| 1. |
- Jernberg, Cecilia
(författare)
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Use of microbiomics to study human impacts on complex microbial communities
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The study of bacterial communities in nature is currently a challenge. The majority of bacteria in clinical and environmental samples have not yet been cultured and therefore we cannot fully understand their roles in nature and how the ecological balance in a specific microbial ecosystem can be disrupted. For example, exposure to pollutants in soil and antibiotics in the human gut can have large consequences on microbial populations but the magnitude of these impacts is difficult to assess. In this thesis, a combination of molecular techniques, microbiomics, were used to assess complex microbial communities in soil and the human gut. One goal of this thesis was to study the impact of the toxic compound, 4-chlorophenol, on the soil microbiota. In addition, a specific 4-chlorophenol degrading bacterium, Arthrobacter chlorophenolicus, was monitored in soil. In order to monitor the cells they were chromosomally tagged with marker genes encoding either the green fluorescent protein (the gfp gene) or firefly luciferase (the luc gene). During degradation of high levels of 4-chlorophenol in soil, total cells counts of A. chlorophenolicus cells could be measured by flow cytometry (GFP protein) and the metabolic activity could be measured by lurninometry (luciferase activity). In addition, the relative abundance of A. chlorophenolicus in soil could be measured by terminal restriction fragment length polymorphism (T-RFLP) and a higher relative abundance was detected in soil contaminated with 4chlorophenol compared with non-treated soil. The impacts of 4-chlorophenol and A. chlorophenolicus on the dominant members of the soil microbiota were also assessed by T-RFLP. Another goal of this thesis was to study the impact of a short term antibiotic administration in a long term perspective, using either clindamycin, in a two year study or a triple therapy for eradication of Helicobacter pylori containing clarithromycin and metronidazole, in a four year study, on the human fecal microbiota. Both the total bacterial community and specific populations, i.e. Bacteroides spp. and Enterococcus spp., were monitored by T-RFLP. The Bacteroides populations never returned to their pre-treatment composition after clindamycin exposure during the two year study period. Selection and persistence of resistant Bacteroides clones up to two years after treatment was furthermore detected. In the four year study, Enterococcus populations increased as a response to the clarithromycin and metronidazole treatment. An increase in the levels of antibiotic resistance genes, specific erm genes, conferring resistance to macrolides and lincosamides were detected for up to 2 and 4 years after both types of antibiotic treatments in the respective studies. It was also possible to specifically monitor two probiotic Lactobacillus strains and their transient colonization by T-RFLP. In conclusion, the use of a polyphasic approach with complementary analytical tools made it possible to obtain a comprehensive picture of complex microbial communities. In addition, specific bacteria of interest in complex soil and fecal samples could be monitored using microbiomics approaches.
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| 2. |
- Eiler, Alexander, 1976-
(författare)
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The Niches of Bacterial Populations in Productive Waters : Examples from Coastal Waters and Four Eutrophic Lakes
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Recent research in microbial ecology has focused on how aquatic bacterial communities are assembled. Only a few of these studies follow a “Gleasonian” approach where the roles of single bacterial populations are in focus. In this thesis, novel molecular tools were used to describe the distribution and evolutionary relationships of microbes in productive aquatic environments. Many new phylogenetic groups of bacteria were identified, likely representing bacterial populations restricted to productive freshwaters. I also addressed the dynamics and functional role of individual bacterial populations in eutrophic lakes and brackish environments with a focus on either biogeochemically significant or potentially pathogenic representatives. Flavobacteria blooms were observed, on occasions characterized by high heterotrophic production. In addition to high temporal dynamics microbial community composition and function differed on the spatial scale, as exemplified by free-living and Cyanobacteria-associated habitats. At the community scale, microbial processes, such as biomass production and substrate uptake could be predicted from the presence and absence of individual bacterial populations. I also studied the niches of potentially pathogenic Vibrio populations in various coastal waters. Using a novel culture-independent method, a V. cholerae population was detected along the entire Swedish coastline. Results from an environmental survey and a laboratory mesocosm experiment reveal that phytoplankton-derived dissolved organic matter enhance the growth of V. cholerae and other Vibrio spp. and hence create a largely overlooked niche for these heterotrophic bacteria. This thesis and future work on the role of individual bacterial populations will facilitate predictions of biogeochemical cycles and the distribution of bacteria in the context of global climate change and local eutrophication.
