cumA , a Gene Encoding a Multicopper Oxidase, Is Involved in Mn 2+ Oxidation in Pseudomonas putida GB-1

ABSTRACT Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn 2+ . Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA ) encoding a protein homologous to multicopper oxidases. Addition of Cu 2+ increased the Mn 2+ -oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu 2+ . A second open reading frame (designated cumB ) is located downstream from cumA . Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum . A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB . This was confirmed by site-specific gene replacement in cumB . The cumB mutation did not affect the Mn 2+ -oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn 2+ and that CumB is required for optimal growth of P. putida GB-1-002.