Characterization of a novel MIIA domain-containing protein (MdcE) in Bradyrhizobium spp.

Abstract

Several genes coding for proteins with metal ion-inducible autocleavage (MIIA) domains were identified in type III secretion system tts gene clusters from draft genomes of recently isolated Bradyrhizobium spp. MIIA domains have been first described in the effectors NopE1 and NopE2 of Bradyrhizobium diazoefficiens USDA 110. All identified genes are preceded by tts box promoter motifs. The identified proteins contain one or two MIIA domains. A phylogenetic analysis of 35 MIIA domain sequences from 16 Bradyrhizobium strains revealed four groups. The protein from Bradyrhizobium sp. LmjC strain contains a single MIIA domain and was designated MdcE (MdcE_LmjC). It was expressed as a fusion to maltose-binding protein (MalE) in E. coli and subsequently purified by affinity chromatography. Recombinant MalE-MdcE_LmjC-Strep protein exhibited autocleavage in the presence of Ca²⁺, Cu²⁺, Cd²⁺ and Mn²⁺, but not by Mg²⁺, Ni²⁺ or Co²⁺. Site-directed mutagenesis at the predicted cleavage site abolished autocleavage activity of MdcE_LmjC. An LmjC mdcE⁻ mutant was impaired in the ability to nodulate Lupinus angustifolius and Macroptilium atropurpureum.