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Applied and Environmental Microbiology, November 2005, p. 6808-6815, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6808-6815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

Diana Hernández-Romero,1 Francisco Solano,2 and Antonio Sanchez-Amat1*

Department of Genetics and Microbiology,1 Department of Biochemistry and Molecular Biology B, University of Murcia, 30100 Murcia, Spain2

Received 20 March 2005/ Accepted 19 July 2005

Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to L-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.

* Corresponding author. Mailing address: Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain. Phone: 34 968364955. Fax: 34 968363963. E-mail: antonio{at}um.es.

Applied and Environmental Microbiology, November 2005, p. 6808-6815, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6808-6815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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