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 recombination events from sacB inactivation.
The catalase gene katG
from M. tuberculosis
can be used as a counterselectable marker when cloned into fast growing mycobacteria such as M. smegmatis
, because its affinity to the drug isoniazid (INH) is approximately 20 times higher than that of the katG
from M. smegmatis
(Dubnau et al., 1996
). However, the katG
gene is large (2.2KB), and therefore less convenient to use in cloning. Also, this technique does not apply to slow growing mycobacteria, unless the katG
gene was previously deleted, which would necessitate working with an INH resistant M. tuberculosis
Mutations in the rpsL
gene, encoding ribosomal protein S12, can confer resistance to streptomycin, and sometimes to other aminoglycosides as well (Bohman et al., 1984
). Complementing such bacteria with rpsL+
copy restores dominant sensitivity to streptomycin, and was used for negative selection in several bacteria, including S. pneumoniae
(Sung et al., 2001
), Streptomyces (Hosted and Baltz, 1997
) and M. smegmatis
(Sander et al., 1995
). However, as is the case for katG
, the use of this method requires working with a clinically-relevant antibiotic resistant strain, in this case streptomycin.
The E. coli galK gene is only 1.3KB long, which makes it easier than katG or sacB for cloning and genetic manipulation. We show here that both in fast and slow growing mycobacteria, selection on 0.2% 2-DOG is highly effective with approximately one background colony for every 105 CFU. For the preparation of deletion mutants in the 2 step allelic exchange method () we show that selection on 0.5% 2-DOG is at least as good, if not better, than use of 5% sucrose in selecting for precise second recombination (as measured by ratio of precise recombination to spontaneous 2-DOG or sucrose resistance). Most important, a strategy combining sacB/sucrose and galK/2-DOG (at 0.2%) was 100% effective in selecting for recombination events. A main drawback of the 2 step allelic exchange system using a single counterselection marker is a rate of spontaneous marker inactivation, leading for a need to re-screen for hygromycin sensitivity. The use of galK with sacB together eliminates the need for the counter screening on hygromycin, thus saving one week and considerable labor when working with M. smegmatis, and potentially at least 4–5 weeks of time and labor when working in M. tuberculosis. Using the galK/sacB system, 2-DOG/sucrose resistant colonies can be directly screened by Southern analysis, as our experiments indicate that nearly 100% of these colonies will be precise recombinants. A potential limitation of galK and 2-DOG use is its price – approximately 50 USD for 1 gram. However, given the strict selection, only a small number of plates needs to be used for every experiment, making the use of 2-DOG worthwhile, especially considering the time and labor saved.
In summary, we have documented the utility of galK/2-DOG as a counterselectable marker system in mycobacteria. This system further expands the available tools for genetic manipulation of mycobacteria.