Optimization of the 5-bromo-2′-deoxyuridine selection and its application for the isolation of nitrate respiration-deficient mutants of Haloferax volcanii

Abstract

The 5-bromo-2′-deoxyuridine (BrdU) selection is a negative selection procedure for the isolation of `loss of function' mutants. The principle is similar to the penicillin selection, which cannot be used with penicillin-resistant species, e.g. the whole domain of Archaea. The BrdU selection was described previously as a three-step procedure for the isolation of phototrophically negative mutants of the archaeon Halobacterium spec. GRB, with a 10 2-fold enrichment of mutants in each round (Soppa, J., Oesterhelt, D. 1989. J. Biol. Chem. 264, 13 043–13 046). To optimize the procedure, a model system was developed making use of arginine fermentative growth and two different H. spec. GRA mutants. Optimization of different parameters resulted in a more than 10-fold increase in efficiency of the selection. The optimized BrdU selection was applied for the isolation of nitrate respiration-deficient mutants of Haloferax volcanii after UV mutagenesis of the wildtype. Two rounds of the BrdU selection yielded cell mixtures that contained between 3 and 11% mutants. Mutants of three different phenotypes were selected, i.e. mutants unable to grow via nitrate respiration, mutants with a reduced growth rate, and mutants with a late onset of growth. The data underscore that the BrdU selection is a general method for the isolation of `loss of function' mutants in haloarchaea.