Bacteria are ubiquitous to marine living surfaces, taking on a broad spectrum of roles from mutualistic to pathogenic. Despite their universality, much remains unknown about their basic ecology and interactions with higher organisms. To address this gap, this thesis firstly examines the bacterial communities associated with three co-occurring marine eukaryote hosts from temperate Australia: the demosponge Cymbastela concentrica, the subtidal red macroalga Delisea pulchra and the intertidal green macroalga Ulva australis. Molecular characterisation of the bacterial communities was undertaken using 16S rRNA gene library analysis to compare within-host (alpha) and between-host (beta) diversity for the three microbial communities. This study highlights the potentially substantial contribution host-associated microorganisms could have on marine microbial diversity. The remaining focus for this thesis was on the bacterial community associated with D. pulchra. This alga produces a suite of biologically active secondary metabolites (furanones) that non-toxically inhibit acyl homoserine lactone (AHL)-driven quorum sensing in bacteria, affecting a range of phenotypes including colonisation and virulence traits. The ecology of D. pulchra???s epiphytic bacteria was investigated using a mechanistic approach to explain bacterial colonisation patterns. In particular, concepts and models of ecological succession founded in eukaryote ecology were investigated. The thesis concludes with a study investigating the effect of furanones and elevated temperature on bacteria-induced disease and thallus bleaching of D. pulchra. In the presence of furanones colonisation and infection of two Roseobacter isolates from D. pulchra???s epiphytic bacterial community were inhibited. Ruegeria strain R11 was demonstrated to have temperature regulated virulence, which caused thallus bleaching in furanone-free algae. The implications of elevated sea temperatures resulting from global warming for algal health are discussed.

Bacteria play important roles in the transformation of nutrients in wetlands, but few studies have examined parameters affecting variation in bacterial community composition between wetlands. We compared the composition of eubacterial and denitrifying bacterial biofilm communities in 32 agricultural constructed wetlands in southern Sweden, and the extent to which wetland environmental parameters could explain the observed variation. Structure and richness of the eubacterial 16S rRNA gene and three denitrifying bacterial enzyme genes (nirK, nirS and nosZ), analysed by molecular fingerprinting methods, varied among the constructed wetlands, which could be partly explained by different environmental parameters. Results from the enzyme gene analyses were also compared to determine whether the practice of using a single denitrifying bacterial gene could characterize the overall community composition of denitrifying bacteria. We found that nirK was more diverse than both nirS and the nosZ, and the band structure and richness of the three genes were not related to the sam environmental parameters. This suggests that using a single enzyme gene may not suffice to characterize the community composition of denitrifying bacteria in constructed agricultural wetlands. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

