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Applied and Environmental Microbiology, April 1999, p. 1762-1768, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

cumA, a Gene Encoding a Multicopper Oxidase, Is Involved in Mn2+ Oxidation in Pseudomonas putida GB-1

Geert-Jan Brouwers,1,* Johannes P. M. de Vrind,1 Paul L. A. M. Corstjens,1 Pierre Cornelis,2 Christine Baysse,2 and Elisabeth W. de Vrind-de Jong1

Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 R.A. Leiden, The Netherlands1 and Flanders Interuniversity Institute of Biotechnology, Department of Immunology, Paracytology and Ultrastructure, Vrije Universiteit Brussel, B-1640 Sint Genesius Rode, Belgium2

Received 21 August 1998/Accepted 15 January 1999

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 (designated cumA) encoding a protein homologous to multicopper 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 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 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.

* Corresponding author. Mailing address: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 R.A. Leiden, The Netherlands. Phone: 31 71 5274707. Fax: 31 71 5274537. E-mail: brouwers{at}chem.leidenuniv.nl.

Applied and Environmental Microbiology, April 1999, p. 1762-1768, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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