Development of an Sce-I mutagenesis system for Burkholderia cepacia complex strains.

The Burkholderia cepacia complex (Bcc) consists of at least 20 phenotypically similar but genotypically distinct Gram-negative bacteria that are ubiquitous in nature, are capable of promoting plant growth and biodegradation of pollutants, but that also are highly antibiotic resistant and produce damaging effects towards plants, fungi, and humans. To study these genetically recalcitrant bacteria in detail, molecular tools are required that work efficiently with the many strains and species of the Bcc. One mutagenesis strategy that has been used effectively to analyze the genes of Burkholderia cenocepacia is based upon the activity of the Sce-I restriction enzyme. Unfortunately, this system is limited in its applicability to many members of the Bcc. Therefore, we undertook the expansion of this system to create an Sce-I mutagenesis system that could be used with many different species and strains of the Bcc, including members of the B. cenocepacia IIIB Midwest clones. We demonstrated the use of this system by clean-deleting the lipo-oligosaccharide (LOS) inner core biosynthesis gene waaC, to create a B. cenocepacia PC184 strain variant with truncated LOS. This enhanced mutagenesis system can be used to analyze a wide range of Burkholderia and other Gram-negative bacteria.