Purification and Characterization of Regulatory Factors of the Mercury Resistance Determinants From TnMERI1

• Main Authors: Ching-Yi Chen, 陳靖怡
• Other Authors: Chieh-Chen Huang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/64206166999259330225

碩士 === 國立中興大學 === 生命科學系 === 91 === Bacillus megaterium strain MB1 of Gram-positive bacteria confers a broad-spectrum mercury resistance was isolated from sediment of Minamata Bay, Japan. The mercury resistance module is encoded in a chromosomal class II transposon, TnMERI1. The complete genetic structure of the mercury resistance module is O/PmerB3-merB3-O/PmerR1- merR1-merE-like-merT-merP-merA-O/PmerR2-merR2-merB2-merB1. The mer operon differs from that of Gram-negative bacteria by not only containing three proposed operator/promoter regions (O/PmerB3, O/PmerR1 and O/PmerR2) and two regulatory genes (merR1 and merR2) but also three organomercurial lyase genes (merB3, merB2 and merB1). Both of regulatory genes were expressed in E. coli by using a T7 RNA polymerase/promoter expression system (pET system). And the proteins were then purified by an affinity chromatography with Heparin-Sepharose column. MerR1 could be eluted with the NaCl concentration around 0.9 M while MerR2 could be eluted with the NaCl concentration around 0.5 M. Gel retardation assay showed that both MerR1 and MerR2 could bind the three proposed operator/promoter regions, but how the Hg2+ ion could affect the binding properties of the regulators need forward study.