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| 3. |
- Maraha, Ninwe
(författare)
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Physiological status of bacteria used for environmental applications
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several bacteria have properties of interest for biotechnological applications, such as bioremediation of pollutants and biocontrol of plant pathogens. In order to perform their intended tasks in the environment the cells need to remain viable and active. Therefore, the aim of this thesis was to use a combination of molecular approaches to determine the physiological status of specific bacterial populations in soil. Complementary experiments were done in pure cultures to gain a better understanding of specific physiological states, such as bacterial dormancy. In some studies, the bacteria were tagged with the following marker genes to enable them to be specifically detected in soil: gfp (encoding the green fluorescent protein, GFP), luxAB (encoding bacterial luciferase) or luc (encoding eukaryotic luciferase). Viability stains, 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), were used to stain active and dead cells, respectively. The marker-gene tagged cells were incubated in soil under different conditions and the number of GFP fluorescent and stained cells was enumerated by flow cytometry at specified sampling periods. Luciferase activity was used to monitor metabolic activity of the population. In addition, the number of culturable cells was determined by selective plate counting and compared to the results obtained by flow cytometry. Finally, in one study, proteomics was used to elucidate which proteins were expressed under different nutrient conditions. The physiological status of Arthrobacter chlorophenolicus A6 (a chlorophenol degrading bacterium) was investigated after introduction into soil incubated at different temperatures, 5 and 28 °C. The majority of the A6 population remained metabolically active after 20 days of incubation in soil at 5 °C. However, there was a fraction of the GFP-fluorescent A6 population that was not stained with CTC or PI, presumably indicating a subfraction of dormant cells that were alive but inactive. By contrast, after the same period of incubation at 28 °C, the majority of the cells died. The ability of A. chlorophenolicus A6 to enter a state of dormancy during incubation at cold temperatures, makes this strain a good candidate for treating chlorophenol contaminated soil in temperate climates. Two Pseudomonas fluorescens strains, proposed for improving crop yields, were also studied. Pseudomonas fluoresens A506 is used to reduce frost damage to plants and Pseudomonas fluorescens SBW25 is a plant growth promoting bacterium. First, a GFPtagged variant of the A506 strain was studied to determine whether GFP could be used to detect the cells when they were viable but non-culturable (VBNC). The results showed that GFP tagged cells could be detected even in a V13NC state as long as the cell membrane was intact. The SBW25 strain was studied in pure cultures and in soil to determine the physiological status of the cells under different nutritional conditions, using many of the approaches described above for A6. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI, indicating that most of the cells were presumably dormant. In soil, a subpopulation of the SBW25 cell population died. However, approximately 60% of the population in soil apparently entered a state of dormancy, similar to that observed under starvation conditions in pure cultures. Several differences were found in the proteins that were expressed when SBW25 was incubated under nutrient rich conditions compared to starvation conditions. These differences provide a clue as to what proteins enable SBW25 to survive starvation and dormant states.
