Counterselection System for Geobacillus kaustophilus HTA426 through Disruption ofpyrFandpyrR

Abstract

ABSTRACTCounterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system forGeobacillus kaustophilusHTA426, established through simultaneous disruption of thepyrFandpyrRgenes. ThepyrFgene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented withpyrFwas prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, thepyrRgene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrFΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented withpyrFwas prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested thatpyrFcould serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed inG. kaustophilusand conferred new functions on the thermophile. This report describes the first establishment of apyrF-based counterselection system in aBacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression inG. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.