Adaptation and acclimatization to ocean acidification in marine ectotherms: an in situ transplant experiment with polychaetes at a shallow CO₂ vent system

Metabolic rate determines the physiological and life-history performances of ectotherms. Thus, the extent to which such rates are sensitive and plastic to environmental perturbation is central to an organism's ability to function in a changing environment. Little is known of long-term metabolic plasticity and potential for metabolic adaptation in marine ectotherms exposed to elevated pCO₂. Consequently, we carried out a series of in situ transplant experiments using a number of tolerant and sensitive polychaete species living around a natural CO₂ vent system. Here, we show that a marine metazoan (i.e. Platynereis dumerilii) was able to adapt to chronic and elevated levels of pCO₂. The vent population of P. dumerilii was physiologically and genetically different from nearby populations that experience low pCO₂, as well as smaller in body size. By contrast, different populations of Amphiglena mediterranea showed marked physiological plasticity indicating that adaptation or acclimatization are both viable strategies for the successful colonization of elevated pCO₂ environments. In addition, sensitive species showed either a reduced or increased metabolism when exposed acutely to elevated pCO₂. Our findings may help explain, from a metabolic perspective, the occurrence of past mass extinction, as well as shed light on alternative pathways of resilience in species facing ongoing ocean acidification

The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats

The Mediterranean Sea is a marine biodiversity hot spot. Here we combined an extensive literature analysis with expert opinions to update publicly available estimates of major taxa in this marine ecosystem and to revise and update several species lists. We also assessed overall spatial and temporal patterns of species diversity and identified major changes and threats. Our results listed approximately 17,000 marine species occurring in the Mediterranean Sea. However, our estimates of marine diversity are still incomplete as yet—undescribed species will be added in the future. Diversity for microbes is substantially underestimated, and the deep-sea areas and portions of the southern and eastern region are still poorly known. In addition, the invasion of alien species is a crucial factor that will continue to change the biodiversity of the Mediterranean, mainly in its eastern basin that can spread rapidly northwards and westwards due to the warming of the Mediterranean Sea. Spatial patterns showed a general decrease in biodiversity from northwestern to southeastern regions following a gradient of production, with some exceptions and caution due to gaps in our knowledge of the biota along the southern and eastern rims. Biodiversity was also generally higher in coastal areas and continental shelves, and decreases with depth. Temporal trends indicated that overexploitation and habitat loss have been the main human drivers of historical changes in biodiversity. At present, habitat loss and degradation, followed by fishing impacts, pollution, climate change, eutrophication, and the establishment of alien species are the most important threats and affect the greatest number of taxonomic groups. All these impacts are expected to grow in importance in the future, especially climate change and habitat degradation. The spatial identification of hot spots highlighted the ecological importance of most of the western Mediterranean shelves (and in particular, the Strait of Gibraltar and the adjacent Alboran Sea), western African coast, the Adriatic, and the Aegean Sea, which show high concentrations of endangered, threatened, or vulnerable species. The Levantine Basin, severely impacted by the invasion of species, is endangered as well

Proteomics and biotechnologies: new methods for glycoproteome analysis

Biological systems are made up of a plethora of organic components. Since nowadays attention has been focused on two important classes of bioinformative molecules, nucleic acids and proteins, whose large scale study has led to the rise of the so called “omics” sciences, genomics and proteomics. In particular the study of the proteome implies not only the protein complement of a given cell, but even the study on a high throughput scale of proteins post translational modifications, interactions, and functions. More than 50% of mammalian proteins are glycosylated and this observation has led to the conclusion that sugars attachment broadens variability among gene products. Glycans generally cover cellular surfaces, ranging from viruses to the most complex multicellular organisms and they can be considered as a molecular code that dictates to cells how to communicate with each other. The wide range of important biological processes mediated by carbohydrates has given origin to glycomics, the large scale study of the whole set of glycans of an organism. This PhD thesis targeted the development of methodological platforms for the study of glycoproteins and glycoconjugates by the integration of affinity chromatography strategies together with high performance liquid chromatography and mass spectrometry. My first steps in the study of glycosylation were focused on the development of enrichment, derivatization, and mass spectrometry procedures, that were applied to the study of the protein content of egg. Peptides of egg glycoproteins, bearing N-glycosylation sites, were captured by Concanavalin A affinity chromatography and detected by LC-MS/MS after deglycosylation. Oligosaccharides were analyzed by MALDI-MS/MS before and after dansylhydrazine derivatization of the reducing end. This derivatization was introduced to enhance oligosaccharides fragmentation characteristics. Attempts to achieve more structural information on glycans were carried out using multi-stage mass spectrometry on MALDI-LTQ-Orbitrap. A proteomic approach was employed to investigate the molecular bases of myocarditis, a group of diseases that have in common the inflammation of the heart. In this case, the study of glycosylation anomalies in the sera sample of myocarditis-affected patients came after a wider study including both the analysis of free peptides and a general overview on protein content by 2D gel electrophoresis. This study represents a starting point for further studies aiming at the screening of proteic biomarkers for this pathological status and involving different fields of clinical investigations, opening up new opportunities for therapeutics and early diagnostics. Glycoproteins-based therapeutics are generally produced in mammalian host systems, but plants can be considered a valuable alternative for their ability to produce homogeneously glycosylated recombinant proteins. The possibility of exploiting plants as host systems and the lack of consistent information concerning glycosylation in the plant model system Arabidopsis thaliana encouraged me to investigate its glycoproteome, using the strategies previously developed. The last part of my work was devoted to the study of innate immunity mechanisms triggered by lipopolysaccharides, bacterial glycoconjugates located on cell surfaces. The full exploitation of glycoconjugates potential for industrial biotechnological and pharmaceutical applications requires a deep knowledge of the immunological mechanisms at the base of host-pathogen recognition. The goal of the project was the development of a strategy to capture LPS-interacting proteins and glycoproteins in human serum, considering that some of these mechanisms are still unknown in non-human biological systems

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

BACKGROUND: The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. RESULTS: Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis–Menten kinetics with a K(M) of 9.13 mg/ml and a v(max) of 3469 μM min(-1). The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. CONCLUSIONS: In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion