Identification and molecular analysis of the Leptothrix discophora SS-1 mofA gene, a gene putatively encoding a manganese-oxidizing protein with copper domains

A small amount of a manganese-oxidizing protein was purified from spent culture medium of Leptothrix discophora strain SS-1 and used to raise antibodies (alpha MOF). Expression libraries of L. discophora were constructed in lambda gt11 and screened with alpha MOF. DNA inserts from the resulting alpha MOF-positive lambda gt11 clones were used to isolate the corresponding gene (called mofA) from a genomic library. The mofA gene was mapped in the center of a NcoI-EcoRI restriction fragment 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. Transcription 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 open readingframe of 4986 bp preceded by a potential promoter region and 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 termination site. Part of a second open readingframe (ORF) was found instead. 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 similarity to bilirubin oxidase from Myrothecium verrucaria and phenoxazinone synthase from Streptomyces antibioticus. These homologous domains contain the consensus sequences presumed to code for copper domains.