Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle

In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141.