cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidationin Pseudomonas putida GB-1

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide seq uence analysis of the transposon insertion site of a nonoxidizing mutant re vealed a gene (designated cumA) encoding a protein homologous to multicoppe r oxidases. Addition of Cu2+ increased the Mn2+-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 Cu2+. A second open rea ding 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 mut ation did not affect the Mn2+-oxidizing ability of the organism but resulte d in decreased growth. In summary, our data indicate that the multicopper o xidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.