Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea Patricia Lucas-Elio a , Pilar Hernandez a , Antonio Sanchez-Amat a , Francisco Solano b, * a Department of Genetics and Microbiology, Faculty of Biology, School of Medicine, University of Murcia, 30100 Murcia, Spain b Department of Biochemistry and Molecular Biology B, School of Medicine, University of Murcia, 30100 Murcia, Spain Received 23 June 2004; received in revised form 25 October 2004; accepted 3 November 2004 Available online 26 November 2004 Abstract This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea , a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140–170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS- PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Pigmentation; Antibacterial protein 1. Introduction Marinomonas mediterranea is a melanogenic marine bacterium isolated from the Mediterranean Sea at the Murcia coast. It was the first prokaryotic cell found to express both laccase and tyrosinase activities [1]. These activities are due to the expression in this microorganism of two different PPOs. One of them is a membrane-bound multipotent laccase able to oxidize a wide range of substrates characteristic for tyrosinases and/or laccases [2]. The other PPO is a classical SDS-activated tyrosinase, being the main enzyme involved in melanin synthesis [3]. Mutants in the different genes coding for these enzymes have been isolated. The strain T101 does not show multipotent laccase activity [4]. The strains ng56 [1] and T105 [3] mutants are amelanotic and lack tyrosinase activity, although they are affected in different loci of the same operon [5]. All these enzymatic activities are growth phase-regulated and they have a common sensor histidine kinase regulator, PpoS, which seems to exert its action at posttranscriptional level [6]. The biosynthetic aspects of melanin have been thor- oughly studied mainly in fungi [7,8]. A number of possible functions have been proposed for this pigment, including a pathogenic and phytopatogenic role. However, most of the reports claim a protective function for this pigment consid- 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.11.002 Abbreviations: CFU, colony forming units; CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; DHI, 5,6-dihydroxyIndole; DHN, 1,8- dihydroxynaphthalene; LB, Luria-Bertani medium; MBC, minimal bacter- icidal concentration; MIC, minimal inhibitory concentration; MMC, complex marine medium; MMM, minimum marine medium; PPO, polyphenol oxidase * Corresponding author. Tel.: +34 968 367 194; fax: +34 968 364150. E-mail address: psolano@um.es (F. Solano). Biochimica et Biophysica Acta 1721 (2005) 193 – 203 http://www.elsevier.com/locate/bba