|
| |
|
|
Bacterial interactions and secondary metabolites in plant and soil environment
Rapoport, Daria ; Marečková, Markéta (advisor) ; Lovecká, Petra (referee) ; Brabcová, Vendula (referee)
Phylum Actinobacteriota represents one of the major phyla in plant and soil environments. Known for the ability to produce numerous secondary metabolites, Actinobacteriota may affect other bacteria and plants in various ways. The thesis aimed at assessing the connection between the production of secondary metabolites and interactions of actinobacteria in plant and soil environments. In the first part, we assessed how rare actinobacteria from acid soil affect cultivable soil bacteria. We isolated a collection of actinobacteria from acid soil dominated by previously uncultivable lineage and isolated a representative described as a new family Treboniaceae and multiple other potentially novel species of known genera. The genome and metabolome analysis of Trebonia kvetii, sp.nov. gen.nov., demonstrated its ability to produce unusual and potentially novel bioactive metabolites. We designed an experiment where a single strain was co-cultivated with the total soil bacterial community of the same soil in a set up, where a free exchange of diffusible metabolites was allowed between them. Three actinobacteria strains coming from distinct lineages were selected for this interaction. Each strain significantly and specifically affected cultivable bacteria as well as the metabolite pool, part of which was induced...
|
|
|
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.
|
|
|
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.
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie
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...
|
|
|
Actinobacteria communities in natural and anthropogenic environments
Faitová, Andrea ; Marečková, Markéta (advisor) ; Slaninová Kyselková, Martina (referee) ; Yurado Lobo, Valme (referee)
10 Abstract Actinobacteria are important bacterial group participating in various ecosystem processes particularly in the decomposition of complex organic compounds. Their abilities enable them to surviving in harsh conditions of oligotrophic habitats like lakes, deserts, cave walls or recalcitrant and resistant litter in soil, where Actinobacteria often dominate. Although certain biotic and abiotic factors were recognized to modulate Actinobacteria incidence in such habitats, the influence of anthropogenic pressure on their communities is scarcely known. The main objective of this thesis is therefore to determine differences of Actinobacteria communities under the direct (the human visitors changing microenvironment of caves, part 1) and indirect (climate change factors like altered precipitation or plant litter quality, part 2) anthropogenic influence in two habitats, plant litter in soil and cave walls, where Actinobacteria play important roles and dominate. In a first part of the thesis we monitored Actinobacteria communities in French limestone caves walls differently affected by humans (pristine versus anthropized caves). For identification of important species like potential pathogens or pigments producing Actinobacteria using amplicon sequencing of environmental DNA (Illumina MiSeq), we firstly used...
|
guest ::
