IDENTIFICATION AND MOLECULAR ANALYSIS OF THE LEPTOTHRIX DISCOPHORA SS-1 MOFA GENE, A GENE PUTATIVELY ENCODING A MANGANESE-OXIDIZING PROTEINWITH COPPER DOMAINS

A small amount of a manganese-oxidizing protein was purified from spen t culture medium of Leptothrix discophora strain SS-1 and used to rais e antibodies (alpha MOF). Expression libraries of L. discophora were c onstructed in lambda gt11 and screened with alpha MOF. DNA inserts fro m the resulting alpha MOF-positive lambda gt11 clones were used to iso late the corresponding gene (called mofA) from a genomic library. The mofA gene was mapped in the center of a NcoI-EcoRI restriction fragmen t of 7262 bp. This fragment was cloned in a low-copy broad-host vector . The resulting plasmid (pGBM31) was used in an in vitro transcription -translation experiment with an Escherichia coli S30 extract. Transcri ption of the mofA gene could be initiated on its own putative promoter region. Translation resulted in a protein of approximately 180 kD as determined by gel electrophoresis. DNA sequence analysis revealed an o pen readingframe of 4986 bp preceded by a potential promoter region an d a ribosome binding site. The translation product, consisting of 1662 amino acids (174.3 kD) contains an N-terminal signal peptide. The DNA sequence downstream from the mofA gene revealed no transcription term ination site. Part of a second open readingframe (ORF) was found inste ad. The translation product of this ORF (called mofB) also contains an N-terminal signal peptide. Comparison of the primary structure of the mofA encoded protein with other proteins revealed domains with simila rity to bilirubin oxidase from Myrothecium verrucaria and phenoxazinon e synthase from Streptomyces antibioticus. These homologous domains co ntain the consensus sequences presumed to code for copper domains.