Reassessment of the Microbial Role in Mn-Fe Nodule Genesis in Andean Paleosols

The presence of Mn-Fe nodules in the epipedons (surface horizons) of paleosols of presumed Upper Neogene age in the northwestern Venezuelan Andes have been interpreted as products of inorganic oxidation and reduction processes operating over the full range of glacial and interglacial cycles that affected paleosol morphogenesis. New microscopic/chemical data from combined SEM-EDS-FIB analyses of representative Mn-Fe nodules indicate microbes play an important role in Mn/Fe precipitation leading to their genesis in alpine Mollisols (Argiustolls). Although the prevailing new data are based mainly on fossil forms of filamentous bacteria and fungi and other biogenic pseudomorphs that may represent the former resident bacteria, the presence of extant microbes must await field experiments/collection, followed by a molecular microbiology approach to determine the biological drivers of metal precipitation. As in other terrestrial niche environments, microbes are seen here to play a role, perhaps a key one, in the morphogenesis of paleosols of importance in upper Neogene paleoenvironmental reconstruction.

Biocontrol agents promote growth of potato pathogens, depending on environmental conditions

There is a pressing need to understand and optimize biological control so as to avoid over-reliance on the synthetic chemical pesticides that can damage environmental and human health. This study focused on interactions between a novel biocontrol-strain, Bacillus sp. JC12GB43, and potato-pathogenic Phytophthora and Fusarium species. In assays carried out in vitro and on the potato tuber, the bacterium was capable of near-complete inhibition of pathogens. This Bacillus was sufficiently xerotolerant (water activity limit for growth = 0.928) to out-perform Phytophthora infestans (~0.960) and challenge Fusarium coeruleum (~0.847) and Fusarium sambucinum (~0.860) towards the lower limits of their growth windows. Under some conditions, however, strain JC12GB43 stimulated proliferation of the pathogens: for instance, Fusarium coeruleum growth-rate was increased under chaotropic conditions in vitro (132 mM urea) by >100% and on tubers (2-M glycerol) by up to 570%. Culture-based assays involving macromolecule-stabilizing (kosmotropic) compatible solutes provided proof-of-principle that the Bacillus may provide kosmotropic metabolites to the plant pathogen under conditions that destabilize macromolecular systems of the fungal cell. Whilst unprecedented, this finding is consistent with earlier reports that fungi can utilize metabolites derived from bacterial cells. Unless the antimicrobial activities of candidate biocontrol strains are assayed over a full range of field-relevant parameters, biocontrol agents may promote plant pathogen infections and thereby reduce crop yields. These findings indicate that biocontrol activity, therefore, ought to be regarded as a mode-of-behaviour (dependent on prevailing conditions) rather than an inherent property of a bacterial strain.

Mineralogy, chemistry and biological contingents of an early-middle Miocene Antarctic paleosol and its relevance as a Martian analogue

Fossil mesofauna and bacteria recovered from a paleosol in a moraine situated adjacent to the inland ice, Antarctica, and dating to the earliest glacial event in the Antarctic Dry Valleys opens several questions. The most important relates to understanding of the mineralogy and chemistry of the weathered substrate habitat in which Coleoptera apparently thrived at some point in the Early/Middle Miocene and perhaps earlier. Here, Coleoptera remains are only located in one of six horizons in a paleosol formed in moraine deposited during the alpine glacial event (> 15 Ma). A tendency for quartz to decrease upward in the section may be a detrital effect or a product of dissolution in the early stage of profile morphogenesis when climate was presumably milder and the depositing glacier of temperate type. Discontinuous distributions of smectite, laumontite, and hexahydrite may have provided nutrients and water to mesofauna and bacteria during the early stage of biotic colonization of the profile. Because the mesofauna were members of burrowing Coleoptera species, future work should assess the degree to which the organisms occupied other sites in the Dry Valleys in the past. Whereas there is no reasonable expectations of finding Coleoptera/insect remains on Mars, the chemistry and mineralogy of the paleosol is within a life expectancy window for the presence of microorganisms, principally bacteria and fungi. Thus, parameters discussed here within this Antarctic paleosol could provide an analogue to identifying similar fossil or life-bearing weathered regolith on Mars.

Arbuscular mycorrhizal fungal hyphae reduce soil erosion by surface water flow in a greenhouse experiment

The role of arbuscular mycorrhizal fungi (AMF) in resisting surface flow soil erosion has never been tested experimentally. We set up a full factorial greenhouse experiment using Achillea millefolium with treatments consisting of addition of AMF inoculum and non-microbial filtrate, non-AMF inoculum and microbial filtrate, AMF inoculum and microbial filtrate, and non-AMF inoculum and non-microbial filtrate (control) which were subjected to a constant shear stress in the form of surface water flow to quantify the soil detachment rate through time. We found that soil loss can be explained by the combined effect of roots and AMF extraradical hyphae and we could disentangle the unique effect of AMF hyphal length, which significantly reduced soil loss, highlighting their potential importance in riparian systems.

The influence of substrate texture on early biological colonization

Biological colonization of stone is a major concern in the preservation and presentation of cultural heritage. Colonization is typically associated with unpleasant soiling, and varying degrees of biodeterioration. A better understanding of why organisms grow where they do, will aid in
developing preventative, and treatment methods for biosoiling of cultural heritage. Sandstone exposure trials were set up at nine different locations across Northern Ireland to investigate the influences of local climate, local environmental,and micro-climatic factors on the early stages (up to 21 months) of biological colonization.
Results showed that, green and yellow soiling occurred on tooled stone surfaces, whereas darkening occurred preferentially on smooth surfaces. It is likely that different populations of organisms occur on these surfaces with green algae occurring on tooled surfaces due to slower drying rates (i.e. prolonged moisture retention), and cyanobacteria and fungi thriving on smooth surfaces due to their ability to withstand moisture fluctuation.

Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species

Fungi of the genus Aspergillus are widespread in the environment. Some Aspergillus species, most commonly Aspergillus fumigatus, may lead to a variety of allergic reactions and life-threatening systemic infections in humans. Invasive aspergillosis occurs primarily in patients with severe immunodeficiency, and has dramatically increased in recent years. There are several factors at play that contribute to aspergillosis, including both fungus and host-related factors such as strain virulence and host pulmonary structure/immune status, respectively. The environmental tenacity of Aspergilllus, its dominance in diverse microbial communities/habitats, and its ability to navigate the ecophysiological and biophysical challenges of host infection are attributable, in large part, to a robust stress-tolerance biology and exceptional capacity to generate cell-available energy. Aspects of its stress metabolism, ecology, interactions with diverse animal hosts, clinical presentations and treatment regimens have been well-studied over the past years. Here, we synthesize these findings in relation to the way in which some Aspergillus species have become successful opportunistic pathogens of human- and other animal hosts. We focus on the biophysical capabilities of Aspergillus pathogens, key aspects of their ecophysiology and the flexibility to undergo a sexual cycle or form cryptic species. Additionally, recent advances in diagnosis of the disease are discussed as well as implications in relation to questions that have yet to be resolved.

CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal

Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Review of the genus Bursaphelenchus Fuchs, 1937 (Nematoda, Aphelenchida) with some conclusions on the host-parasite and vector-parasite evolution.

INTRODUCTION AND GOALS: Genus Bursaphelenchus includes several pests of the world importance for the rural economy, the most dangerous are the Bursaphelenchus xylophilus (the pinewood nematode caused decline of the pine trees in south Asia and in one spot area in Europe, Portugal, Peninsula de Setubal) and the Bursaphelenchus cocophilus, causing the decline of coco-palm plantations in Carribean and Latin American regions. The peculiarity of the host-parasite association of the genus that the nematode life cycle includes three trophic components: plant (mostly a tree), insect vector and a fungus. Goals of the presentation is to list all species of the world fauna and all efficient diagnostic characters, then create the identification tool and analyze the similarity of species and possible ways and causes of the host-parasite evolution of the group. RESULTS: Complete list of species with synonymy and a catalogue of all efficient diagnostic characters with their states, selected from papers of the most experienced taxonomists of the genus, are given for the genus Bursaphelenchus. List of known records of Bursaphelenchus species with names of natural vectors and plants and their families is given (for world pests the most important groups of trees and insects are listed). The tabular, traditional and computer-aided keys are presented. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on all efficient taxonomic characters and separately on the spicule characters only, are given. Discussion whether the species groups are natural or purely diagnostic ones is based on the relationships dendrograms and the vector and associated plant ranges of Bursaphelenchus species; the xylophilus species group (B. xylophilus, B. abruptus, B. baujardi, B. conicaudatus, B. eroshenkii, B. fraudulentus, B. kolymensis, B. luxuriosae; B. mucronatus), the hunti group (B. hunti, B. seani, B. kevini and B. fungivorus) are probably the natural ones. CONCLUSIONS: The parasitic nematode association includes three trophic components: plant, insect vector and fungus. The initial insect-plant complex Scolytidae-Pinaceae is changeable and only in rare occasions the change of the preferred vector to Cerambycidae (the xylophilus group), Hymenoptera (the hunti group) led to formation of the natural species-groups. From the analysis it is clear that although the vector range is changeable it is comparatively more important for the evolution of the genus Bursaphelenchus than associations with plants at the family level. Data on the fungi species (3rd component in natural Bursaphelenchus associations) are insufficient for the detailed comparative analysis.

Antioxidant,antimicrobial and toxicological properties of Schinus molle L. essential oils

Ethnopharmacological relevance: Schinus molle L. has been used in folk medicine as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural as well as antispasmodic and analgesic; however, there are few studies of pharmacological and toxicological properties of S. molle essential oils. Aim of the study: The aim of this study was to evaluate the antioxidant and antimicrobial activities of S. molle leaf and fruit essential oils, correlated with their chemical composition and evaluate their acute toxicity. Materials and methods: The chemical composition of S. molle leaf and fruit essential oils were evaluated by GC-FID and GC-MS. Antioxidant properties were determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and β-carotene/linoleic acid methods. Antimicrobial properties were evaluated by the agar disc diffusion method and minimal inhibitory concentration assay. Toxicity in Artemia salina and acute toxicity with behavioural screening in mice were evaluated. Results: The dominant compounds found in leaf and fruit essential oils (EOs) were monoterpene hydrocarbons, namely -phellandrene, β-phellandrene, β-myrcene, limonene and α-pinene. EOs showed low scavenging antioxidant activity by the DPPH free radical method and a higher activity by the β-carotene/linoleic acid method. Antimicrobial activity of EOs was observed for Gram+, Gram– pathogenic bacteria and food spoilage fungi. EOs showed cytotoxicity for Artemia salina and lower toxicity in Swiss mice. Conclusions: The result showed that EOs of leaves and fruits of S. molle demonstrated antioxidant and antimicrobial properties, suggesting their potential use in food or pharmaceutical industries.

ANTIOXIDANT, ANTIMICROBIAL AND TOXICOLOGICAL PROPERTIES OF SCHINUS MOLLE L. ESSENTIAL OILS

Schinus molle L. is commonly known as pink pepper or American pepper, of Anacardiaceae family, from subtropical regions of South America, introduced and naturalized in South Europe, including Portugal. In folk medicine, plant extracts and essential oil has related as having antibacterial, antiviral, antifungal, anti-inflammatory, antitumoral, antispasmodic, analgesic and antidepressive properties. The aim of present study was to evaluate the chemical composition and biological activities of essential oil extracted from leaves and fruits of S. molle. For this purpose, the essential oils were analyzed by gas chromatography (GC/FID) and antioxidant properties were evaluated by the free radical DPPH and by system β-carotene/linoleic acid methods. The antimicrobial activities were screened against pathogenic bacteria and fungi and food spoiling fungi by the disc diffusion assay and minimal inhibitory concentration (MIC) was determined for sensitive strains. Toxicity of essential oils were carried out by the brine shrimp mortality test (EC50) and acute lethal dose (DL50) determination after oral administration in Swiss mice The major components in leaf essential oil were α-phellandrene, β-phellandrene and limonene, while myrcene, α-phellandrene and 1,8-cineole are the main components in the fruit essential oil. The essential oils of leaf and fruit of S. molle showed antioxidant activity through the two mechanisms: the ability to capture free radicals and protection of lipid peroxidation. These oils exhibited also a broad microbial activity spectrum, against pathogenic bacteria Gram-positive and Gram-negative and Candida spp. The fruit essential oil showed high cytotoxicity against Artemia salina. Essential oils of leaves and fruits of S. molle showed significant antioxidant and microbial properties, so the studies continue to clarify more in deep its toxicity, including hepatotoxicity and nephrotoxicity, and to evaluate its medicinal or nutraceutical potential.

Brazilian savanna forest : conservation, medicinal reservoir and bioprospecting

This study aimed to analyse the Brazilian savanna forest from a Legal Reserve (LR) area from a perspective of conservation, reservoir of organic carbon and medicinal biomass for a prospective use of native medicinal plants. An ethnobotanical and ethnopharmacological survey was carried out close to a community settled in the rural area in the south of Tocantins, being selected 9 of the most cited species (cajuí- Anacardium othonianum; inharé-Brosimum gaudichaudii; jatobá-Hymenaeae courbaril; jenipapo-Genipa americana, aroeira-Myracrodruon urundeuva; negramina-Siparuna guianensis; barbatimão- Stryphnodendron obovatum; assa peixe-Vernonia brasiliana, embaúba-Cecropia pachystachya). Crude foliar extracts were subjected to a preliminary phytochemical prospection and triage of secondary metabolites with antimicrobial activity of potential interest in health and familiar agriculture. Phenolic compounds, terpenes and flavonoids were detected in the extracts of most species, which suggests the presence of antimicrobial, antioxidant and anti-insect activities. It was evident the need to better know the LR as a reservoir of medicinal biomass in an area under ecological tension where 35% (610ha) of the property is LR and should be protected by law. Therefore, a forest inventory of live woody species was performed using the allometric or indirect method. This identified a rare remnant of Semidecidual Seasonal Forest amidst the largest world savannah, the Cerrado biome. An analysis of the forest average productivity per basal area (m².ha), aerial live biomass (ton.ha-1) and carbon stock was carried out. The forest fragment was considered relatively rich in species and diversity, although showing signs of disturbance and dominance by a few species. Its horizontal structure suggests biotic regeneration conditions. It is an important reservoir of medicinal plants. Of the families (57.5%) presenting medicinal species, 19 from a total of 33 are represented in the area and contain 44% (27) of the total species (61) and 63% (432) of the total individuals catalogued. Medicinal species have ecological importance for the equilibrium of the local flora and represent 80% of the 10 species with higher Importance Value Index (IVI): Tetragastris altissima, Chrysophyllum marginatum, Oenocarpus distichus, Sclerolobium paniculatum, Simarouba versicolor, Alibertia macrophylla, Siparuna guianensis, Maprounea guianensis, Licania parvifolia e Physocalymma scaberrimum. Medicinal productivity was high for this type of phytophysionomy: 183,2 ton. ha-1 of biomass and 91,51 ton. ha-1 of carbon representing 66% of the total biomass and carbon of this Cerrado forest. From this stage S. guianensis (Siparunaceae) was selected for performing bioassays in order to verify its biological activity against microorganisms of health and agricultural relevance. This is a native aromatic medicinal plant recommended as priority for conservation, with local popular medicinal validation and availability of medicinal feedstock (3300 Kg.ha-1), with the foliar fraction giving 38Kg/ha of crude extract and 5L/ha of essential oil. Foliar crude extracts and essential oil were obtained and tested in vitro using a disk diffusion bioassay. Different concentrations of these natural products were tested against gram-positive bacteria (Staphylococcus aureus ATCC 29213), gram-negative bacteria (Escherichia coli ATCC 25922 and ATCC 35218; Pseudomonas aeruginosa ATCC 10145) and fungi (Candida albicans ATCC 6258 e Fusarium oxysporum). The essential oil inhibited the growth of S. aureus in its crude concentration (380μg.mL-1), as well as diluted to half (190μg.mL-1) and a quarter strength (95μg.mL-1). It’s likely that such action is due to sesquiterpenes major components, such as bisabolol and bisabolene (10.35%), measured by gas chromatography (GC-MS, GC-FID). Extracts did not exhibit any antimicrobial activity against the microorganisms tested. The native medicinal plants prospective market is an alternative that favours the conservation of biodiversity while generating benefits for the development of sustainable family productive activities within local ecosystems instead of the current inappropriate uses. This strengthens conservation policies of Legal Reserve in rural settlements and is in agreement with public policy on global warming and climate changes.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Assessing the effectiveness of chemical treatment with nanomaterials in improving the quality of different industrial effluents

