The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

O principal objetivo do trabalho foi estimar a possibilidade de sobrevivência de micro-organismos extremófilos na superfície de exoplanetas conhecidos, assim como na superfície de seus eventuais satélites naturais. Foi utilizado um modelo que simula a atmosfera terrestre primordial, composta principalmente por nitrogênio, água e dióxido de carbono. E em se tratando de extremófilos, esses cálculos não foram limitados à Zona Habitável dos sistemas planetários, pois esse conceito foi estendido para uma região mais ampla, a Zona Extremófila, onde a vida pode existir. Extremófilos são micro-organismos terrestres que vivem sob condições extremas de temperatura, nível de radiação, umidade, pressão, salinidade, pH, etc. . Eles são candidatos naturais para habitarem meios ditos extraterrestres onde tais condições são eventualmente encontradas. Alguns exemplos desses ambientes em nosso sistema solar são: Marte, Titã (satélite de Saturno) e Europa (satélite de Júpiter). Há algumas centenas de planetas orbitando outras estrelas (exoplanetas) e a maioria deles são gigantes gasosos, em particular Hot Jupiters. A temperatura superficial de um planeta depende fortemente de seu albedo, de sua distância orbital, de condições geodinâmicas intrínsecas, além do tipo espectral de sua estrela hospedeira. A estimativa dessa temperatura foi obtida considerando o ciclo silicato-carbono e um balanço de energia global, que contribuiram para se obter estimativas da pressão parcial atmosférica devido ao dióxido de carbono e da temperatura média, na superfície dos planetas e/ou de seus satélites hipotéticos. Os eventuais satélites naturais de planetas gigantes podem abrigar vida e essa possibilidade foi testada através da análise das condições de estabilidade orbital desses corpos celestes. Os resultados deste trabalho deverão fornecer subsídios para a hipótese da panspermia. / The main objective of this study is to estimate the chance of survival of microorganisms (extremophiles) on the surface of known exoplanets, as well as on the surface of its potential natural satellites. We used a model that simulates the primordial atmosphere composed by, primarily, nitrogen, water and carbon dioxide. And when it comes to extremophiles, these calculations were not limited to the Habitable Zone of planetary systems, since this concept was extended to a wider region, the Extremophile Zone, where life can exist. Extremophiles are terrestrial microorganisms living under extreme conditions of temperature, light level, humidity, pressure, salinity, pH, etc ... They are natural candidates for living in habitats considered extraterrestrials where such conditions are encountered eventually. Examples of such environments in our solar system are: Mars, Titan (moon of Saturn) and Europe (satellite of Jupiter). There are hundreds of planets orbiting other stars (exoplanets) and most of them are gas giants, particularly Hot Jupiters. The surface temperature of a planet/moon depends heavily on its albedo, its orbital distance, of geodynamic conditions intrinsic, in addition to the spectral type of their host star. The estimate of this temperature was obtained considering the carbon-silicate cycle and a global energy balance, which contributed to obtain estimates of the partial pressure due to atmospheric CO2 and the average temperature on the surface of planets and/or their hypothetical satellites. Natural satellites of giant planets may harbor life, and this possibility was tested by analyzing the conditions of orbital stability of these heavenly bodies. The results of this study should provide support for the hypothesis of panspermia.

