In the Boreal region, the length of day and night varies extremely over the year and organisms are exposed to different diel (24 h) fluctuations of light and temperature. Among them are pelagic populations of bacteria that are ubiquitous in freshwater ecosystems. The structure of prokaryotic assemblages in lakes is regulated by both abiotic and biotic parameters known to have diel patterns; yet, knowledge on changes of the active bacterial community composition (aBCC) over diel cycles is limited, especially at short temporal scales. Here, measurements of physicochemical parameters, nutrient levels and chlorophyll a concentrations, characterization of the carbon pool and 16S rRNA sequencing were used to elucidate the aBCC in a peat bog and the oligotrophic lake Klocka in Jämtland county, Sweden. I show that the activity of bacterioplankton communities remained relatively stable at 6-h intervals and did not follow any diel patterns during an uneven light regime period in June. However, the activity of peat bog communities changed in a cyclic pattern over three diel cycles during an even light period in September, whereas diel changes did not substantially differentiate between sampling periods and among depths in Klocka. The results of the thesis provide valuable insights into the importance of diel cycles in bacterial diversity and community dynamics in lentic habitats.

Coral reefs are under considerable decline. The framework builders in coral reefs are scleractinian corals, which comprise so-called holobionts, consisting of cnidarian host, algal symbionts (genus Symbiodinium), and other associated microbes. Corals are commonly considered stenohaline osmoconformers, possessing limited capability to adjust to salinity changes. However, corals differ in their ability to cope with different salinities. The underlying mechanisms have not yet been addressed. To further understand putative mechanisms involved, I examined coral holobiont osmoregulation conducting a range of experiments on the coral Fungia granulosa. In my research F. granulosa from the Red Sea exhibited pronounced physiological reactions (decreased photosynthesis, cessation of calcification) upon short-term incubations (4 h) to high salinity (55). However, during a 29-day in situ salinity transect experiment, coral holobiont photosynthesis was unimpaired under high salinity (49) indicating acclimatization. F. granulosa microbiome changes after the 29-day high salinity exposure aligned with a bacterial community restructuring that putatively supports the coral salinity acclimatization (osmolyte synthesis, nutrient fixation/cycling). Long-term incubations (7 d) of cultured Symbiodinium exhibited cell growth even at ‘extreme’ salinity levels of 25 and 55. Metabolic profiles of four Symbiodinium strains exposed to increased (55) and decreased (25) salinities for 4 h indicated distinct carbohydrates and amino acids to be putatively involved in the osmoadjustment. Importantly, under high salinity the osmolyte floridoside was consistently increased. This could be corroborated in the coral model Aiptasia and in corals from the Persian/Arabian Gulf, where floridoside was also markedly increased upon short- (15 h) and long-term (>24 months) exposure to high salinity, confirming an important role of floridoside in the osmoadjustment of cnidarian holobionts. This thesis demonstrates osmoacclimatization of F. granulosa and osmoadjustment of cultured Symbiodinium. All three main compartments (i.e. coral host, Symbiodinium, bacteria) seem to contribute to the coral holobionts salinity adjustment. However, the exact mechanisms of coral host and bacteria contribution remain to be determined. Floridoside likely constitutes a conserved osmolyte increasing the salinity resilience of Symbiodinium and also of the cnidarian/coral holobiont. Floridoside further possess’ antioxidative properties, possibly providing a protection from reactive oxygen species formation as a result of salinity stress or/and other environmental stressors.