Microbes have a remarkable ability to adapt to a host of environmental stressors, including low temperature, high pressure and osmotic stresses. The adaptations of resistant microbes to low temperatures are varied, and may include the accumulation of solutes to maintain osmotic balance, the production of antifreeze proteins (AFPs) or ice nucleation proteins (INPs) to manipulate ice growth or formation. AFPs depress the freezing point, inhibit ice recrystallization, and have been reported to inhibit or delay the growth of gas hydrates. Conversely, INPs precipitate ice formation at relatively high subzero temperatures. Collectively, these activities can be described as ‘ice-association’ activities. Here, ice-affinity and/or freeze-thaw cycling were used to either select for isolates with ice association properties or to assess the low temperature resistance of microbial consortia derived from various environments. Ice-affinity successfully selected psychrotolerant microbes from cultured temperate and boreal soils, some of which had been previously reported in glaciers and Arctic/Antarctic sites. Many of the recovered microbes demonstrated ice-association activities. Freeze-thaw selection also greatly decreased the abundance and diversity of consortia from distinct sites, and allowed the recovery of individual isolates, many of which demonstrated ice-association. Freeze-thaw selection was also used to assess the role of cross-tolerance between osmotic and freeze-thaw stresses, based on the common challenge of desiccation. Microbial consortia from lakes with varying degrees of salinity were subjected to freeze-thaw stress, and the consortia from more saline lakes tended to show greater low temperature resistance. While few of the recovered microbes demonstrated ice-association activities, those from the more saline lakes tended to contain a higher intracellular solute concentration and were more likely to form biofilms. This underscores the diversity of resistance strategies and supports the notion of cross-tolerance. To determine if these selective regimes would have applications for hydrate growth inhibition, microbes derived from an oil well sample were subjected to freeze-thaw stress. Selection reduced microbial abundance, shifted the diversity, and resulted in the recovery of microbes with some ice-association activity. Taken together, this thesis demonstrates that the application of low temperature stress can be used to successfully investigate stress resistance mechanisms within microbial communities from distinct environments. / Thesis (Ph.D, Biology) -- Queen's University, 2010-09-21 15:58:14.932

Fungi play important roles in various environments. Some of them infect plants and cause economically important diseases. However, many fungal pathogens cause similar symptoms or are even spread asymptomatically, making it difficult to identify them morphologically. Therefore, culture-independent, sequence-based diagnostic methods that can detect and identify fungi independently of the symptoms that they cause are desirable. Whole genome metagenomic sequencing has the potential to enable rapid diagnosis of plant diseases without culturing pathogens and designing pathogen-specific probes. In my study, the MinION nanopore sequencer, a portable single‐molecule sequencing platform developed by Oxford Nanopore Technologies, was employed to detect the fungus Calonectria pseudonaviculata (Cps), the causal agent of the devastating boxwood blight disease of the popular ornamental boxwood (Buxus spp.). Various DNA extraction methods and computational tools were compared. Detection was sensitive with an extremely low false positive rate for most methods. Therefore, metagenomic sequencing is a promising technology that could be implemented in routine diagnostics of fungal diseases. Other fungi may play important roles in the atmosphere because of their ice nucleation activity (INA). INA is the capacity of some particles to induce ice formation above the temperature that pure water freezes (-38°C). Importantly, INPs affect the ratio of ice crystals to liquid droplets in clouds, which in turn affects Earth's radiation balance and the intensity and frequency of precipitation. A few fungal species can produce ice nucleating particles (INPs) that cause ice formation at temperatures ≥ –10°C and they may be present in clouds. Two such fungal genera are Fusarium and Mortierella but little is known about their INPs and the genetic basis of their INA. In my study, F. avenaceum and M. alpina were examined in detail. INPs of both species were characterized and it was found that strains within both species varied in regards to the strength of INA. Whole genome sequencing and comparative genomic studies were then performed to identify putative INA genes. Differential expression analyses at different growth temperatures were also performed. INP properties of the two species shared