>Magister Scientiae - MSc / Microorganisms of the class Actinobacteria and domain Archaea are interesting from a biotechnological perspective owing to their metabolic attributes as producers of secondary metabolites and resilience under harsh environmental conditions respectively. Lake Magadi is a soda lake well studied in terms of its geology and limnology. Research attention has also been drawn to the microbial populations which thrive in this unique habitat but currently there are no reports on the assessment of its microflora using molecular methods. This study aimed to assess the actinobacterial and archaeal communities within Lake Magadi, Kenya a hypersaline –highly alkaline habitat using metagenomic methods as a preliminary study to identify potential candidates for exploitative biology Samples from two sites dubbed Lake Magadi station 2 (LM2) and Lake Magadi salt pan 4 (LMS4) within the Lake Magadi were analyzed using the 16S rRNA gene as a phylogenetic marker. Cluster analysis of taxon-specific 16S rDNA PCR-DGGE profiles revealed moderately heterogeneous actinobacterial and archaeal populations across the sample sites under investigation which is probably a reflection of the differences in abiotic conditions at the study sites. This observation was also confirmed from the multi-dimensional scaling (MDS) plot. PCR-based clonal libraries of actinobacterial and archaeal communities of both study sites retrieved a total of thirty-two clones (twenty actinobacterial and twelve archaeal) were sequenced. Analysis of the sequences revealed cultured and uncultured signatures of microorganisms typical of hypersaline and or highly alkaline niches. A few (3) sequences presented novelty (<96%) in identities with any previously identified organism. It was concluded that the species dominance at site LMS4 [situated within the salt flats of Lake Magadi and site for exploration of trona and its mineralized extensions (nacholite and gayllusite)] is likely to be dictated by anthropogenic stress since most of the microbial signals associated with the study site are typical of saline and or alkaline environmental samples exposed to especially mining but also agricultural and waste management practices. Isolation studies also revealed previously identified strains peculiar to hypersaline brines and sediments. The strains retrieved were affiliated to the taxonomically diverse genus Bacillus and Halomonas sp. The true applications and potential opportunities these isolates have for biotechnology have been well documented. Observations made from the culture dependent and culture independent methods suggests strongly that study site LMS4 is subjected to environmental conditions more severe than at site LM2. This study is a guide for future studies as it provides primary information on the haloalkaliphilic representatives of the actinobacteria phylum and domain Archaea within the soda lake environment. It can serve as a pedestal for investigation into the molecular machinery that supports the haloalkaliphilic lifestyles of inhabiting microorganisms and consequently give leads as to how they can be commercially exploited.

Microorganisms with an ability to thrive in harsh environments are referred as “extremophiles”. With advances in biotechnology, interest has grown in the extremophile research because of their unique macromolecules’ characteristics due to their growth environments. Over last decade, researchers have isolated many extremophiles from environments like volcano, salt lakes, hydrothermal vents, deep oceans, Antarctica glaciers etc. Macromolecules of these extremophiles are responsible for their survival in extreme environments. In this research work we have isolated lipid molecules from three different microorganisms. 1) GWE1 strain, a thermophilic bacterium, isolated from dark crusty material from sterilization ovens. 2) 7L strain, a thermophilic bacterium, isolated from Chilean Copahue Volcano. 3) I1P strain, a facultative anaerobe of the family Enterobacteriaceae, recently isolated from Antarctica. Complex lipid arrangement and/or type in the cell membrane are known to affect thermostability of microorganisms and efforts were made to understand the chemical nature of the polar lipids of membrane. In this work, we extracted total lipids from cell membrane, separated them by TLC into various fractions and characterize the lipid structures of fractions with analytical tools such as 1H, 13C, 31P and 2D NMR spectroscopy, ATR-FTIR spectroscopy and MSn spectrometry. In GWE1 strain, we were able to identify glycerophosphoethanolamine, glycerophosphate, glycerophosphoglycerol and cardiolipin lipid classes and an unknown glycerophospholipid class with novel MS/MS spectra pattern. We have also noticed the presence of saturated iso-branched fatty acids with NMR spectra in individual lipid classes. In case of I1P strain, we have identified glycerophosphoglycerol, glycerophosphoethanolamine, glycerophosphate, and acyl glycerophosphoglycerol lipid classes with unsaturated fatty acids in their structure, which could be one of the many reasons for survivability at lower temperatures. In case of 7L strain, we were able to identify glycerophosphoglycerol, cardiolipin, glycerophosphoethanolamine and glycerophosphate lipid classes with saturated iso branched fatty acids. FAME analysis revealed iso-15:0 (52.29 %) and iso-17:0 (18.64 %) as major fatty acyl chains. We did not observe major difference in polar head group composition of lipid classes between thermophiles (GWE1 and 7L) compare to psychrophiles (I1P). Major difference among these three strains was in fatty acid composition of lipid molecule. Both thermophiles showed presence of lipids with long chain saturated fatty acids while I1P showed presence of lipid molecule with unsaturated fatty acid chain. Lipids made of unsaturated fatty acids have lower melting points and they introduce kink in the cell membrane structure. At lower temperatures, these effects allow membrane to maintain fluidity and its functionality, which in turn allows the microorganism to grow at lower temperature. Lipids made with saturated iso branched fatty acid chain have higher melting points and they pack together densely in cell membrane. At high temperature because of higher melting point and dense packing, membrane fluidity is not affected and this effect allows microorganism to grow at the higher temperature. We believe that change in fatty acid composition is one of the many reasons for these microorganisms to survive the extreme condition. Thermostability of the other macromolecules (DNA, enzyme) of these extremophiles is not studied in this dissertation. / Chemistry

