CLONING AND CHARACTERIZATION OF THE AZURIN ISO-1 GENE, CONCERNED WITHTHE ELECTRON-TRANSPORT CHAIN INVOLVED IN METHYLAMINE METHANOL OXIDATION IN THE OBLIGATE METHYLOTROPH METHYLOMONAS SP. STRAIN-J/

Two azurin-type blue copper proteins, which is concerned with the elec tron transport chain involved in methylamine/methanol oxidation, have been found in the obligate methylotroph Methylomonas sp. strain J. The azurin iso-l gene was cloned and sequenced to analyze the role in the electron transport chain. PCR products synthesized with primers based on the N- and C-terminal amino acid sequences of azurin iso-l were us ed as probes for cloning. One complete open reading frame (the azurin iso-l gene) and one partial orf (orfl) were found in a cloned Eco105I- HindIII fragment, pMAZ3, with a total of 1066 bp. The gene encoded 148 amino acid residues. The amino acid sequence after Ala-21, deduced fr om the nucleotide sequence, was identical to that of the azurin iso-l protein. The gene was in a region separate from the mau gene cluster i n the chromosome. Escherichia coli expressed azurin iso-l. The results of northern blotting analysis suggested that expression of the azurin iso-l gene is regulated by a complex regulatory network controlling o xidation of methylamine or methanol in this strain; for example, coppe r ions affected the expression of the azurin iso-l gene.