title: Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle.
abstract: 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. In addition to Thermus and Micrococcus, sensitivity to BCI substrate cleavage was observed for other Gram-negative and Gram-positive species belonging to various bacterial phyla, including representatives of the genera Staphylococcus, Bacillus, Corynebacterium, Rhodococcus, Paracoccus and Xanthomonas. Thus, the toxicity of indoxyl derivative accumulation upon BCI substrate cleavage can be used for selection purposes in a broad range of microorganisms.
authors.affiliation: Lehrstuhl für Mikrobiologie, Technische Universität München, Emil-Ramann-Straße 4, D-85354 Freising-Weihenstephan, Germany.
authors.forename: Angel
authors.initials: A
authors.lastname: Angelov
authors.forename: Haijuan
authors.initials: H
authors.lastname: Li
authors.forename: Andreas
authors.initials: A
authors.lastname: Geissler
authors.forename: Benedikt
authors.initials: B
authors.lastname: Leis
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liebl
mesh_terms: D003470:Culture Media / Q000737:chemistry; D005827:Genetics, Microbial / Q000379:methods*; D007211:Indoles / Q000378:metabolism* / Q000633:toxicity*; D050296:Microbial Viability / Q000187:drug effects; D016982:Micrococcus luteus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D012641:Selection, Genetic; D016963:Thermus thermophilus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D003470:Culture Media; D007211:Indoles; C034082:indoxyl
keywords: Clean deletion; Counterselection; Indoxyl toxicity