similarities, both appearing to consist of secreted aggregates larger than 30 kDa. Low temperatures induced INA in both species. Lists of candidate INA genes were identified based on their presence in the strains with the strongest INA and/or induction of their expression at low temperatures and because they either encode secreted proteins or enzymes that produce other molecules known to have INA in other organisms. These genes can now be characterized further to help identify the fungal INA genes in both species. This can be expected to help increase our understanding of the role of fungal INA in the atmosphere. / Doctor of Philosophy / Fungi are important to life on Earth and play roles in the environments that surround us. On the one hand, fungi can make plants sick and some plant diseases may even cause economic losses to farmers. If the cause of a disease can be identified accurately in an early stage before symptoms develop, disease transmission may be prevented and plants may be protected from disease. However, it is a challenge to find out which fungus causes which disease since symptoms of different fungal diseases look very similar. Typically, we have to wait for plants to become very sick or we have to isolate the fungus that causes a disease to identity it, which may be time-consuming and not lead to precise identification. DNA sequencing technologies have the potential to lead to more sensitive, faster, and more accurate disease diagnosis and, therefore, may help prevent disease outbreaks. In my study, the MinION nanopore sequencer, a small portable device, was used to detect the fungus causing boxwood blight on boxwood. By loading the DNA of unhealthy boxwood on the device, the boxwood blight pathogen was identified within a very short time. Thus, this method is a promising diagnostic method that may be applied to detect other plant fungal diseases as well. On the other hand, fungi may affect Earth's climate by affecting how many water droplets in clouds are frozen, which in turn affects Earth's temperature and how often and how much it rains and snows. Fungi may affect the freezing of water droplets in clouds since some of them have ice nucleation activity (INA), which is the capacity to catalyze ice formation at a higher temperature than the temperature at which pure water freezes (-38°C), and they may be present in clouds. So far, INA has only been found in a few fungi, including the species Fusarium avenaceum and Mortierella alpina, but the mechanism of their INA is poorly understood. In my study, multiple F. avenaceum and M. alpina strains were examined in detail. Two approaches were used. First, strains in each species were compared with each other to find out how strong their INA is. Once it was found that they differed in their strength of INA, their genomes were sequenced and compared to find genes present in the most active strains and missing from the least active strains since it is these genes that may contribute to INA. It was also found that both fungal species had stronger INA when they were grown at lower temperatures. Therefore, the expression of their genes between higher and lower temperatures was compared to find the genes that were more highly expressed at lower temperatures since it is these genes that may cause INA. Based on previous studies, fungal INPs may either consist of secreted proteins or be the products of biosynthetic gene clusters. Therefore, the list of potential genes was reduced by looking for genes encoding either secreted proteins or biosynthetic gene clusters. The list of these potential INA genes will make it easier to identify the INA genes in F. avenaceum and M. alpina and determine the role of fungi in affecting the weather and climate on Earth.

Pendant longtemps, les microorganismes présents dans l’atmosphère n’ont été considérés qu’en tant que particules inertes subissant les conditions hostiles de cet environnement. Cependant, de récentes études mettant en évidence la présence de microorganismes métaboliquement actifs dans la phase aqueuse des nuages incitent à s’interroger sur le rôle que ces organismes pourraient avoir sur les processus physiques et chimiques des nuages. En effet, la formation de gouttelettes de nuage ou de cristaux de glace à des températures supérieures à -36°C nécessite la présence de particules dites « noyaux de condensation » ou « noyaux glaçogènes », dont les bactéries pourraient être des représentantes. De plus, plusieurs travaux ont révélé une importance potentielle des microorganismes dans la transformation de la matière organique dans les nuages. L’objectif de ces travaux de thèse a donc été d’étudier les interactions réciproques entre les microorganismes et les conditions physico-chimiques des nuages. Dans un premier temps, les composantes physico-chimiques et microbiologiques ont été caractérisées au moyen de prélèvements nuageux au sommet du puy de Dôme (1465 m, France) et