E. W. de Vrind-de Jong

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 (αMOF). Expression libraries of L. discophora were constructed in λgtll and screened with αMOF. DNA inserts from the resulting αMOF‐positive λgt11 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 reading frame of 4986 bp preceded by a potential promoter region and a ribosome binding site. The translation product, consisting of ...

Stimulation of Mn2+ Oxidation in Leptothrix discophora SS-1 by Cu2+ and Sequence Analysis of the Region Flanking the Gene Encoding Putative Multicopper Oxidase MofA

The effect of Cu2+ on the Mn2+-oxidizing activity secreted by Leptothrix discophora strain SS-1 was studied. Cu2+ stimulated the activity of spent medium from stationary cultures when added during growth of the bacteria, although cell yield decreased with increasing Cu2+ concentrations. The stimulation was tentatively explained by involvement of the putative multicopper oxidase MofA in Mn2+ oxidation. Cu2+ did not enhance the activity when added directly to spent medium, possibly because the ions had to be incorporated during synthesis of the oxidizing factor. Sequence analysis of the region downstream from the mofA gene resulted in identification of mofB, which encodes a protein with a potential peptidyl-prolyl-cis-trans isomerase site, and mofC, which encodes a protein with a potential heme-binding site. The possibility that mofA, mofB, and mofC belong to one operon, and the possible functions of MofB and MofC, are discussed.The effect of Cu2+ on the Mn2+-oxidizing activity secreted by Leptothrix discophora strain SS-1 was studied. Cu2+ stimulated the activity of spent medium from stationary cultures when added during growth of the bacteria, although cell yield decreased with increasing Cu2+ concentrations. The stimulation was tentatively explained by involvement of the putative multicopper oxidase MofA in Mn2+ oxidation. Cu2+ did not enhance the activity when added directly to spent medium, possibly because the ions had to be incorporated during synthesis of the oxidizing factor. Sequence analysis of the region downstream from the mofA gene resulted in identification of mofB, which encodes a protein with a potential peptidyl-prolyl-cis-trans isomerase site, and mofC, which encodes a protein with a potential heme-binding site. The possibility that mofA, mofB, and mofC belong to one operon, and the possible functions of MofB and MofC, are discussed.