Understanding microbial evolution lies at the heart of microbiology and environmental sciences. Numerous studies have been dedicated to elucidating the underlying mechanisms that create microbial genetic diversity and adaptation. However, due to technical limitations such as the high level of uncultured cells in almost every natural habitat, most of current knowledge is primarily based on axenic cultures grown under laboratory conditions, which typically do not simulate well the natural environment. How well the knowledge from isolates translates to in-situ processes and natural microbial communities remains essentially speculative. The recent development of culture-independent genomic techniques (aka metagenomics) provides possibilities to bypass some of these limitations and provide new insights into microbial evolution in-situ. To date, most of metagenomic studies have been focused on a few reduced-diversity model communities, e.g., acid mine drainage. Highly complex communities such as those of soil and sediment habitats remain comparatively less understood. Furthermore, a great power of metagenomics, which has not been fully capitalized yet, is the ability to follow the evolution of natural microbial communities over time and environmental perturbations, i.e., times-series metagenomics. Although the recent developments in DNA sequencing technologies have enabled (inexpensive) time-series studies, the bioinformatics approaches to analyze the resulting data have clearly fallen behind. Taken together, to scale up metagenomics for complex community studies, three major challenges remain: 1) the difficulty to process and analyze massive short read sequencing data, often at the terabyte level; 2) the difficulty to effectively assemble genomes from complex metagenomes; and 3) the lack of methods for tracking genotypes and mutational events such as horizontal gene transfer (HGT) through time. Therefore, developing efficient bioinformatics approaches to address these challenges represents an important and timely issue. This thesis aimed to develop novel bioinformatics pipelines and algorithms for high performance computing, and, subsequently, apply these tools to natural microbial communities to generate quantitative insights into the relative importance of the molecular mechanisms creating or maintaining microbial diversity. The tools are not specific to a particular habitat or group of organisms and thus, can be broadly used to advance our understanding of microbial evolution in different settings. In particular, the comparative whole-genome analysis of 24 Escherichia isolates form various habitats, including human and non-human associated habitats such as freshwater ecosystems and beaches, showed that organisms with more similar ecologies tend to exchange more genes, which has important implications for the prokaryotic species concept. To more directly test these findings from isolates and quantify the patterns of genetic exchange among co-occurring populations, three years of time-series metagenomics data from planktonic samples from Lake Lanier (Atlanta, GA) were analyzed. For this, it was first important to develop bioinformatics algorithms to robustly assemble population genomes from complex community metagenomes, identify the phylogenetic affiliation of assembled genome and contig sequences, and detect horizontal gene transfer among these sequences. Using these novel algorithms, in situ bacterial lineage evolution was quantitatively assessed, especially with respect to whether or not ecologically distinct lineages evolve according to the recently proposed fragmented speciation model (Retchless and Lawrence, Science 2008). Evidence in support of this model was rarely observed. Instead, it appeared that rampant HGT disseminated ecologically important genes within the population, maintaining intra-population diversity. By expanding the previous approaches to include methods to assess differential gene abundance and selection pressure between samples, it was possible to quantify how soil microbial communities respond to a decade of warming by 2 0C, which simulated the predicted effects of climate change. It was found that the heated communities showed significant shifts in composition and predicted metabolism, reflecting the release of additional soil carbon compared to the unheated (control) communities, and these shifts were community-wide as opposed to being attributable to a few taxa. These findings indicated that the microbial communities of temperate grassland soils play important roles in mediating the feedback responses to climate change. Collectively, the findings presented here advance our understanding of the modes and tempo of microbial community adaptation to environmental perturbations and have important implications for better modeling the microbial diversity on the planet. The bioinformatics algorithms and approaches developed as part of this thesis are expected to facilitate future genomic and metagenomic studies across the fields of microbiology, ecology, evolution and engineering.

Phytostabilization is an emerging technology for the remediation of mine tailings sites. In arid and semiarid environments, mine tailings disposal sites are a major source of environmental pollution as they are subject to eolian dispersion and water erosion. Mine tailings are acidic to neutral, high in metal content, and nutrient poor. Furthermore, these sites remain unvegetated even after decades of no additional mining activity. In arid and semiarid regions, climatic variables such as high winds, salinity, and drought exacerbate the problem. The Klondyke mine tailings site is a model site for studying plant establishment in mine tailings within semiarid regions. It was a lead and zinc ore- processing operation from 1948 to 1958 and is similar in physicochemical characteristics to other acidic pyritic mine tailings.In a greenhouse study, a native drought tolerant halophyte, Atriplex lentiformis (Torr.) S. Wats., was evaluated for its potential as a phytostabilization candidate in compost-amended tailings from the Klondyke site. Germination, plant growth, and metal uptake of A. lentiformis were examined, and the microbial community was monitored by enumeration of autotrophic iron- and sulfur-oxidizing bacteria as well as heterotrophic bacteria. Results demonstrated that with 10 to 15% compost addition, growth of A. lentiformis was not affected and shoot metal concentrations were generally not a concern for foraging animals. Furthermore, the heterotrophic bacterial community is severely stressed but recovers with compost addition and successful plant growth. Therefore, A. lentiformis is a good candidate for phytostabilization of mine tailings with compost amendments.Poor revegetation of mine tailings has been attributed to the microbial community involved in acidifying tailings; however, no thorough microbial studies have been conducted. The second study characterizes the bacterial community of the Klondyke site and compares it to an offsite control sample. Results demonstrate that the heterotrophic community is indicative of soil health as it has a positive relationship with pH, phylotype richness, and diversity. Also, the mine tailings contain an unexplored diversity of acidophiles that are important in maintaining acidity and thus, metal bioavailability. Therefore, the bacterial community in mine tailings should be monitored in phytostabilization studies to evaluate restoration.

