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

Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by 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 sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability.     

Dimethoxyphenol oxidase activity of different microbial blue multicopper proteins

2,6-Dimethoxyphenol is a versatile substrate for Pyricularia oryzae laccase, PpoA from Marinomonas mediterranea, phenoxazinone synthase from Streptomyces antibioticus and mammalian ceruloplasmin. In addition, in cellular extracts of microorganisms expressing other blue multicopper proteins with no enzymatic activity previously described, such as Escherichia coli (copper resistance CueO), Pseudomonas syringae and Xanthomonas campestris (copper resistance CopA), Bacillus subtilis (sporulation protein CotA) and Saccharomyces cerevisiae (iron transporter Fet3p), laccase activity is detected under appropriate conditions. This oxidase activity can be spectrophotometrically followed by the oxidation of 2,6-dimethoxyphenol. Specific staining after SDS-PAGE is also possible for some of these proteins. This detection assay can facilitate the study of the multiple functions that such proteins seem to carry out in a variety of microorganisms.     

Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea

Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli. Its predicted amino acid sequence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal laccases. In addition, two additional putative copper-binding sites near its N-terminus are also present. Recombinant expression in E. coli of this protein clearly demonstrates its multipotent capability, showing both laccase-like and tyrosinase-like activities. This is the first prokaryotic laccase sequenced and the first PPO showing such multipotent catalytic activity. The expression of several truncated products indicates that the four copper-binding sites typical of blue multicopper proteins are essential for the laccase activity of this enzyme. However, the last two of these sites are not necessary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histidine-rich clusters close to the N-terminus of the protein.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Cloning and molecular characterization of a SDS-activated tyrosinase from Marinomonas mediterranea

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper-binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS-dependent conformational change that facilitates the substrate accessibility to the dicopper active site.     

A novel type of lysine oxidase: L-lysine-epsilon-oxidase

The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas-Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193-203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493-2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of free L-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H2O2, namely lysine-alpha-oxidase (LalphaO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-epsilon-oxidase (lod for the gene) instead of marinocine. Lod shows high stereospecificity for free L-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by beta-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site.     

The antimicrobial activity of marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity

Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned.     

Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea

The incorporation of fungal laccases into novel applications has been delayed mainly due to their intrinsic sensitivity towards halides and alkaline conditions. In order to explore new sources of enzymes we evaluated the multipotent polyphenol oxidase PPO1 from the marine bacterium Marinomonas mediterranea. Here we report that, in contrast to its fungal counterparts, PPO1 remained functional above neutral pH presenting high specificity for phenolic compounds, in particular for methoxyl-substituted mono-phenols and catechols. These properties, in addition to its tolerance towards chloride (up to 1 M) and its elevated redox potential at neutral pH (0.9 V), suggest this enzyme may be an interesting candidate for specific applications such as the Amperometric determination of phenolic compounds and bio-fuel cells.     

Location and catalytic characteristics of a multipotent bacterial polyphenol oxidase.

The melanogenic marine bacterium Marinomonas mediterranea contains a multipotent polyphenol oxidase (PPO) able to oxidize substrates characteristic for tyrosinase and laccase. Thus, this enzyme shows tyrosine hydroxylase activity and it catalyzes the oxidation of a wide variety of o-diphenol as well as o-methoxy-activated phenols. The study of its sensitivity to different inhibitors also revealed intermediate features between laccase and tyrosinase. It is similar to tyrosinases in its sensitivity to tropolone, but it resembles laccases in its resistance to cinnamic acid and phenylthiourea, and in its sensitivity to fluoride anion. This enzyme is mostly membrane-bound and can be solubilized either by non-ionic detergent or lipase treatments of the membrane. The expression of this enzymatic activity is growth-phase regulated, reaching a maximum in the stationary phase of bacterial growth, but L-tyrosine, Cu(II) ions, or 2,5-xylidine do not induce it. This enzyme can be separated from a second PPO form by gel permeation chromatography. The second PPO is located in the soluble fraction and shows a sodium dodecyl sulfate (SDS)-activated action on the characteristic substrates for tyrosinase, L-tyrosine, and L-dopa, but it does not show activity towards laccase-specific substrates. The involvement of the multipotent PPO in melanogenesis and its relationship with the SDS-activated form and with the alternative functions proposed for multicopper oxidases in other microorganisms are discussed.     