No / This paper draws a comparison between the 700-year-old historically reported will-o'-the-wisp phenomenon and the more recent discovery of extremophilic colonization of hostile environments; both have been observed as present in isolated, stressed environmental regions and originating from biological phenomena. However, whereas extremophilic activity can be understood in terms of a survival strategy based upon the synthesis of specific suites of protective biochemicals which are designed to control biogeologically the stressed habitats and to provide protection against the extreme environments, the analytical techniques that have proved so successful for the illumination of these survival strategies of extremophiles and which are now being miniaturized for in-field studies and for extraterrestrial exploration have not been applied to a clarification or evaluation of the phenomenon of will-o'-the-wisp. The reason is simply that the will-o'-the-wispsightings have now disappeared completely. Tantalizingly, all of the most reasonable physico-chemical and biological explanations for the will-o'-the-wisp phenomenon proved to be unsatisfactory in some respect and it is clear that, just as in the case of extremophilic colonization, will-o'-the-wisp would benefit from a modern rigorous analytical study which would produce the data from which the potentially novel biological behaviour could be characterized and which would help a better understanding to be made of our natural world.

Human ́s constant need for metals requires unsustainable mining and refining of metalore. As a result, highly contaminated wastewaters are discharged in the environmentcompromising the nearby habitat together with all its life forms. Microbial fuel cells arebioelectrochemical systems (BES’s) that use microorganisms to convert organic andinorganic matter, producing electricity as the final product. This technology has shownto have great potential for application for bioremediation of wastewaters. This thesisdescribes the gene expression and the taxonomical abundance of an acidophilic,electricity generating community that was used to treat mining wastewaters in amicrobial fuel cell. A complete metatranscriptomics analysis has been performed onduplicate MFC anode acidophilic microbial community generating electricity frominorganic sulfur compounds (ISC) oxidation at extremely low pH. The analysis showsthat the most expressed genus is Ferroplasma-like, the genus Acidithiobacillus-like isfollowing along with Sulfobacillus-like and Thermoplasma-like. Some of the generaexpressed show behaviours never described before suggesting that potentially, newspecies have been selected. The reactions of the sulfur pathway are regulated mostly bytwo genera: Acidithiobacillus-like during the disproportionation of tetrathionate, andFerroplasma-like by expressing the hdr gene that catalyses the reaction from elementalsulfur to sulfite, the sulfite is then converted to sulfate. The hdr gene has not previouslybeen found in F. acidarmanus-like suggesting that the specie might have been selectedfor this trait. Acidithiobacillus-like genus has a bigger role for the energy conservationand the electron transport in the sample, however the data are not sufficient to point outwhich gene has the major role in the process. The CO2 fixation in the chamber wasconsiderably low as a result from a significant carboxysomes production, bacterialcompartiments involved in the carbon dioxide fixation. The transcripts abundanceregarding the metal resistance genes have shown low expression suggesting that thecells were not under stress. This result is indicated by the synthesis of a transcriptionalrepressor protein that had prevented a significant production of metal resistanceenzymes. Likewise, the pH homeostasis plot does not show vast transcripts abundances,indicating that the cells were thriving under conditions not far from the optimum.

