Cloning and identification of a two-component signal-transducing regulatory system from Bacteroides fragilis.

A DNA fragment was cloned from Bacteroides fragilis that bestowed low-level tetracycline resistance to Escherichia coli strains harbouring the cloned fragment on a multicopy plasmid. The tetracycline resistance determinant was localized to a 4.3kb Bg/II-PstI subfragment of the original clone. DNA sequence analysis of this fragment revealed that it contained an operon encoding two proteins: one of 519 amino acids, RprX, and a second of 236 amino acids, RprY. Protein sequence analysis revealed that the two proteins shared sequence identity with a family of multicomponent signal-transducing regulatory proteins identified from many diverse bacterial genera. RprX shared identity with the first component of the regulatory system, the histidine protein kinase receptor (for example EnvZ, PhoR, CheA, and VirA). RprY shared identity with the second member of the regulatory protein pair, the regulatory response protein (for example OmpR, PhoB, CheY, and VirG). Expression of these proteins from a multicopy plasmid vector in E. coli resulted in a decrease in the level of the outer membrane porin protein OmpF and an increase in the level of the outer membrane porin protein OmpC. The decrease in OmpF levels correlates with, and may be the cause of, the increased tetracycline resistance. Regulation of the levels of OmpF and OmpC is normally controlled by a multicomponent signal-transducing regulatory pair of proteins, EnvZ and OmpR. The effect RprX and RprY have on OmpF expression is mediated at the level of transcription. Thus, RprX and RprY may be interfering with the normal regulation of OmpF by OmpR and EnvZ.