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The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Masák, Jan (referee) ; Obšilová, Veronika (referee)
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...
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Soil microbial communities in agroecosystems and natural habitats contributing to resistance and resilience of the soil environment
Sarikhani, Ensyeh ; Kopecký, Jan (advisor) ; Demnerová, Kateřina (referee) ; Větrovský, Tomáš (referee)
Ensyeh Sarikhani Soil microbial communities in agroecosystems and natural habitats contributing to resistance and resilience of the soil environment. Summary The control of common scab of potatoes (CS) includes resistant varieties (cultivars), precise fertilization, increase of soil moisture, and chemical treatments. Yet, these management practices do not have common or reproducible results at differing sites. A monitoring study was done in 32 sites to evaluate the relation between CS and biological/chemical soil parameters. Correlations were observed between scab severity and content of nutrients such as Fe, N, and Ca in soil and periderm, and between disease severity and abundance of actinobacteria and total bacteria, together with the pathogenicity determinant, txtB gene (biosynthetic gene of thaxtomin) in both soil and periderm of potatoes. The findings led to novel conclusions, which can help to understand relationships applicable in scab control. Peat and DTPA chelated iron were supplemented to pots filled with soil conducive for CS in order to determine the effects of soil organic matter, iron and pH on CS development. The results were compared with data obtained for a suppressive soil from a nearby field with naturally low CS severity. Both peat and iron supplements decreased CS and the combination...
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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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Antibiotic rezistance genes in soil actinobacteria
Patrmanová, Tereza ; Kopecký, Jan (advisor) ; Lichá, Irena (referee)
Actinobacteria are important members of the soil ecosystems, where they are involved in organic matter decomposition. It is worth mentioning that their secondary metabolism allows them to produce a variety of different compounds. These compounds include antibiotics, among them aminoglycosides have a place in clinical practice. These antibiotics are significant due to a broad spectrum of activities against both gram-negative and gram-positive bacteria. However, their use currently carries a risk, mainly their toxicity and development of antibiotic resistance in bacteria. Resistance is the cause of low effectiveness of some of those antibiotics. Actinobacteria as aminoglycoside producers must protect themselves from these compounds, so a variety of resistance types was developed, out of which enzymatic inactivation is the most studied one. Actinobacteria have evolved several mechanisms, which contribute to a resistance to the agents with antimicrobial effects. Genes encoding antibiotic resistance are abundant in soil environment. Their variability is influenced by many factors, especially the selection of bacteria in soil contaminated with antibiotics and also with strains originating from human and animal waste. Significant role has a horizontal gene transfer, which allows distribution of resistence...
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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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Enviromental DNA-inexhaustible source of unique bacterial genes
Culka, Martin ; Černá, Věra (advisor) ; Vaněk, Ondřej (referee)
Search for new enzymes or variants of known ones is now a hot issue in enzymological research. The classical culture-based approach, however, often fails when applied on environmental samples, because they contain uncultured microorganisms at most. For this reason, a new approach has been developed - the metagenomics. This approach is based on direct isolation of total DNA (RNA) from specific environment and its subsequent sequence-based (genotype) or function-based (phenotype) analysis. In this work, the metagenomic approach has been used to find new forms of penicillin G acylase, the enzyme that catalyze cleavage or formation of acyl - β-lactam nucleus bond and is used in industry for synthesis of semi-synthetic β-lactam antibiotics, in eleven samples from 4.5 m soil horizon. Sequence analysis of PCR amplicons on metagenomic templates revealed nucleotide sequences of major part of potential structural penicillin acylase genes. After translation it has been found that the sequences are most homologous to penicillin amidase from Conexibacter woesei. Further perspective of this metagenomic study is amplification of at least one complete structural gene of environmental penicillin acylase. However, the cloned regions of the gene can also be used to create hybrid penicillin acylases using gene shuffling...
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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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