Studies on gene ARR expression

[摘要] ABSTRACTRifampicin is a major chemotherapeutic agent used against mycobacterial andnocardial infections. High level resistance is primarily due to mutationalalterations in the rpoB gene encoding the β subunit of RNA polymerase. Whenchallenged, these bacteria may inactivate rifampicin by one of four mechanisms:decomposition, ADP-ribosylation, glucosylation and phosphorylation. ADPribosylationoccurs in many mycobacterial pathogens but nothing is known aboutthe properties of the enzyme responsible. Consequently mutational analysis maybe used to explore structure-function relationships in this protein.Three mutants with changes in the open reading frame were selected and studied.The altered arr gene in pMG1 was obtained by in vivo selection whilst in pMG2and pMG4 by in vitro mutagenesis. The mutated arr gene in pMG1 and pMG2conferred resistance to 50 μg/ml of rifampicin while in pMG4 to 200 μg/ml. Thissuggested that alterations near the N-terminus resulted in lowered activity becauseof closer proximity to the active site. This is the first successful report of inducedarr gene expression. This over-expression of the Arr ADP-ribosyltransferase andits mutants assisted in their later purification by metal affinity chromatography.

[发布机构] University of the Witwatersrand