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第17775号 / 理博第3898号 / 新制||理||1562(附属図書館) / 30582 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 准教授 奥田 昇, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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A imensa diversidade de micro-organismos no solo leva a uma inevitável riqueza de interações entre espécies. Neste intuito, esse projeto é inovador na identificação dos cogumelos da Mata Atlântica, e na descrição da comunidade bacteriana associada às suas micosferas. Usando corpos de frutificação dos fungos (cogumelos) como indicadores para sistemas ricos em nutrientes, as amostras foram coletadas para investigar as interações entre os fungos (maioria do domínio Basidiomycota) e as bactérias presentes no solo em volta das micélios fúngicos (ambiente micosférico). As análises foram feitas com técnicas independentes de cultivo (análise PCR-DGGE, sequenciamento Sanger de fragmentos de ITS/18S e pirosequenciamento de tags da região V4 de 16S DNAr). As famílias fúngicas Marasmiaceae e Lepiotaceae, do domínio Basidiomycota foram as mais abundantes entre os cogumelos amostrados (13 e 5 cogumelos, respectivamente), e estavam presentes entre todas as três parcelas de estudo. As demais amostras foram alocadas dentro das famílias Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae e Strophariaceae, além de dois do domínio Ascomycota. Baseado na análise de DGGE, é bastante claro que existe uma grande diferença na comunidade bacteriana (de toda a comunidade bacteriana, de ?- proteobacteria e de ?-proteobacteria) entre os solos associados ou não com os corpos de frutificação. Os dados de pirosequenciamento indicaram que dentro dos tratamentos as amostras se agruparam baseado nas famílias fúngicas ou no substrato onde os cocumeglos ocorrem (solo ou serrapilheira), sendo clara em algumas micosferas as alterações na ocorrência de grupos microbianos. O grupo de UTOs mais induzidas na região da micosfera foi composto dos grupos Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3 e Spartobacteria gênero incertae sedis. Isto indica que existe um processo de seleção para bactérias específicas dependendo das diversas variáveis e fatores ambientais presentes no microhabitat micosfera. / The immense microbial diversity in the soil leads to inevitable richness in inter-species interactions. For this reason, this is a novel project that focuses on identifying mushrooms and the associated bacterial community with their mycospheres in the Brazilian Atlantic Rainforest. Using fungal fruiting bodies (mushrooms) as indicators of nutrient-rich systems, samples were taken to investigate the interactions between fungi (mostly of the domain Basidiomycota) and bacteria present in the soil surrounding fungal mycelia (mycosphere environment). The analyses were conducted using culture-independent techniques, where isolating DNA of bacteria and/or mushrooms attempts to provide information on microbial functionality. These culture-independent analyses (PCR-DGGE, Sanger sequencing of ITS/18S fragments, and pyrosequencing of tags from the V4 region of 16S rDNA) generated extensive data on the bacterial diversity selected for in the presence of fungal structures in the soil. The fungal families Maramiaceae and Lepiotaceae, of the domain Basidiomycota were the most abundant among the mushrooms sampled (13 and 5 mushrooms, respectively) and were present in all of the three sampling sites. The rest of the mushrooms were found to be within the families Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae, and Strophariaceae, in addition to two of the domain Ascomycota. Based on DGGE analysis, it is clear that there is a great difference in the bacterial community (entire bacterial community, of ?- proteobacteria and of ?-proteobacteria) between all fungal fruiting body-associated and non-associated soils. The pyrosequencing data indicated that within each treatment group, the samples did not separate according to fungal families or substrate where the mushrooms were found (in the soil or amoung the leaf litter). However, some mycospheres exhibited clearly altered bacterial communities. The group of OTUs most induced in the mycosphere region consisted of Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3, and Spartobacteria genus incertae sedis. This indicates that there is a selection process for specific bacteria depending on a wide range of variable and environmental factors acting in the mycosphere microhabitat.