Bacteria are ubiquitous to marine living surfaces, taking on a broad spectrum of roles from mutualistic to pathogenic. Despite their universality, much remains unknown about their basic ecology and interactions with higher organisms. To address this gap, this thesis firstly examines the bacterial communities associated with three co-occurring marine eukaryote hosts from temperate Australia: the demosponge Cymbastela concentrica, the subtidal red macroalga Delisea pulchra and the intertidal green macroalga Ulva australis. Molecular characterisation of the bacterial communities was undertaken using 16S rRNA gene library analysis to compare within-host (alpha) and between-host (beta) diversity for the three microbial communities. This study highlights the potentially substantial contribution host-associated microorganisms could have on marine microbial diversity. The remaining focus for this thesis was on the bacterial community associated with D. pulchra. This alga produces a suite of biologically active secondary metabolites (furanones) that non-toxically inhibit acyl homoserine lactone (AHL)-driven quorum sensing in bacteria, affecting a range of phenotypes including colonisation and virulence traits. The ecology of D. pulchra???s epiphytic bacteria was investigated using a mechanistic approach to explain bacterial colonisation patterns. In particular, concepts and models of ecological succession founded in eukaryote ecology were investigated. The thesis concludes with a study investigating the effect of furanones and elevated temperature on bacteria-induced disease and thallus bleaching of D. pulchra. In the presence of furanones colonisation and infection of two Roseobacter isolates from D. pulchra???s epiphytic bacterial community were inhibited. Ruegeria strain R11 was demonstrated to have temperature regulated virulence, which caused thallus bleaching in furanone-free algae. The implications of elevated sea temperatures resulting from global warming for algal health are discussed.

abstract: Biological soil crusts (BSCs), topsoil microbial assemblages typical of arid land ecosystems, provide essential ecosystem services such as soil fertilization and stabilization against erosion. Cyanobacteria and lichens, sometimes mosses, drive BSC as primary producers, but metabolic activity is restricted to periods of hydration associated with precipitation. Climate models for the SW United States predict changes in precipitation frequency as a major outcome of global warming, even if models differ on the sign and magnitude of the change. BSC organisms are clearly well adapted to withstand desiccation and prolonged drought, but it is unknown if and how an alteration of the precipitation frequency may impact community composition, diversity, and ecosystem functions. To test this, we set up a BSC microcosm experiment with variable precipitation frequency treatments using a local, cyanobacteria-dominated, early-succession BSC maintained under controlled conditions in a greenhouse. Precipitation pulse size was kept constant but 11 different drought intervals were imposed, ranging between 416 to 3 days, during a period of 416 days. At the end of the experiments, bacterial community composition was analyzed by pyrosequencing of the 16s rRNA genes in the community, and a battery of functional assays were used to evaluate carbon and nitrogen cycling potentials. While changes in community composition were neither marked nor consistent at the Phylum level, there was a significant trend of decreased diversity with increasing precipitation frequency, and we detected particular bacterial phylotypes that responded to the frequency of precipitation in a consistent manner (either positively or negatively). A significant trend of increased respiration with increasingly long drought period was detected, but BSC could recover quickly from this effect. Gross photosynthesis, nitrification and denitrification remained essentially impervious to treatment. These results are consistent with the notion that BSC community structure adjustments sufficed to provide significant functional resilience, and allow us to predict that future alterations in precipitation frequency are unlikely to result in severe impacts to BSC biology or ecological relevance. / Dissertation/Thesis / M.S. Biology 2013