Na Antártica e no Ártico, a vida microbiana é presente e diversificada, tendo se adaptado a condições similares às encontradas em mundos que despertam interesse à astrobiologia. Este estudo investigou as alterações fisiológicas e fenotípicas do extremófilo psicrotrófico Exiguobacterium antarcticum B7 sob diferentes temperaturas. Foram utilizadas técnicas de eletroforese de proteínas em duas dimensões associada a espectrometria de massa, juntamente com técnicas de microscopia. E. antarcticum foi também submetida a condições físicas encontradas em Marte e no ambiente interplanetário. Seguindo evidências de que micro-organismos psicrotróficos podem também ser resistentes a períodos prolongados de dessecação, foi investigada a presença de linhagens microbianas resistentes a anidrobiose em solos da Antártica e permafrost do Ártico. Por fim, voltando-se à ecologia microbiana de solos polares permanentemente congelados, foi estudada a diversidade microbiana de três estratos de permafrost do ártico canadense com o uso de sequenciamento de DNA de nova geração. / In Antarctica and the Arctic, microbial life is present and diverse, having adapted to low temperature and low humidity conditions, similar to the worlds that arouse more interest to astrobiology. This study investigated the physiological changes of the psychrotrophic extremophile Exiguobacterium antarcticum B7 at different temperatures. Two dimensional protein electrophoresis techniques was used for proteome analysis in conjunction with mass spectrometry, as well as microscopy techniques. E. antarcticum was also subjected to physical parameters found on Mars and in the interplanetary environment. Following evidence that psychrotrophic microorganisms may also be resistant to prolonged periods of desiccation, it was investigated the presence of microbial strains resistant to anhydrobiosis on Antarctic soils and Arctic permafrost. At last, turning the attention to the microbial ecology of permanently frozen polar soils, it was studied the bacterial diversity of three Arctic permafrost strata with next-generation high throughput DNA sequencing.

The biodiversity of two distinct marine environments was observed to describe the biogeocomplexity of these extreme ecological systems. A shallow-water hydrothermal vent in Papua New Guinea served as a study of a thermophilic ecosystem influenced by arsenic rich vent fluids while a 60 m deep offshore primarily anoxic karst sink served as a study of an anaerobic sulfur-influenced habitat. Both environments support unique biological communities that are influenced by the physical and chemical pressures imposed on them by the harsh conditions of these systems. In Tutum Bay, Ambitle Isle, Papua New Guinea, a transect was created from a shallow hydrothermal vent that extended 120 m away from the vent. Previous studies have shown that the geochemistry of the system is heavily influenced by arsenic which is toxic to most organisms. In this study, macro- and meiofauna were collected and scored and combined with bacterial sequence data collected along the length of the transect. It was found that near vent sites harbored biological communities more similar than sites further from the vent. Many species were found only at sites near the hydrothermal vent. Near-vent communities were less diverse than those away from the vent, and biodiversity generally increased as distance from the vent increased. Distinct correlations between thermophilic organisms and temperature were observed. The metabolic repertoire of the microbial communities suggests that many strategies are used to obtain energy and carbon. The relative abundance of bacteria containing genes to reduce arsenic was comparable to those able to reduce sulfur compounds. Primary production appeared to be a mix of chemo- and phototrophy. Food webs and association analysis suggest a complex interplay between macrofaunal, meiofaunal and bacterial communities. While the system is heavily influenced by arsenic, no specific correlation between the relative abundance of arsenic metabolizing organisms and the amount of arsenic in the system could be drawn. This is likely due to the fact that most of the arsenic produced by the system is readily adsorbed onto iron oxyhydroxides, reducing the arsenic's bioavailability. The anoxic conditions at Jewfish sink provide a different hurdle than the hot arsenic conditions found in Papua New Guinea. The anoxic conditions are shared by other pit features found in karst geography, but the metabolic processes between Jewfish sink and these other karst habitats are different. The blue holes and black holes of the Bahamas are some of the most well-studied of these karstic pits. In these features, which are large circular pits with diameters of over 300 m, light and sulfur are used as a means of energy acquisition. Jewfish sink, having an opening only 6 m in diameter, is light restricted compared to these systems. As a result, the strategy of organisms dwelling in the anoxic conditions of the sink is different than those found at the well-studied holes in the Bahamas. Geochemical measurements were recorded over two time periods spanning a combined total of 6 years. The anoxic bottom waters of Jewfish sink remain stable and contained high levels of sulfide throughout most of the seasons studies. Sequence analysis of prokaryotes within the sink showed that sulfur reducers had the highest relative abundance compared to other functional guilds. To monitor the changes of the microbial communities within the sink, bacterial communities were examined at 4 depths within the sink at 9 different intervals over a period of 685 days. Denaturing Gradient Gel Electrophoresis (DGGE) was used to fingerprint 16s rRNA bacterial communities and dissimilatory sulfite reducing communities by targeting the 16s rRNA bacterial gene and the dsr gene associated with dissimilatory sulfite reducing bacteria and archaea. The lowest depth studied within the sink (40 m) remained stable chemically and biologically until a turnover event occurred within the second winter of the study. This turnover event disrupted the biological communities at 40 m and led to a reestablished community comprised of different species that those found prior to the event. Upper waters within the sink show that clines establish themselves seasonally and partition zones that confine bacterial communities that are more similar to each other within these zones while excluding bacterial communities that are outside of these zones. Oxygenated water was shown to not contain prokaryotes containing the dsr gene. As the oxycline changed seasonally, dissimilatory sulfite reducing prokaryotes containing the dsr gene remained in the anoxic zone and required time to reestablish themselves whenever oxygenated water displaced them.

