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The role of F420-dependent oxidoreductases in actinobacteria
Kekrt, Lukáš ; Kameník, Zdeněk (advisor) ; Palyzová, Andrea (referee)
Deazaflavin F420 is an unusual cofactor involved in oxidoreduction reactions in the cells of some microorganisms. The role of F420/F420H2-dependent oxidoreductases has been extensively described in the case of central archaeal metabolism, particularly those of methanogens. In contrast, our knowledge of these enzymes in actinobacteria is limited. This work focuses on the characterization of selected actinobacterial oxidoreductases from luciferase-like hydride transferase family, which putatively use the F420 cofactor. Specifically, Apd6 biosynthetic proteins and their sequence homologs were studied. Three recombinant proteins were prepared and purified and their enzymatic activity was tested in the presence of F420 and a set of putative substrates by means of in vitro reactions. The reaction products were monitored by liquid chromatography with UV and mass spectrometry detection. Some of the expected reaction products were detected in in vitro reactions, confirming that the proteins were catalytically active. Furthermore, one of the proteins surprisingly exhibited an unusual reaction specificity. Key words: Specialized metabolism, actinobacteria, F420 cofactor, redox reactions, reaction specificity, 4-alkyl-L-proline derivates.
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Microbial communities of soils affected by long-term heavy metal contamination
Průchová, Pavla ; Kopecký, Jan (advisor) ; Matyska Lišková, Petra (referee)
This work is focused on microbial communities living in the soil affected by long-term exposure to heavy metals. The soil was sampled at two sites with different levels of contamination near Příbram. In the samples, respiration rate was measured in vitro after addition of carbon sources and at different levels of cadmium, one of the contaminating metals found in the soil. After the incubation with carbon sources, soil samples were collected for environmental DNA isolation. Gene coding for 16S rRNA in Actinobacteria was amplified from the environmental DNA samples and the amplicon composition was assessed by terminal restriction fragment length polymorphism analysis. The resulting profiles were used to compare actinobacterial communities in both groups of soil samples and in individual treatments. The analysis showed a clear distinction between the two sites differing in the contamination level and shifts in the community composition of various intesity depending on the added substrate.
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Gene pool of the secondary metabolism in soil bacterial communities
Patrmanová, Tereza ; Kopecký, Jan (advisor) ; Brabcová, Vendula (referee)
The need for new antibiotics and other biologically active compounds is the reason for an increased interest in secondary metabolites of soil bacteria. The phylum Actinobacteria has the dominant position in the soil environment thanks to the potential of producing a broad spectrum of antibiotics and the presence of a number of defense mechanisms preventing the effects of antibiotics. The aim of this thesis was to determine the number of copies of selected secondary metabolic genes in the soils of two sites using designed primers and primers from literature. The design of effective new primers for the detection of selected genes in the soil environment was not achieved in this work, and therefore only primers from literature that had been verified for their specificity were used. In samples taken from soil profiles of two sites, abundances of bacteria, actinobacteria, type II polyketide synthase genes and Erm methyltransferase genes mediating resistance to MLSB antibiotics (macrolides, lincosamides and streptogramins B) were determined by digital PCR. The comparison of the determined copy numbers gave an information about the structure of the bacterial community and the relative abundance of bacteria carrying selected secondary metabolic genes depending on the soil condition changes due to the...
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Gene pool of the secondary metabolism in soil bacterial communities
Patrmanová, Tereza ; Kopecký, Jan (advisor) ; Brabcová, Vendula (referee)
The need for new antibiotics and other biologically active compounds is the reason for an increased interest in secondary metabolites of soil bacteria. The phylum Actinobacteria has the dominant position in the soil environment thanks to the potential of producing a broad spectrum of antibiotics and the presence of a number of defense mechanisms preventing the effects of antibiotics. The aim of this thesis was to determine the number of copies of selected secondary metabolic genes in the soils of two sites using designed primers and primers from literature. The design of effective new primers for the detection of selected genes in the soil environment was not achieved in this work, and therefore only primers from literature that had been verified for their specificity were used. In samples taken from soil profiles of two sites, abundances of bacteria, actinobacteria, type II polyketide synthase genes and Erm methyltransferase genes mediating resistance to MLSB antibiotics (macrolides, lincosamides and streptogramins B) were determined by digital PCR. The comparison of the determined copy numbers gave an information about the structure of the bacterial community and the relative abundance of bacteria carrying selected secondary metabolic genes depending on the soil condition changes due to the...
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Izolace specifických druhů bakterií z permafrostu
RODOVÁ, Ivana
This work is mainly focused on the isolation and characterization of bacteria from Arctic permafrost, which can utilize aliphatic and aromatic carbohydrates. Previous work showed that mainly Proteobacteria and Actinobacteria dominate in permafrost soils. Therefore, this work was mainly focused on these two groups. Aliphatic and aromatic carbohydrates are utilized by very specialized group of bacteria which use unique enzymatic apparatus. Thanks to this ability these bacteria play crucial role in carbon cycle in specific cryoturbated soil horizons typical for Arctic permafrost soils, where high amount of aliphatic and aromatic compounds was confirmed. These pure isolates may also be used in treating contaminated areas by oil or other pollutants.
