Interactions between Two MerR Regulators and Three Operator/Promoter Regions in the Mercury Resistance Module of Bacillus megaterium

Abstract

The mercury resistance module of Bacillus transposon TnMERI1 is regulated by three operator/promoter regions (O/P_merB3, O/P_merR1, and O/P_merR2) and two regulatory proteins (MerR1 and MerR2) encoded by the module itself. To clarify the roles of MerR1 and MerR2 in the regulatory mechanism, both proteins were overexpressed and purified. MerR1 bound the regulatory regions O/P_merB3 and O/P_merR1, with a preference for O/P_merB3 as measured on in vitro gel shift assays. However, MerR2 bound O/P_merR2, as revealed by gel shift and restriction endonuclease protection assays. The transcriptional start sites of O/P_merB3 and O/P_merR2 were determined by rapid amplification of 5′-cDNA ends (5′-RACE) in the TnMERI1 original host, Bacillus megaterium strain MB1. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays showed that O/P_merB3 and O/P_merR1 were induced in the presence of Hg²⁺ but not O/P_merR2. It was concluded that MerR1 regulates O/P_merB3 and O/P_merR1, while MerR2 regulates O/P_merR2.