A correta adoção de medidas de manejo de resistência de insetos-pragas é motivo de preocupação constante, inclusive para as novas tecnologias disponíveis, como as plantas geneticamente modificadas para a expressão de toxinas da bactéria Bacillus thuringiensis (Bt). Portanto, para manter a eficiência das plantas-Bt comercialmente disponíveis, é preconizada a manutenção de áreas de refúgio (livres de plantas Bt) para evitar a rápida seleção de insetos resistentes. No caso de insetos polífagos, a determinação das áreas de refúgio pode levar em consideração a contribuição que fontes não-comerciais e/ou não-transgênicas têm na produção de adultos colonizadores que não sofreram seleção para o evento de transgenia de interesse. A identificação da fonte alimentar pode determinar a procedência de insetos e tornar mais eficiente e segura a implementação de zonas de refúgio e a determinação dos riscos de desenvolvimento de resistência pelo inseto-praga alvo. O objetivo deste trabalho foi o de avaliar o potencial de dois marcadores biológicos, a assinatura isotópica e a microbiota intestinal, para a identificação da fonte de alimento do imaturo pela análise de adultos de Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), como subsídio para o monitoramento da origem de insetos migrantes em áreas de cultivo Bt para a adoção de estratégias adequadas de manejo de resistência. A análise isotópica de adultos foi realizada utilizando-se insetos criados em 12 plantas hospedeiras distintas, seis de metabolismo C3 e seis de metabolismo C4. Parte dos adultos provenientes dessa criação foram liofilizados, macerados e submetidos à análise isotópica para a determinação dos níveis de ?13C e ?15N. Amostras das plantas utilizadas na alimentação desses insetos também foram submetidas ao mesmo processo de análise. A outra parte dos adultos recémemergidos de S. frugiperda foi utilizada para a determinação da microbiota intestinal pela análise de região do gene do 16S rRNA após sequenciamento em plataforma 454. Os resultados da análise isotópica de carbono para as plantas utilizadas como fonte alimentar apresentaram médias entre -31,37? e -25,07? para plantas C3 e entre -13,03? e -12,26? para plantas C4. Adultos de S. frugiperda oriundos de lagartas criadas em plantas do grupo C3 apresentaram média de ?13C entre - 30,36? e -23,72?, enquanto aqueles do grupo C4 apresentaram média entre - 18,25? e -13,28?. O sequenciamento da microbiota via metagenômica produziu 126,970 sequências com cerca de 421 pb. O alimento influenciou substancialmente a diversidade da microbiota intestinal associada ao intestino de adultos, independentemente do metabolismo fotossintético da planta hospedeira. Análises comparativas de diversidade em que a abundância relativa dos diferentes componentes foi considerada (Unifrac ponderado) permitiu a distinção de praticamente todas as microbiotas. Foram identificadas várias UTOs exclusivamente associadas à microbiota intestinal de adultos provenientes de cada fonte de alimento, mas a maioria apresentou abundância relativa extremamente baixa. Apenas as UTOs 1199 e 2255 foram exclusivamente associados ao milho com abundância relativa superior a 2,5%. / The correct insect resistant management has been a topic of constant concern, even for the newer technologies available, such as genetically modified crops expressing Bacillus thuringiensis (Bt) toxins. The use of refuge areas with non-Bt crops to avoid the fast selection for resistant insects is proposed for maintaining the efficiency of Bt-crops. The implementation of refuge areas for polyphagous insects can consider non-commercial and non-Bt crops as sources of susceptible insects. The identification of the food source used during immature development can make the implementation of refuge areas safer and more efficient and allow for better estimates of risk assessment for insect resistance development. The objective of this study is to determine the potential use of, the isotopic signature and gut microbiota of adults as biological markers to allow for the identification of the food source during the larval stage of Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae). Adults were obtained from larval rearing on 12 food sources, six host-plants with a C3 photosynthetic metabolism and six host-plants with a C4 metabolism. Part of the adults obtained and the food source used during their immature development were lyophilized and macerated, and subjected to ?13C e ?15N isotopic analysis. The remaining adults of S. frugiperda were used to determine the composition of the gut microbiota by metagenomic analysis of the V1-V3 region of the 16S rRNA gene using a 454 sequencing platform. The carbon isotopic signatures obtained for the hostplants used as food source were between -31.37? and -25.07? for C3 plants, and - 13.03? and -12.26? for C4 plants. Adults of S. frugiperda obtained from larval rearing on C3 plants had a carbon isotopic signature between -30.36? and -23.72?, while those from C4 host-plants has between -18.25? and -13.28?. The metagenomic sequencing yielded 126,970 reads with an average of 421bp. The larval food source substantially influenced the diversity of the adult gut microbiota regardless of the plant\'s photosynthesis metabolism. Comparative analysis among gut microbiota in which the relative abundance was taken into account (weight- Unifrac) allowed the discrimination of the majority of the communities. Several OTUs were identified as exclusive to the adult gut microbiota from different food sources, but most of these OTUs were minor components of the community. OTUs 1199 and 2255, both exclusively associated with corn, were the only ones to represent at least 2.5% of their community.