Industrial activities are the major sources of pollution in all environments. Depending on the type of industry, various levels of organic and inorganic pollutants are being continuously discharged into the environment. Although, several kinds of physical, chemical, biological or the combination of methods have been proposed and applied to minimize the impact of industrial effluents, few have proved to be totally effective in terms of removal rates of several contaminants, toxicity reduction or amelioration of physical and chemical properties. Hence, it is imperative to develop new and innovative methodologies for industrial wastewater treatment. In this context nanotechnology arises announcing the offer of new possibilities for the treatment of wastewaters mainly based on the enhanced physical and chemical proprieties of nanomaterials (NMs), which can remarkably increase their adsorption and oxidation potential. Although applications of NMs may bring benefits, their widespread use will also contribute for their introduction into the environment and concerns have been raised about the intentional use of these materials. Further, the same properties that make NMs so appealing can also be responsible for producing ecotoxicological effects. In a first stage, with the objective of selecting NMs for the treatment of organic and inorganic effluents we first assessed the potential toxicity of nanoparticles of nickel oxide (NiO) with two different sizes (100 and 10-20 nm), titanium dioxide (TiO2, < 25 nm) and iron oxide (Fe2O3, ≈ 85x425 nm). The ecotoxicological assessment was performed with a battery of assays using aquatic organisms from different trophic levels. Since TiO2 and Fe2O3 were the NMs that presented lower risks to the aquatic systems, they were selected for the second stage of this work. Thus, the two NMs pre-selected were tested for the treatment of olive mill wastewater (OMW). They were used as catalyst in photodegradation systems (TiO2/UV, Fe2O3/UV, TiO2/H2O2/UV and Fe2O3/H2O2/UV). The treatments with TiO2 or Fe2O3 combined with H2O2 were the most efficient in ameliorating some chemical properties of the effluent. Regarding the toxicity to V. fischeri the highest reduction was recorded for the H2O2/UV system, without NMs. Afterwards a sequential treatment using photocatalytic oxidation with NMs and degradation with white-rot fungi was applied to OMW. This new approach increased the reduction of chemical oxygen demand, phenolic content and ecotoxicity to V. fischeri. However, no reduction in color and aromatic compounds was achieved after 21 days of biological treatment. The photodegradation systems were also applied to treat the kraft pulp mill and mining effluents. For the organic effluent the combination NMs and H2O2 had the best performances in reduction the chemical parameters as well in terms of toxicity reduction. However, for the mine effluent the best (TiO2/UV and Fe2O3/UV) were only able to significantly remove three metals (Zn, Al and Cd). Nonetheless the treatments were able of reducing the toxicity of the effluent. As a final stage, the toxicity of solid wastes formed during wastewater treatment with NMs was assessed with Chironomus riparius larvae, a representative species of the sediment compartment. Certain solid wastes showed the potential to negatively affect C. riparius survival and growth, depending on the type of effluent treated. This work also brings new insights to the use of NMs for the treatment of industrial wastewaters. Although some potential applications have been announced, many evaluations have to be performed before the upscaling of the chemical treatments with NMs.

Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae

Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.