Sabe-se que humanos, plantas e animais são colonizados por uma elevada diversidade de microrganismos e que esses organismos eucariotos dependem destes microrganismos para manutenção do seu desenvolvimento. Usando dois modelos de associação microrganismo-hospedeiro, foi testado a hipótese de que hospedeiros pertencentes a domínios da vida distintos, apesar de suas particularidades estruturais, genotípicas, filogenéticas e fisiológicas, compartilham similaridades nos modos de associação com a comunidade bacteriana. Sendo assim, o objetivo do presente trabalho foi mapear a comunidade bacteriana associada a plantas do gênero Anthurium endêmicas e/ou não. Paralelamente, mapear a comunidade bacteriana associada a gêneros distintos de cianobactérias, ao longo da curva de crescimento e quando esta é submetida a condições de cultivo distintas. Como resultados, primeiramente, foi observado que plantas Anthurium alcatrazense endêmicas da Ilha apresentam riqueza e diversidade menor que as plantas da espécie Anthurium loefgrenii coletada na ilha de Alcatrazes e também menor que as plantas Anthurium intermedium e Anthurium pentaphyllum coletadas na região de continente. Também foi observado que a estrutura da comunidade bacteriana associada as plantas de A. alcatrazense é distinta quando comparada com as plantas coletadas no continente e também da própria ilha de Alcatrazes. Essa dissimilaridade foi principalmente representada por OTUs afiliadas à Betaproteobacteria e Gammaproteobacteria. Esses resultados sugerem especificidade microrganismo-hospedeiro. Considerando a associação cianobactéria e bactérias heterotróficas, os resultados demonstraram que a comunidade bacteriana associada é especifica de acordo com o gênero de cianobactéria, composta principalmente por classes apresentando abundância relativa de sequencias distintas como, Betaproteobacteria, Gammaproteobacteria, Flavobacteria e Cytophagia. Por outro lado, foi possível observar que ao longo das fases de multiplicação da linhagem Microcystis aeruginosa, ocorre uma sucessão de grupos bacterianos, sendo principalmente representado pela variação da abundância de Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria e Flavobacteria relativo a fase estacionaria de multiplicação. Quando submetida em condições de cultivo distintas, foi possível observar que variações nas taxas de multiplicação da cianobactéria influenciaram uma modulação da estrutura da comunidade bacteriana associada, desta forma sugerindo que rápidas alterações na estrutura da comunidade bacteriana associada a M. aeruginosa, é resultado de processos de auto-regulação entre cianobactéria e bactérias heterotróficas associadas. De forma geral, pode-se sugerir que hospedeiros distintos apresentam padrões de associações com as bactérias similares, podendo estas similaridades sugerir estratégias para um melhor entendimento e manejo dos ecossistemas. / It is known that humans, plants and animals are colonized by a high diversity of microorganisms and that these eukaryotic organisms depend on these microorganisms to maintain their development. Using two microorganism-host association models, we hypothesized that hosts belonging to distinct domains of life, despite their structural, genotypic, phylogenetic and physiological particularities, share similarities in the modes of association with the bacterial community. Thus, the objective of this work was to map the bacterial community associated with plants of the genus Anthurium endemic and / or not. In parallel, map the bacterial community associated with distinct genera of cyanobacteria, along the growth curve of and when it is submitted to different culture conditions. In this context, we observed that Anthurium alcatrazense plants endemic to the Island, present less richness and diversity than the plants of the species Anthurium loefgrenii collected in the island of Alcatrazes and smaller than the plants Anthurium intermedium and Anthurium penthaphyllum collected in the continent. We found that the structure of the bacterial community associated with the plants of A. alcatrazense is distinct when compared to the plants collected in the continent and island of Alcatrazes itself. This dissimilarity was mainly represented by OTUs affiliated with Betaproteobacteria and Gammaproteobacteria. These results suggest microorganism-host specificity. Considering the association cyanobacteria and heterotrophic bacteria, the results demonstrated that the associated bacterial community is specific according to the genus of cyanobacteria, composed mainly by abundance distinct from those of classes, Betaproteobacteria, Gammaproteobacteria, Flavobacteria and Cytophagia. On the other hand, it was possible to observe that during the multiplication stages of the Microcystis aeruginosa strain, a succession of bacterial groups occurs, mainly represented by the variation of the abundance of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Flavobacteria relative to the stationary phase of multiplication. When submitted under different culture conditions, it was possible to observe that variations in cyanobacteria multiplication rates influenced a modulation of the associated bacterial community structure, thus suggesting that rapid changes in the bacterial community structure associated with M. aeruginosa is a result of processes of self-regulation between cyanobacteria and associated heterotrophic bacteria. In general, distinct hosts show patterns of associations with similar bacteria, and these similarities may suggest strategies for a better understanding and management of ecosystems.

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, although there are two core bacterial phyla, Firmicutes and Proteobacteria. Feedstock, facility, and fermentation technology may explain the difference in community structure between different facilities. Lactic acid, the most important environmental variable that influences bacterial community structure grouping, could be utilized as an indicator of bacterial contamination. We also identified genes responsible for the multiple antibiotic-resistance phenotype of an Enterobacter cloacae strain isolated from a bioethanol fermentation facility. We performed PCR assays and revealed the presence of canonical genes encoding resistance to penicillin and erythromycin. However, a gene encoding resistance to virginiamycin was not detected.