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| 4. |
- Nordin, Karolina, 1972-
(författare)
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4-chlorophenol biodegradation by Arthrobacter chlorophenolicus A6
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A microorganism was isolated which could grow on unusually high concentrations of the toxic pollutant 4-chlorophenol. Taxonomic studies showed that the microorganism constituted a novel species within the genus Arthrobacter and it was named Arthrobacter chlorophenolicus A6. A. chlorophenolicus A6 was chromosomally tagged with either the gfp gene, encoding the green fluorescent protein (GFP), or the luc gene, encoding firefly luciferase. When the tagged cells were inoculated into 4-chlorophenol contaminated soil they could completely remove 175 µg/g 4-chlorophenol within 10 days, whereas no loss of 4-chlorophenol was observed in the uninoculated control microcosms. During these experiments the gfp and luc marker genes allowed monitoring of cell number and metabolic status.When A. chlorophenolicus A6 was grown on mixtures of phenolic compounds, the strain exhibited a preference for 4-nitrophenol over 4-chlorophenol, which in turn was preferred over phenol. Analysis of growth and degradation data indicated that the same enzyme system was used for removal of 4-chlorophenol and 4-nitrophenol. However, degradation of unbstituted phenol appeared to be mediated by another or an additional enzyme system. The luc-tagged A. chlorophenolicus A6 gave valuable information about growth, substrate depletion and toxicity of the phenolic compounds in substrate mixtures. The 4-chlorophenol degradation pathway in A. chlorophenolicus A6 was elucidated. The metabolic intermediate subject to ring cleavage was found to be hydroxyquinol and two different pathway branches led from 4-chlorophenol to hydroxyquinol. A gene cluster involved in 4-chlorophenol degradation was cloned from A. chlorophenolicus A6. The cluster contained two functional hydroxyquinol 1,2-dioxygenase genes and a number of other open reading frames presumed to encode enzymes involved in 4-chlorophenol catabolism. Analysis of the DNA sequence suggested that the gene cluster had partly been assembled by horizontal gene transfer.In summary, 4-chlorophenol degradation by A. chlorophenolicus A6 was studied from a number of angles. This organism has several interesting and useful traits such as the ability to degrade high concentrations of 4-chlorophenol and other phenols alone and in mixtures, an unusual and effective 4-chlorophenol degradation pathway and demonstrated ability to remove 4-chlorophenol from contaminated soil.
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| 5. |
- Timmusk, Salme, 1959-
(författare)
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Mechanism of Action of the Plant Growth Promoting Bacterium Paenibacillus polymyxa
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Paenibacillus polymyxa belongs to the group of plant growth promoting rhizobacteria (PGPR). Activities associated with P. polymyxa-treatment of plants in earlier experiments include, e.g., nitrogen fixation, soil phosphorus solubilization, production of antibiotics, auxin, chitinase, and hydrolytic enzymes, as well as promotion of increased soil porosity. My thesis work showed that, in stationary phase, P. polymyxa released the plant hormone cytokinin isopentenyladenine, in concentrations of about 1.5 nM.In a gnotobiotic system with Arabidopsis thaliana as a model plant, it was shown that P. polymyxa-inoculation protects plants; challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) showed that pre-inoculated plants were significantly more resistant than control plants. By RNA-differential display on RNA from P. polymyxa-treated or control plants, changes in gene expression were tested. One mRNA, encoding ERD15 (drought stress-responsive gene) showed a strong inoculation-dependent increase in abundance. In addition, several biotic stress-related genes were also activated by P. polymyxa. Antagonism towards the fungal pathogens Phytophthora palmivora and Pythium aphanidermatum was studied. P. polymyxa counteracted the colonization of zoospores of both oomycetes on A. thaliana roots, and survival rates of plants treated with P. polymyxa were much higher when challenged by P. aphanidermatum. Using a green fluorescent protein-tagged isolate of P. polymyxa, colonization of A. thaliana roots was investigated. Two main conclusions can be drawn. Firstly, the bacterium enters the root tissue (but not leaves) and is abundantly present in intercellular spaces. Secondly, the root becomes severely damaged, indicating that – under some conditions – P. polymyxa is a "deleterious bacterium", and in others it promotes growth. Based on work presented in my thesis, I argue that a balance between the activities of a PGPR, the genetic background and physiological state of a plant, and the environmental conditions employed in test systems, ultimately determines the resulting effect.
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