The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.

Soluble dietary fibers (SDF) are fermented more than insoluble dietary fibers (IDF), but their effect on colonic bacterial community structure and function remains unclear. Thus, bacterial community composition and function in the colon of BALB/c mice (n = 7) fed with a high level (approximately 20%) of typical SDF, oat-derived beta-glucan (G), microcrystalline cellulose (M) as IDF, or their mixture (GM), were compared. Mice in group G showed a lowest average feed intake (p < 0.05) but no change on the average body weight gain (p > 0.05) compared to other groups, which may be associated with the highest concentration of colonic propionate (p < 0.05) in these mice. The bacterial alpha-diversity of group G was significantly lower than other groups (p < 0.01). In group G, the relative abundance of bacteria belonging to the phylum Bacteroidetes was significantly increased, whereas bacteria from the phylum Firmicutes were significantly decreased (p < 0.01). The core bacteria for different treatments showed distinct differences. Bacteroides, Dehalobacterium, and Prevotella, including known acetogens and carbohydrate fermenting organisms, were significantly increased in relative abundance in group G. In contrast, Adlercreutzia, Odoribacter, and Coprococcus were significantly more abundant in group M, whereas Oscillospira, Desulfovibrio, and Ruminoccaceae, typical hydrogenotrophs equipped with multiple carbohydrate active enzymes, were remarkably enriched in group GM (p < 0.05). The relative abundance of bacteria from the three classes of Proteobacteria, Betaproteobacteria, Gammaproteobacteria (including Enterobacteriaceae) and Deltaproteobacteria, were significantly more abundant in group G, indicating a higher ratio of conditional pathogenic bacteria in mice fed dietary beta-glucan in current study. The predicted colonic microbial function showed an enrichment of "Energy metabolism" and "Carbohydrate metabolism" pathways in mice from group G and M, suggesting that the altered bacterial community in the colon of mice with the two dietary fibers probably resulted in a more efficient degradation of dietary polysaccharides. Our result suggests that the influence of dietary beta-glucan (SDF) on colonic bacterial community of mice was more extensively than MCC (IDF). Co-supplementation of the two fibers may help to increase the bacterial diversity and reduce the conditional pathogens in the colon of mice.

Deep-sea corals have long been regarded as cold-water coral; however a reevaluation of their habitat limitations has been suggested after the discovery of deep-sea coral in the Red Sea where temperatures exceed 20˚C. To gain further insight into the biology of deep-sea corals at these temperatures, the work in this PhD employed a holotranscriptomic approach, looking at coral animal host and bacterial symbiont gene expression in Dendrophyllia sp., Eguchipsammia fistula, and Rhizotrochus sp. sampled from the deep Red Sea. Bacterial community composition was analyzed via amplicon-based 16S surveys and cultured bacterial strains were subjected to bioprospecting in order to gauge the pharmaceutical potential of coralassociated microbes. Coral host transcriptome data suggest that coral can employ mitochondrial hypometabolism, anaerobic glycolysis, and surface cilia to enhance mass transport rates to manage the low oxygen and highly oligotrophic Red Sea waters. In the microbial community associated with these corals, ribokinases and retron-type reverse transcriptases are abundantly expressed. In its first application to deep-sea coral associated microbial communities, 16S-based next-generation sequencing found that a single operational taxonomic unit can comprise the majority of sequence reads and that a large number of low abundance populations are present, which cannot be visualized with first generation sequencing. Bioactivity testing of selected bacterial isolates was surveyed over 100 cytological parameters with high content screening, covering several major organelles and key proteins involved in a variety of signaling cascades. Some of these cytological profiles were similar to those of several reference pharmacologically active compounds, which suggest that the bacteria isolates produce compounds with similar mechanisms of action as the reference compounds. The sum of this work offers several mechanisms by which Red Sea deep-sea corals cope with environmental conditions in which no other deep-sea corals have yet to be reported. These deep-sea coral are associated with rich microbial communities, which produce molecules that induce bioactivity. The aggregate of this work provides direction for future research of Red Sea deep-sea coral and highlights the potential pharmacological benefit of conserving these species and their unique ecosystem.