Na Antártica e no Ártico, a vida microbiana é presente e diversificada, tendo se adaptado a condições similares às encontradas em mundos que despertam interesse à astrobiologia. Este estudo investigou as alterações fisiológicas e fenotípicas do extremófilo psicrotrófico Exiguobacterium antarcticum B7 sob diferentes temperaturas. Foram utilizadas técnicas de eletroforese de proteínas em duas dimensões associada a espectrometria de massa, juntamente com técnicas de microscopia. E. antarcticum foi também submetida a condições físicas encontradas em Marte e no ambiente interplanetário. Seguindo evidências de que micro-organismos psicrotróficos podem também ser resistentes a períodos prolongados de dessecação, foi investigada a presença de linhagens microbianas resistentes a anidrobiose em solos da Antártica e permafrost do Ártico. Por fim, voltando-se à ecologia microbiana de solos polares permanentemente congelados, foi estudada a diversidade microbiana de três estratos de permafrost do ártico canadense com o uso de sequenciamento de DNA de nova geração. / In Antarctica and the Arctic, microbial life is present and diverse, having adapted to low temperature and low humidity conditions, similar to the worlds that arouse more interest to astrobiology. This study investigated the physiological changes of the psychrotrophic extremophile Exiguobacterium antarcticum B7 at different temperatures. Two dimensional protein electrophoresis techniques was used for proteome analysis in conjunction with mass spectrometry, as well as microscopy techniques. E. antarcticum was also subjected to physical parameters found on Mars and in the interplanetary environment. Following evidence that psychrotrophic microorganisms may also be resistant to prolonged periods of desiccation, it was investigated the presence of microbial strains resistant to anhydrobiosis on Antarctic soils and Arctic permafrost. At last, turning the attention to the microbial ecology of permanently frozen polar soils, it was studied the bacterial diversity of three Arctic permafrost strata with next-generation high throughput DNA sequencing.

Environmental gradients exist at global and local scales and the variable conditions they encompass allow the coexistence of different microbial assemblages. Studying gradients and the selection forces they enclose can reveal the spatial succession and interactions of microorganisms and, therefore, how they are assembled in functionally stable communities. By combining high-throughput sequencing technology and laboratory experimental approaches, I investigated the factors that influence the microbial community assemblages in two types of environmental gradients in the Red Sea. I have studied the communities in the chemoclines occurring at the transition zones along the interfaces between seawater and the Deep Hypersaline Anoxic Brines (DHABs) at the bottom of the Red Sea. Across these chemoclines salinity increases of 5-10 times respect to the overlying seawater. I compared the microbial community diversity and metabolisms in the chemoclines of five different DHABs, finding different microbial community compositions due to the different DHABs characteristics, but the same succession of metabolisms along the five interfaces. From the Suakin Deep brine, I assembled the genome of a novel bacterial phylum and revealed the metabolic features that allow this organism to cope with the challenging variable conditions along the chemocline. In an alternative environmental system, I studied the effect of different thermal regimes on the microbiome of coastal sediment exposed to different yearly ranges of temperature variation. Sediment bacterial communities living under larger temperature variations are more flexible and can grow under a larger range of thermal conditions than communities experiencing narrower temperature ranges. My results highlight the large metabolic flexibility of microorganisms and their capacity to efficiently self-organize in complex functional assemblages under extreme ranges of environmental conditions.