Marinomonas mediterranea MMB-1 transposon mutagenesis: Isolation of a multipotent polyphenol oxidase mutant

Marinomonas mediterranea is a melanogenic marine bacterium expressing a mul tifunctional polyphenol oxidase (PPO) able to oxidize substrates characteri stic for laccases and tyrosinases, as well as produce a classical tyrosinas e. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO ac tivity. Transposon mutagenesis has been applied for the first time to M. me diterranea by using different minitransposons loaded in R6K-based suicide d elivery vectors mobilizable by conjugation. Higher frequencies of insertion s were obtained by using mini-Tn10 derivatives encoding kanamycin or gentam ycin resistance. After applying this protocol, a multifunctional PPO-negati ve mutant was obtained. By using the antibiotic resistance cassette as a ma rker, flanking regions were cloned. Then the wild-type gene was amplified b y PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper binding sit es of other blue copper proteins, including fungal laccases. In addition, i t shows some extra copper-binding sites that might be related to its multip otent enzymatic capability.