First report of toxic Cylindrospermopsis raciborskii and Raphidiopsis mediterranea (Cyanoprokaryota) in Egyptian fresh waters

Cylindrospermopsis raciborskii, a potentially toxic and highly adaptable freshwater cyanobacterium, was believed to have been misidentified in the Nile at the end of the 19th century. This study reports the presence of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea for the first time in Egyptian fresh waters since that time. Cylindrospermopsis raciborskii appeared in the El-Dowyrat fish pond during May 2002, when bottom waters reached, as a result of climatic change, sufficiently high temperatures to allow the germination of its akinetes in the sediments. Both C. raciborskii and R. mediterranea showed seasonal variations, with highest densities recorded in August of each year. The count of the two species correlated positively with pH, temperature and conductance, and negatively with nutrients, during the study period. The densities of C. raciborskii and R. mediterranea varied significantly along the depth profile of this pond, with peaks obtained at 1 and 0.5m, respectively. Isolates of C. raciborskii and R. mediterranea from this pond exhibited toxicity to Artemia salina, Daphnia magna and mice. Cylindrospermopsis raciborskii extracts had hepatotoxic effects on mice, but R. mediterranea extracts showed neurotoxic effects on mice. The identification of toxic C. raciborskii and R. mediterranea in this pond should be considered during the monitoring of cyanobacteria in drinking and recreational water sources in Egypt.     

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea

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.     

Cyclopina (Copepoda, Cyclopoida) from Brazilian sandy beaches

Five new species of Cyclopina Claus are described and C. mediterranea Steuer is redescribed from Brazilian sandy beaches. Cyclopina curoli sp. n. can be very abundant in coarse sandy shows. It is related to C. laurentica Mocholls and C. crassisserosa Herbst with which it shares a similar structure of Leg 5 in the female. Cyclopina dorae sp. n. is most closely related to C. meditarranea Steuer. Cyclopina arenosa sp. n. and Cyclopina yutimaete sp. n. are reminiscent of C. pontica Monchenko, the only other species of Cyclopina that possess a 2-segmented endopod in leg 1. Cyclopina caissara sp. n. is distinguished from all other congeneric species by having a very reduced inner spine in the leg 5 exopod of both male and female. This species shares with C. schneideri Scott and C. schneideri and C. brevifurca Sars the 12–segmented antennule. Taxonomical problems related to C. schneideri and C. brevifurea are discussed. Cyclopina mediterranea Steuer, 1940 is recorded for the first time from the American continent.     

A new taxon in the Infundibulicybe gibba complex (Basidiomycota, Agaricales, Tricholomataceae) from Sardinia (Italy)

A new species of Infundibulicybe (viz. I. mediterranea sp. nov.) is described from Sardinia based both on morphological and molecular ITS data. The species, a close ally of I. gibba, differs from the latter in the darker tinges of the basidiomata, the stipe, which is nearly concolorous with the pileus, and smaller basidiospores. Drawings of the main micro-morphological features as well as a color photograph of fresh basidiomata in situ are provided.     

Flanking genes of an essential gene give information about the evolution of metazoa

We collected as much information as possible on new lamin genes and their flanking genes. The number of lamin genes varies from 1 to 4 depending more or less on the phylogenetic position of the species. Strong genome drift is recognised by fewer and unusually placed introns and a change in flanking genes. This applies to the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the urochordate Ciona intestinalis, the annelid Capitella teleta and the planaria Schmidtea mediterranea. In contrast stable genomes show astonishing conservation of the flanking genes. These are identical in the sea anemone Nematostella vectensis and the cephalochordate Branchiostoma floridae lamin B1 gene. Even in the lamin B1 genes from Xenopus tropicalis and man one of the flanking genes is conserved. Finally our analysis forms the basis for a molecular analysis of metazoan phylogeny.     

