Identification of an operon involved in tyrosinase activity and melanin synthesis in Marinomonas mediterranea

The genomic region of Marinomonas mediterranea containing the genes required for tyrosinase activity and melanin synthesis has been cloned by marker rescue using the-transposon-generated, amelanogenic strain T105. Five OR-Fs, two incomplete and three complete, have been sequenced in the genomic region where the transposon was inserted. RT-PCR analysis indicates that ORF 3, coding for tyrosinase, and ORF4, coding for a protein of 250 amino acids, are in the same transcriptional unit, constituting an operon whose promoter region has been determined by 5'-RACE. This operon has been sequenced in the wild-type and several mutant strains, indicating that both ORFs are required for expression of tyrosinase activity and melanin synthesis. The nitrosoguanidine generated, amelanogenic mutant ng56, shows a nonsense mutation in ORT3 coding for the tyrosinase. On the other hand, in the strain T105 the transposon is inserted in ORF4. The product of this gene is related to copper metabolism, since the addition of this metal ion to cell extracts or culture media partially restores melanin synthesis and tyrosinase activity in the strain T105. However, it does not show significant sequence similarity to previously characterized metallochaperones and hence may be an example of a new kind of those proteins. The operon has been denoted as ppoB, taking into consideration that ppoA denotes the M. mediterranea gene coding for the previously cloned polyphenol oxidase with laccase activity. This is the first demonstration of the tyrosinase gene forming part of an operon in a Gram-negative bacterium. (C) 2004 Elsevier B.V. All rights reserved.