (2-DOG) in both M. smegmatis and M. tuberculosis. We tested the utility of E. coli galK as a counterselectable marker in mycobacterial recombination, both alone and in combination with sacB. We found that 0.5% 2-DOG effectively selected recombinants that had lost the galK marker with the ratio of galK loss/galK mutational inactivation of approximately 1:4. When we combined galK and sacB as dual counterselectable markers and selected for dual marker loss on 0.2% 2-DOG/5% sucrose, 98.6–100% of sucrose/2-DOG resistant clones had undergone recombination, indicating that the frequency of mutational inactivation of both markers was lower than the recombination frequency. These results establish a new counterselectable marker system for use in mycobacteria that can shorten the time to generate unmarked mutations in M. smegmatis and M. tuberculosis.

Introduction

Counterselectable markers, i.e. genes whose presence can be selected against, are a powerful tool in molecular microbiology and cell biology. Uses of counterselectable markers in genetics include selection of recombination products, selection for plasmid loss, and as reporter genes (Reyrat et al., 1998). Despite advances in recent years in techniques for genetic manipulation of mycobacteria, several deficiencies still remain. Although several reliable methods are available to disrupt chromosomal genes with markers conferring antimicrobial resistance, allelic exchange of unmarked deletion mutations or amino acid substitution alleles is still laborious. One frequently used method for construction of unmarked mutations utilizes a two step strategy in which resolution of a tandem chromosomal duplication (produced through recombination of a suicide plasmid at the locus of interest) is selected through loss of a counterselectable marker. Two markers have been widely used for this purpose: rpsL and sacB. rpsL has been useful, but requires a background strain that is streptomycin resistant, limiting its general utility (Sander et al., 1995). sacB, which confers susceptibility to sucrose (Pelicic et al., 1996a), has proven extremely useful in the genetic manipulations mentioned earlier (Pelicic et al., 1996b, Pavelka and Jacobs, 1999). However, sacB has a frequency of spontaneous inactivating mutations that is higher than that of the desired targeted recombination event. In published use of sacB in Mycobacterium smegmatis recombination, 60–70% of sucrose resistant cells are due to sacB inactivation rather than recombination (Pavelka and Jacobs, 1999). Additional counterselectable markers are therefore needed, either with a lower spontaneous inactivation rate, or for combined use with sacB.

The Escherichia coli (E. coli) galK gene (galK), encoding the enzyme galactokinase, catalyzes the phosphorylation of galactose to galactose-1-phosphate. It also efficiently phosphorylates a galactose analogue, 2-deoxy-galactose (2-DOG). The product of this reaction, 2-deoxy-galactose-1-phosphate, cannot be further metabolized, leading to buildup of toxic levels and cell death. Thus, the constitutive expression of galK in E. coli leads to sensitivity to 2-DOG, but is otherwise nontoxic to the cell, a property that has been used for counterselection in E. coli (Alper and Ames, 1975, Ueki et al., 1996, Warming et al., 2005). The small size of the galK gene is also advantageous in PCR amplification and other genetic manipulations. We therefore attempted to adapt galK for use as a counterselectable marker in mycobacteria, specifically M. smegmatis and Mycobacterium tuberculosis.

Section snippets

Strains and media

M. smegmatis mc2155 was grown in liquid 7H9 media, supplemented with 0.05% Tween 80, 0.5% glycerol and 0.5% dextrose. For solid media, 7H10 was used with same additives but without Tween. M. tuberculosis (Erdman) was grown on 7H10 plates with 10% OADC, 0.5% glycerol. When needed, 2-deoxy-galactose (2-DOG) (Sigma Aldrich, D4407) was added at 0.2 or 0.5%. 5% sucrose was added when needed. Antibiotics used were kanamycin 40 μg/ml for E. coli or 20 μg/ml for mycobacteria, and hygromycin B at 150 μg/ml

Expression of E. coli, but not M. tuberculosis, galK confers sensitivity to 0.2% 2-DOG in M. smegmatis and M. tuberculosis

Although mycobacteria have a native galK gene (Rv0620, MSmeg_3692), it is under tight regulation and requires glutamate for induction (Raychaudhuri et al., 1998). We postulated that constitutive expression of mycobacterial galK might render the bacteria 2-DOG sensitive. To test this idea, we expressed Rv0620 from the constitutive MOP promoter from the attB site on the M. smegmatis chromosome. Importantly, pDB58 has an attP site, by which it can be integrated as a single copy into the chromosome

Discussion

Counterselectable markers are useful tools in molecular microbiology. We show here that expression of E. coli galK confers sensitivity to 2-DOG in fast and slow growing mycobacteria. Until now, Bacillus subtilis sacB has been the most commonly used counterselectable marker in mycobacteria. Although extremely useful, it has the disadvantage of a spontaneous mutation rate that is higher than the rate of rare recombination events, necessitating laborious counterscreening to distinguish genetic

Acknowledgments

This work was supported by NIH grant AI53417 to MSG, NIH grant AI075805 to CLS, and Michael and Ethel L. Cohen Foundation to DB. The authors would like to thank Zully Feliciano for assistance in preparation of the manuscript.

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Present address: Department of Molecular Microbiology, Washington University School of Medicine, Campus Box 8230, 660 S. Euclid Avenue, St. Louis, MO 63110, USA. Tel.: + 1 314 286 0276.

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