des études statistiques ont permis de mettre en avant des corrélations entre les différents paramètres physico-chimiques et/ou biologiques. Puis, cinq souches microbiennes appartenant à des genres microbiens cultivables majeurs dans les nuages ont été soumises à quatre stress rencontrés dans les nuages : la lumière solaire, la présence de peroxyde d’hydrogène, les variations de chocs osmotiques intervenant lors de la formation et de la dissipation des gouttelettes d’eau et les cycles de gel et de dégel. Il a ainsi été mis en évidence que la lumière solaire et le peroxyde d’hydrogène dans des conditions nuageuses n’ont que peu ou pas d’impact sur la viabilité des cellules. A l’inverse, les chocs osmotiques et le gel-dégel peuvent être hautement délétères selon les souches considérées. La troisième partie de ce travail s’est focalisé à mettre en évidence la présence de souches bactériennes glaçogènes dans l’eau de nuage. Sept souches ont ainsi ont été identifiées et décrites, et l’une d’entre elles a été choisie comme modèle pour étudier le comportement de bactéries (survie et activité glaçogène) dans une chambre de simulaion de nuage (AIDA, Allemagne). En parallèle, l’activité glaçogène biologique de l’eau de nuage a été mesurée à partir de prélèvements au puy de Dôme et l’activité glaçogène bactérienne a été estimée. L’ensemble de ces travaux met en avant une sous-estimation jusqu’alors des proportions de bactéries glaçogènes dans les modèles numériques simulant les processus microphysiques d’initiation de la glace et des précipitations dans les nuages. Ces données vont désormais pouvoir être considérées dans de tels modèles. Enfin, afin d’estimer l’étendue de l’importance des microorganismes dans la chimie atmosphérique, il est nécessaire d’avoir recours à des modèles numériques. La dernière étude de cette thèse s’est consacrée à déterminer des constantes cinétiques de biodégradation de trois composés organiques majeurs des nuages par trois souches bactériennes isolées de cet environnement qui pourront servir à paramétrer des modèles numériques. Une première approche simple a permis de confirmer les résultats précédents de l’équipe en mettant en avant une contribution non négligeable des microorganismes dans leur dégradation. / Airborne microorganisms have long been considered as inert, passive particles dealing with hostile conditions. Recent studies highlighting metabolic activity in cloud water raised questions about the role these organisms may play on physical and chemical processes in clouds. Indeed, cloud droplets and ice crystals formation at temperature warmer than -36°C need the presence of particles called “cloud condensation nuclei” or “ice nuclei”. Bacteria could be one of them. In addition, several works revealed a potential importance of microorganisms in organic matter transformation in clouds. The objective of this thesis was to study the reciprocal interactions between microorganisms and physico-chemical conditions in clouds. First, cloud physico-chemical and microbiological compositions were described by cloud sampling at the puy de Dôme station (1465 m, France) and statistical analyses were performed to highlight correlations between physico-chemical and/or biological parameters. Secondly, five microbial strains belonging to genera frequently isolated from cloud water were subjected to four atmospheric stresses: sunlight, hydrogen peroxide, osmotic shocks occurring when water droplets condensate or evaporate and freeze-thaw cycles. Thus, it was pointed that sunlight and hydrogen peroxide at cloud concentration have no or little impact on cell viability. On the opposite, osmotic shocks and freeze-thaw can be highly deleterious depending on the considered strain. The third part of this thesis focused on the detection of ice nucleating bacterial strains in cloud water. Seven strains were thus identified and described, and one of them was selected as a model to study its behavior (survival and ice nucleation activity, INA) in a cloud simulation chamber (AIDA, Germany). In parallel, biological ice nucleation activity was measured directly on cloud samples and bacterial INA was estimated. All these experiments highlighted underestimations of ice nucleation active bacteria in models simulating microphysical processes in clouds. This new dataset may be used as new parameterization in this kind of models. Finally, in order to estimate the bacterial contribution in cloud chemistry, numerical means are needed. Therefore, the last study of this thesis focused on the determination of biological kinetic constants that may be implemented in atmospheric chemistry models. The biodegradation of three major organic compounds encountered in cloud water by three bacterial strains isolated from clouds was measured. A first approach confirmed precedent team results highlighting a considerable contribution of microorganisms on the transformation of these compounds.