A imensa diversidade de micro-organismos no solo leva a uma inevitável riqueza de interações entre espécies. Neste intuito, esse projeto é inovador na identificação dos cogumelos da Mata Atlântica, e na descrição da comunidade bacteriana associada às suas micosferas. Usando corpos de frutificação dos fungos (cogumelos) como indicadores para sistemas ricos em nutrientes, as amostras foram coletadas para investigar as interações entre os fungos (maioria do domínio Basidiomycota) e as bactérias presentes no solo em volta das micélios fúngicos (ambiente micosférico). As análises foram feitas com técnicas independentes de cultivo (análise PCR-DGGE, sequenciamento Sanger de fragmentos de ITS/18S e pirosequenciamento de tags da região V4 de 16S DNAr). As famílias fúngicas Marasmiaceae e Lepiotaceae, do domínio Basidiomycota foram as mais abundantes entre os cogumelos amostrados (13 e 5 cogumelos, respectivamente), e estavam presentes entre todas as três parcelas de estudo. As demais amostras foram alocadas dentro das famílias Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae e Strophariaceae, além de dois do domínio Ascomycota. Baseado na análise de DGGE, é bastante claro que existe uma grande diferença na comunidade bacteriana (de toda a comunidade bacteriana, de ?- proteobacteria e de ?-proteobacteria) entre os solos associados ou não com os corpos de frutificação. Os dados de pirosequenciamento indicaram que dentro dos tratamentos as amostras se agruparam baseado nas famílias fúngicas ou no substrato onde os cocumeglos ocorrem (solo ou serrapilheira), sendo clara em algumas micosferas as alterações na ocorrência de grupos microbianos. O grupo de UTOs mais induzidas na região da micosfera foi composto dos grupos Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3 e Spartobacteria gênero incertae sedis. Isto indica que existe um processo de seleção para bactérias específicas dependendo das diversas variáveis e fatores ambientais presentes no microhabitat micosfera. / The immense microbial diversity in the soil leads to inevitable richness in inter-species interactions. For this reason, this is a novel project that focuses on identifying mushrooms and the associated bacterial community with their mycospheres in the Brazilian Atlantic Rainforest. Using fungal fruiting bodies (mushrooms) as indicators of nutrient-rich systems, samples were taken to investigate the interactions between fungi (mostly of the domain Basidiomycota) and bacteria present in the soil surrounding fungal mycelia (mycosphere environment). The analyses were conducted using culture-independent techniques, where isolating DNA of bacteria and/or mushrooms attempts to provide information on microbial functionality. These culture-independent analyses (PCR-DGGE, Sanger sequencing of ITS/18S fragments, and pyrosequencing of tags from the V4 region of 16S rDNA) generated extensive data on the bacterial diversity selected for in the presence of fungal structures in the soil. The fungal families Maramiaceae and Lepiotaceae, of the domain Basidiomycota were the most abundant among the mushrooms sampled (13 and 5 mushrooms, respectively) and were present in all of the three sampling sites. The rest of the mushrooms were found to be within the families Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae, and Strophariaceae, in addition to two of the domain Ascomycota. Based on DGGE analysis, it is clear that there is a great difference in the bacterial community (entire bacterial community, of ?- proteobacteria and of ?-proteobacteria) between all fungal fruiting body-associated and non-associated soils. The pyrosequencing data indicated that within each treatment group, the samples did not separate according to fungal families or substrate where the mushrooms were found (in the soil or amoung the leaf litter). However, some mycospheres exhibited clearly altered bacterial communities. The group of OTUs most induced in the mycosphere region consisted of Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3, and Spartobacteria genus incertae sedis. This indicates that there is a selection process for specific bacteria depending on a wide range of variable and environmental factors acting in the mycosphere microhabitat.