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Composition and activity of microbial communities in soil contaminated by heavy metals
Průchová, Pavla ; Kopecký, Jan (advisor) ; Zikánová, Blanka (referee)
The thesis focuses on studying changes of microbial communities living in the soil contaminated by heavy metals. Two sites with different degree of contamination were selected in the Příbram area. Respiration was measured in vitro in the soil samples supplemented with various carbon sources and different concentration of cadmium. The respiration showed that even at cadmium concentration of 1000 mg.kg-1 the community is viable and capable of utilization of substrates while increasing the respiration rate. Enviromental DNA from soil samples was isolated and 16S rRNA gene of actinobacteria was amplified. The terminal restriction fragment length polymorphism analysis showed a clear difference between the profiles of both sites. The shifts in the community profiles were observed also after the addition of substrates. The quantification of total bacteria and actinobacteria was performed by quantitative PCR based on amplification of part of the 16S rRNA gene. The more contaminated site contained slightly more bacteria, but almost twice the actinobacteria than the less contaminated one. The sequencing of amplicons of a part of 16S rRNA gene by Illumina showed an increase in proportion of actinobacteria and changes of their community structure in the more contaminated site. The conclusion was made that, high...
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Actinobacteral communities in agricultural soils at sites with occurence of potato common scab.
Daniel, Ondřej ; Kopecký, Jan (advisor) ; Lichá, Irena (referee)
The diploma thesis is focussed on understanding relationships between soil chemical characteristics, actinobacterial communities of agricultural field soils and occurrence of potato common scab, a disease caused by members of the genus Streptomyces. The aim of monitoring study, on thirty-three sites covering main potato- growing regions in the Czech Republic, was to find relationships suitable for prediction of common scab severity. The second part of the thesis compared actinobacterial communities and incidence of Streptomyces harboring a pathogenic determinant, gene txtA (gene of biosynthetic pathway of phytotoxin thaxtomin A), in soils differing in occurrence of common scab. In the screening study, analysis of terminal restriction fragment length polymorphism (T-RFLP) was employed to compare composition of soil actinobacterial communities. Real-time PCR was used to quantify total actinobacteria and streptomycetes harboring txtA gene in soils differing in scab incidence. The screening study revealed negative correlations between the scab severity and (i) available phosphorus in soil and (ii) diversity of actinobaterial community. The results were used to design a model for scab prediction. A qPCR analysis showed difference in numbers of total actinobacteria and the strains harboring txtA gene in...
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Microbial communities of soils affected by long-term heavy metal contamination
Průchová, Pavla ; Kopecký, Jan (advisor) ; Matyska Lišková, Petra (referee)
This work is focused on microbial communities living in the soil affected by long-term exposure to heavy metals. The soil was sampled at two sites with different levels of contamination near Příbram. In the samples, respiration rate was measured in vitro after addition of carbon sources and at different levels of cadmium, one of the contaminating metals found in the soil. After the incubation with carbon sources, soil samples were collected for environmental DNA isolation. Gene coding for 16S rRNA in Actinobacteria was amplified from the environmental DNA samples and the amplicon composition was assessed by terminal restriction fragment length polymorphism analysis. The resulting profiles were used to compare actinobacterial communities in both groups of soil samples and in individual treatments. The analysis showed a clear distinction between the two sites differing in the contamination level and shifts in the community composition of various intesity depending on the added substrate.
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Ecology of Actinobacterial classes of \kur{Micrococcaceae, Streptomycetaceae} and \kur{Nocardiaceae} in terrestric ecosystems
HEJDOVÁ, Barbora
The families Micrococacceae, Nocardiaceae and Streptomycetaceae belong to a large phylum Actinobacteria. This phylum is ecologicaly important and many of its members are mainly found in soil, where they are involved in the carbon cycle and other degradation processes. This capability of degradation of complex bololymers is typical for the genera Arthrobacter and Rhodococcus in the bioremediation process. Other members like Streptomycetes are characterized by the ability to produce antibiotics, which are used in the pharmaceutical industry. In this work, we focuse on metabolism of selected actinobacterilal families, but also on the pathogenicity and to their occurrence in different habitats. We will use these pieces of information to evaluate the abundance and metabolic potential of these families in soil collected from the north-eastern region of Siberia along the Kolyma River (Cherskii, Republic of Sakha, Russia). Sequencing of the samples showed the highest occurrence of Streptomyces and Arthrobacter genera and have also relatively large metabolic potential cellulose and phenolics degradation, mainly in the upper soil layers.
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The importance of Actinobacteria in Arctic soil
BOŠKOVÁ, Hana
This work is aimed for Actinobacteria and describes their importance in Arctic soil. The members of Actinobacteria are known for their ability to decompose complex natural biopolymers and because they are able to live in harsh arctic environment they could play there an important role in organic matter decomposition. The work compares their abundance in different soil horizons with the focus on cryoturbations and determines the influence of temperature on their amount. This work also represents the results of testing pure Actinobacterial isolates for the production of cellulolytic enzymes.
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