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Analysis of secondary metabolites from Streptomyces by Raman spectroscopy in vivo
Mikulová, Adéla ; Petříčková, K. ; Bobek, J. ; Chroňáková, Alica ; Samek, Ota ; Pilát, Zdeněk
The work deals with the study of Raman spectra of secondary metabolites of bacteria of the genus Streptomyces, especially polyene antifungal agents. Purified isolates and commercial standards of these compounds were used to obtain reference spectra from which spectral markers characterizing specific details of the chemical structure were derived. Verification of the observed markers was performed using in silico Raman spectra predictions. Subsequently, these findings were used for the spectroscopic characterization of the composition of secondary metabolites in vivo, directly in bacteria growing on a solid medium. The developed methodologies will help to optimize the conditions for the biotechnological production of antibiotics.
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Preparation of genetically manipulated producers of hybrid manumycins
Krýslová, Dita ; Petříčková, Kateřina (advisor) ; Doubravová, Linda (referee)
Streptomyces are one of the most prolific producers of various secondary metabolites. Manumycin antibiotics represent an important class of these compounds. They belong to a big class of polyketide metabolites. While their antibiotic effect is not very significant, their other biological properties have a big potential in the treatment of inflammations, tumors, etc. They are characterized by two short polyketide chains, which are attached to a central subunit. At the end of the lower polyketide chain, a C5N cyclic unit is frequently attached. This thesis originates from the colabomycin E, an antibiotic, which was discovered by our team. This antibiotic is a new member of manumycin-type metabolites and is produced by Streptomyces aureus SOK1/5-04 strain. Previous studies on the function of individual genes in the biosynthetic gene cluster of colabomycin E inspired us to consider editing of the current biological activity of colabomycin E by replacement of the C5N unit with another, structurally similar bioactive subunit. Due to high structural similarity, we have selected 4,7- dihydroxycoumarin unit of novobiocin, an aminocoumarin-type metabolite produced also by Streptomyces. The 4,7-dihydroxycoumarin unit is pharmacophore with a cancerostatic activity. We expected that the cancerostatic activity...
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Detection of iron-induced proteins and their effect on anthracycline production in submerged culture of Streptomyces coeruleorubidus
MICHALCOVÁ, Zuzana
In this bachelor's thesis, proteins from the group of enzymes - esterases and their possible induction after the addition of iron to the culture medium were analyzed. Furthermore, the effect of the addition of Fe2+ cations on daunomycin production under submersion cultivation conditions of Streptomyces coeruleorubidus culture was evaluated. The work is divided into two parts. The first part introduced the genus actinobacteria called Streptomyces, their occurrence, properties and possible benefits due to the production of secondary metabolites. In the second, experimental part, the use of the Streptomyces coeruleorubidus strain was described. The effect of iron addition was evaluated by HPLC analysis and native polyacrylamide electrophoresis (native PAGE). HPLC analysis showed that the addition of iron in the form of FeSO4 to the medium increased daunomycin production by approximately 21.5-fold. During native PAGE and after staining, proteins, i.e. esterases, most likely from the groups of and esterases, were detected. Those were more or to a greater extent produced by the Streptomyces coeruleorubidus if the medium contained iron.
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Identification and modeling of gene expression regulatory networks during streptomycetes germination
Straková, Eva ; Vohradský, Jiří (advisor) ; Stopka, Pavel (referee) ; Vondrášek, Jiří (referee)
Streptomycetes have been studied mostly as producers of antibiotics and for fundamentals of complex bacterial cell development. Here, transcriptomic and proteomic approaches were applied to systems study of Streptomyces coelicolor germination as a developmental transition from dormancy to the vegetative stage. The time dynamics of the gene expression levels represented by mRNA and intracellular protein accumulation and synthesis were measured throughout 5.5 h of germination at 13 time points by employing both DNA microarray and two-dimensional gel electrophoresis techniques. Using a numerical model of gene expression, genetic networks were reconstructed and functional groups of genes controlled by the sigma factors were identified. Modeling of the regulatory interactions provided a set of parameters allowing simulate kinetics of gene expression control among the sigma factors and their target genes. Particularly regulons of two sigma factors, SigR and HrdD, were identified. The analysis assigned their key role during the germination process. Analysis of global trends in the gene/protein expression revealed that the full capability of regulatory mechanisms responding to the environmental cues is reached within the first hour of germination, and identified the basic gene/protein functional groups...
