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 Mn²⁺. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designatedcumA) encoding a protein homologous to multicopper oxidases. Addition of Cu²⁺ increased the Mn²⁺-oxidizing activity of the P. putidawild 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 reading frame (designatedcumB) is located downstream from cumA. BothcumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 ofBradyrhizobium japonicum. A mutation in orf74resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumAmutant, 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 Mn²⁺-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 Mn²⁺ and that CumB is required for optimal growth of P. putida GB-1-002.