Disentangling taxonomy within the Rhabditis (Pellioditis) marina (Nematoda, Rhabditidae) species complex using molecular and morhological tools

Correct taxonomy is a prerequisite for biological research, but currently it is undergoing a serious crisis, resulting in the neglect of many highly diverse groups of organisms. In nematodes, species delimitation remains problematic due to their high morphological plasticity. Evolutionary approaches using DNA sequences can potentially overcome the problems caused by morphology, but they are also affected by theoretical flaws. A holistic approach with a combination of morphological and molecular methods can therefore produce a straightforward delimitation of species. The present study investigates the taxonomic status of some highly divergent mitochondrial haplotypes in the Rhabditis ( Pellioditis ) marina species complex by using a combination of molecular and morphological tools. We used three molecular markers (COI, ITS, D2D3) and performed phylogenetic analyses. Subsequently, morphometric data from nearly all lineages were analysed with multivariate techniques. We included R. ( P. ) mediterranea and R. (R.) nidrosiensis to infer species status of the observed lineages. The results showed that highly divergent genotypic clusters were accompanied by morphological differences, and we created a graphical polytomous key for future identifications. This study indisputably demonstrates that R. ( P. ) marina and R. ( P. ) mediterranea belong to a huge species complex and that biodiversity in free-living marine nematodes may be seriously underestimated.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 1–15.     

Revision of the cluster-flies of the Pollenia viatica species-group (Diptera: Calliphoridae)

Abstract. The Pollenia viatica species-group ( =pallida species-group) is defined and described. It consists of P.bicolor Robineau-Desvoidy, P.bulgarica Jacentkovsk, P.fulvipalpis Macquart, P.mediterranea Grunin, P.ruficrura Rondani, stat.rev., P.viatica Robineau-Desvoidy, stat.rev. (= P.pallida Rodendorf, syn.n.) and P.ponti sp.n. It is mono-phyletic, mainly on the basis of a synapomorphic ovipositor tip, and the vagabunda species-group is its sister group. A key to all species is provided, the terminalia of both sexes illustrated and all taxa redescribed. Lectotypes are designated for Pollenia bisulca Pandellé, P.ruficrura Rondani and P.viatica Robineau-Desvoidy. Pollenia ruficrura is removed from its current status as a nomen dubium. Pollenia bicolor and P.ponti are western Mediterranean species, the latter also occurs in Czechoslovakia. P.fulvipalpis is endemic to France and Channel Islands. P.ruficrura is known only from Corsica and Italy. P.mediterranea is an eastern Mediterranean species reaching east to Armenia. P.bulgarica is distributed from Hungary to Caucasus. P.viatica is widespread from France, Great Britain and Southern Scandinavia to Central Asia. All species are summer or autumn species, and seem to have only one generation each year. The immature stages and life-cycle of a species from Paris, France, named 'Pollenia rudis' by Keilin (1909, 1915) are tentatively assigned to P. viatica . It survives the winter as a first instar larva within the earthworm Allolobophora chlorotica Savigny.     

A new species of <Emphasis Type="Italic">Prohatschekia</Emphasis> Nunes-Ruivo, 1954 (Copepoda: Hatschekiidae) parasitic on <Emphasis Type="Italic">Scorpaena elongata</Emphasis> (Cadenat) off Algeria

A new species of parasitic copepod, Prohatschekia mediterranea n. sp. (Siphonostomatoida: Hatschekiidae), is described from a scorpaenid fish, Scorpaena elongata, collected off Algeria. This is the seventh species of Prohatschekia Nunes-Ruivo, 1954 to be described and the first record of the genus from the Mediterranean Sea. The new species is most closely related to P. cremouxi Nunes-Ruivo, 1954, known from a congeneric host collected in Senegal. A key is provided to distinguish the new species from other members of the genus.     

Marinomonas aquamarina sp. nov., isolated from oysters and seawater

The characterization of three bacterial strains isolated from cultured oysters and seawater at the Spanish Mediterranean coast has been performed. Strains were phenotypically and genetically characterized and the results led us to identify them as members of the genus Marinomonas. A phylogenetic analysis based on the almost complete 16S rDNA sequences clustered all three strains together (with sequence similarities around 99.8%) in the vicinity of M. communis and M. vaga sequences and distantly related to the other four species of the genus. The most closely related species was M. communis that shared 97.4-97.6% with the Mediterranean strains. DNA-DNA hybridizations were performed to clarify the taxonomic position of our isolates and the results confirmed their specific isolation, with interspecific binding ratios below 59%. We propose the bacteria to constitute a new Marinomonas species, i.e. M. aquamarina and strain 11SM4T (CECT 5080T, CIP 108405T, CCUG 49439T) as the type strain.