Sabe-se que humanos, plantas e animais são colonizados por uma elevada diversidade de microrganismos e que esses organismos eucariotos dependem destes microrganismos para manutenção do seu desenvolvimento. Usando dois modelos de associação microrganismo-hospedeiro, foi testado a hipótese de que hospedeiros pertencentes a domínios da vida distintos, apesar de suas particularidades estruturais, genotípicas, filogenéticas e fisiológicas, compartilham similaridades nos modos de associação com a comunidade bacteriana. Sendo assim, o objetivo do presente trabalho foi mapear a comunidade bacteriana associada a plantas do gênero Anthurium endêmicas e/ou não. Paralelamente, mapear a comunidade bacteriana associada a gêneros distintos de cianobactérias, ao longo da curva de crescimento e quando esta é submetida a condições de cultivo distintas. Como resultados, primeiramente, foi observado que plantas Anthurium alcatrazense endêmicas da Ilha apresentam riqueza e diversidade menor que as plantas da espécie Anthurium loefgrenii coletada na ilha de Alcatrazes e também menor que as plantas Anthurium intermedium e Anthurium pentaphyllum coletadas na região de continente. Também foi observado que a estrutura da comunidade bacteriana associada as plantas de A. alcatrazense é distinta quando comparada com as plantas coletadas no continente e também da própria ilha de Alcatrazes. Essa dissimilaridade foi principalmente representada por OTUs afiliadas à Betaproteobacteria e Gammaproteobacteria. Esses resultados sugerem especificidade microrganismo-hospedeiro. Considerando a associação cianobactéria e bactérias heterotróficas, os resultados demonstraram que a comunidade bacteriana associada é especifica de acordo com o gênero de cianobactéria, composta principalmente por classes apresentando abundância relativa de sequencias distintas como, Betaproteobacteria, Gammaproteobacteria, Flavobacteria e Cytophagia. Por outro lado, foi possível observar que ao longo das fases de multiplicação da linhagem Microcystis aeruginosa, ocorre uma sucessão de grupos bacterianos, sendo principalmente representado pela variação da abundância de Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria e Flavobacteria relativo a fase estacionaria de multiplicação. Quando submetida em condições de cultivo distintas, foi possível observar que variações nas taxas de multiplicação da cianobactéria influenciaram uma modulação da estrutura da comunidade bacteriana associada, desta forma sugerindo que rápidas alterações na estrutura da comunidade bacteriana associada a M. aeruginosa, é resultado de processos de auto-regulação entre cianobactéria e bactérias heterotróficas associadas. De forma geral, pode-se sugerir que hospedeiros distintos apresentam padrões de associações com as bactérias similares, podendo estas similaridades sugerir estratégias para um melhor entendimento e manejo dos ecossistemas. / It is known that humans, plants and animals are colonized by a high diversity of microorganisms and that these eukaryotic organisms depend on these microorganisms to maintain their development. Using two microorganism-host association models, we hypothesized that hosts belonging to distinct domains of life, despite their structural, genotypic, phylogenetic and physiological particularities, share similarities in the modes of association with the bacterial community. Thus, the objective of this work was to map the bacterial community associated with plants of the genus Anthurium endemic and / or not. In parallel, map the bacterial community associated with distinct genera of cyanobacteria, along the growth curve of and when it is submitted to different culture conditions. In this context, we observed that Anthurium alcatrazense plants endemic to the Island, present less richness and diversity than the plants of the species Anthurium loefgrenii collected in the island of Alcatrazes and smaller than the plants Anthurium intermedium and Anthurium penthaphyllum collected in the continent. We found that the structure of the bacterial community associated with the plants of A. alcatrazense is distinct when compared to the plants collected in the continent and island of Alcatrazes itself. This dissimilarity was mainly represented by OTUs affiliated with Betaproteobacteria and Gammaproteobacteria. These results suggest microorganism-host specificity. Considering the association cyanobacteria and heterotrophic bacteria, the results demonstrated that the associated bacterial community is specific according to the genus of cyanobacteria, composed mainly by abundance distinct from those of classes, Betaproteobacteria, Gammaproteobacteria, Flavobacteria and Cytophagia. On the other hand, it was possible to observe that during the multiplication stages of the Microcystis aeruginosa strain, a succession of bacterial groups occurs, mainly represented by the variation of the abundance of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Flavobacteria relative to the stationary phase of multiplication. When submitted under different culture conditions, it was possible to observe that variations in cyanobacteria multiplication rates influenced a modulation of the associated bacterial community structure, thus suggesting that rapid changes in the bacterial community structure associated with M. aeruginosa is a result of processes of self-regulation between cyanobacteria and associated heterotrophic bacteria. In general, distinct hosts show patterns of associations with similar bacteria, and these similarities may suggest strategies for a better understanding and management of ecosystems.