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Mutational analysis of manumycin antibiotics biosynthetic routes
Kolek, Jan ; Petříček, Miroslav (advisor) ; Zikánová, Blanka (referee)
Mutational analysis of manumycin antibiotics biosynthetic routes The manumycin antibiotics are secondary metabolites, which come from a big group of polyketide metabolites. They are produced by bacteria from genus Streptomyces. Manumycin antibiotics are characterized by two linear polyketide chains, which are connected to a central mC7N moiety. The lower chain is often terminated by the C5N moiety. Manumycin metabolites show many biological activities. They have antimicrobial activity, especially against gram-positive bacteria. Next, they posses antifungal, insecticidal or antiinflamatory activities. Manumycins are also potentional anticancer agents. In order to prepare these compounds by the fermentation, the detailed knowledge of their biosynthetic routes is required. Mutational analysis is based on techniques of genetic engineering. Mutational analysis is a useful pool for analysis of biosynthetic pathways of secondary metabolites and the genes, which are involved in these pathways. This knowledge is essential for application of combinatorial biosynthesis for the design of new metabolites. The new hybrid compound could be used in future as new antibiotics or anticancer drugs. Keywords: manumycin, polyketide, streptomyces, asukamycin
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Analysis of the biosynthetic gene cluster encoding biosynthesis of the manumycin antibiotic U-62162, and the ways of its modification.
Urbanová, Daniela ; Petříček, Miroslav (advisor) ; Schierová, Michaela (referee)
Streptomyces is the largest antibiotic-producing genus in the microbial world. Manumycin-type antibiotics are a small group of its metabolites. Their antibiotic activities are not very important but they show biological properties which can be potencially used e. g. to treat inflammation, cancer or Alzheimer's disease. The structure of manumycin compounds is formed by a central unit with connected upper and lower polyketide chain. The lower chain is mostly terminated by so called C5N unit. The substance U-62162 produced by the strain Streptomyces verdensis differs significantly from the other members of the manumycin-type metabolites in the structure of the lower chain which is fully saturated and lacking the C5N unit. The U-62162 biosynthetic gene cluster was sequenced and functions of identified open reading frames were deduced. Heterologous expressions of the cluster showed some genes reguired for the biosynthesis of the upper chain to be encoded on a different part of the chromosome. The insertional inactivation of the vrdER gene confirmed the enoylreductase to be responsible for the saturation of the lower chain. DSBA oxidoreductase, which gene is located at the edge of the cluster, is probably not involved in the biosynthesis. The insertion of genes for the biosynthesis of the C5N unit did...
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Specific features of streptomycetes isolated from human tissues
Kodatová, Anežka ; Petříčková, Kateřina (advisor) ; Zikánová, Blanka (referee)
This bachelor thesis focuses on activities of the bacterial genus Streptomyces in relations to plants, animals and human. Many cases of their symbiotic relationships with plants are known, but there is also a common plant pathogen S. scabies. As a primary soil bacteria, their association with animals is infrequent, but not exceptional. Symbiotic relationships have been observed for example in several insect species. Streptomyces have been repeatedly isolated from the human microbiome, however only a little information about their influence on human body is available. Due to the wide production of secondary metabolites affecting other microorganisms, their role can be assumed as significant. The relationship between human Streptomyces and human could have a parallel in the Streptomyces symbiosis with plants and insects, but it can also have certain effects on pathogenesis. The ability of beta haemolysis was demonstrated in tested strains. In addition, there are cases of serious diseases caused by Streptomyces. Next to the relatively common actinomycosis in the regions of Sudan and India, diseases such as pneumonia or abscess were reported.
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The effect of 6S-like RNAs on physiological differentiation of Streptomyces coelicolor
Burýšková, Barbora ; Bobek, Jan (advisor) ; Branny, Pavel (referee)
The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...
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Analysis and mapping of binding sites of gene expression regulators in the genus of Streptomyces.
Šmídová, Klára ; Bobek, Jan (advisor) ; Krásný, Libor (referee) ; Kopecký, Jan (referee)
Streptomyces are medically important soil-living bacteria that undergo morphological changes from spores to aerial hyphae and are important producers of bioactive compounds including antibiotics. Their gene expression is tightly regulated at the early level of transcription and translation. In the transcriptional control, sigma factors play a central role; the model organism Streptomyces coelicolor possesses astonishing 65 sigma factors. The expression of sigma factors themselves is controlled on the post-transcriptional level through the action of sRNAs that modify their mRNA level. However, only several sigma factors in Streptomyces have known regulons and also their sRNAs-mediated regulation has not been studied so far. According to previously measured gene expression data, we selected several highly expressed sigma factors. Using mutant strains with HA-tagged sigma factors, regulons of two important sigma factors, SigQ and HrdB, were analyzed by ChIP-seq procedure. Other sigma factors were further studied to see if they possess asRNAs, using 5' and 3' RACE method and northern blotting. Our data confirm the essentiality of HrdB sigma factor during the vegetative phase of growth. The other sigma factor, SigQ, has been revealed to be an important regulator of nitrogen metabolism and osmotic...
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