ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms’ role in this extreme environment, the characterization and description of new species is vital.

Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays.

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.

Novel doublet molecules of cecropin A from Drosophila melanogaster were designed and constructed combining the regular (CECdir) with the inverted (CECret) coding sequence of the standard CEC A1 gene resulting in the following configurations: CECdir-CECret and CECret-CECdir. These two recombinant molecules were generated using a three-primer driven PCR reaction yielding composite single functional aminoacidic molecules with the coding sequences of CECdir linked in frame with the coding sequence of CECret and vice versa. In order to obtain these constructions, a retropeptide DNA-coding sequence was chemically synthesized to match the expected polarity of the newly generated CECret sequence. Both doublet antimicrobial peptides (drAMPs) were cloned in the T7 promoter driven expression plasmid pET27b+ and expressed in E. coli BL21 without any fusion protein. Only the former recombinant peptide was expressed and purified from cell extracts and its specific activity against two different bacteria showed to be higher than those displayed by their monomer parental counterparts.

A "defensin-like" antibacterial peptide from Mytilus edulis chilensis, was sub-cloned into a binary vector for expression in plant tissues. The resulting new clone was electroporated into A. tumefaciens to transform tobacco plants. The presence of the construct in transgenic tobacco lines was demonstrated through RT-PCR, Northern and Western blots. Transformed positive plants were selected and grown for challenging. Tobacco leaves were infiltrated with Pseudomonassyringae pv. syringae and visual lesions determined at different times post-exposure. Of seven plants exposed, four gave variable protection up to seven days post-infection while one of them appears to be fully protected. These results suggest that defensin-like antimicrobial peptides from molluscs are a good source to provide resistance of tobacco plants to Pseudomonassyringae pv. syringae.

Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis. 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 µg/µl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Sep-pak C-18® 20% acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules

A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, cationic peptides are the most widespread. Interestingly, the variety and diversity of these peptides seem to be much wider than suspected. In fact, novel classes of peptides with varying chemical propertiescontinue to be isolated from different vertebrate and invertebrate species, as well as from bacteria. To the early characterized peptides, mostly cationic in nature, anionic peptides, aromatic dipeptides, processed forms of oxygen-binding proteins and processed forms of natural structural and functional proteins can now be added, just to name a few. In spite of the astonishing diversity in structure and chemical nature displayed by these molecules, all of them present antimicrobial activity, a condition that has led researchers to consider them as "natural antibiotics" and as such a new and innovative alternative to chemical antibiotics with a promising future as biotechnological tools. A resulting new generation of anti microbial peptides (AMPs) with higher specific activity and wider microbe-range of action could be constructed, and hopefully endogenously expressed in genetically-modified organisms.

Conditions have been optimised to transfect the fish cell line CHSE-214 to measure expression, maintenance and putative chromosomal integration of the reporter gene LUC, spliced into two versions of an expression vector. The first is pCMVL, and the second p103, a novel pCMVL-derived plasmid to which a highly conserved tandem repeat from the salmon genome was added in an inverted configuration flanking the LUC gene to promote its chromosomal integration. A minimal ratio of one to one, lipopolyamine carrier to plasmid DNA, was enough to efficiently transfect the cell line to follow the fate of target DNAs up to five cell passages. In this time-span we demonstrated the maintenance of the foreign DNA in the cells, the concomitant expression of the reporter gene, and a higher stability of p103 over the control plasmid which might suggest a higher potential for integration. Thus, we define an efficient model system for future in vitro evaluation of potential target genes of commercial interest for fish transgenesis