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Streptomycetes surface growth and differentiation on inert microbeads- morphology and proteome study
Tesařová, Eva ; Weiser, Jaroslav (advisor) ; Lichá, Irena (referee)
Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...
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Biosynthesis of propylproline building unit of lincomycin
Jirásková, Petra ; Janata, Jiří (advisor) ; Čejková, Alena (referee) ; Fišer, Radovan (referee)
The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...
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Analysis of streptomycete isolates carrying a cyclizing aminolevulinate synthase gene
Rašmanová, Petra ; Petříčková, Kateřina (advisor) ; Felsberg, Jurgen (referee)
Streptomyces bacteria are well-known producents of many bioactive metabolites. Its secondary metabolism is a source of many important groups of active compounds that are recently investigated by means of many new methods based on bioinformatic analyses of genome data, modern LC-MS techniques, and metabolic modeling methods. This thesis originates from the genetic screening for a specific gene (als) for cyclizing 5-aminolevulinate synthase. Based on earlier studies, we consider this gene as a genetic tag of the producers of secondary metabolites containing the C5N unit (2-amino-3-hydroxycyklopent- 2-enon). Such metabolites include several groups with variable structures and biological activities, which include manumycins as well. Manumycins are small polyketides with a weak antibiotic aktivity, especially against gram-positive bacteria. However, its cancerostatic and anti-inflammatory effects are of greater importance. Streptomyces monomycini BCCO10 1552 and Streptomyces capoamus BCCO10 1636 strains were found positive for the presence of the als gene in the targeted genetic screening. By the als phylogeny, they cluster near the producents of manumycin compounds. This thesis aimed to determine whether these new natural isolates produce any compounds containing C5N unit, and to characterize them in...
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Identification and activation of a cryptic biosynthetic gene cluster for manumycin-type metabolites in Saccharothrix espanaensis DSM44229
Zelenka, Tomáš ; Petříčková, Kateřina (advisor) ; Beranová, Jana (referee)
1 Abstract: Secondary metabolism of Gram-positive soil bacteria from the genus Streptomyces is a inestimable source of natural products including manumycins, which belong to a polyketide group. These products possess weak antimicrobial, but important antiinflammatory, and antitumor activities. Streptomyces sp. offers broad amounts of yet undiscovered antibiotics, potentially utilizable in clinical medicine. This fact makes out of these organisms a promising solution to our present problem with rising antibiotic resistance among microorganisms. Two main ways are applied in this research: There are efforts of prepairing new derivates based on known products and creating various modifications in their structure. Next, new producers are discovered by "genome mining" methods, activation of silent gene clusters, followed by improvements of antibiotic production. One of those silent clusters was found in the Saccharothrix espanaensis DSM44229 strain. The genetic information has been transferred to a heterologous host in order to characterize its product. Cluster activation and production of novel manumycin-type metabolites occurred in the host after the transfer.
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Streptomycetes surface growth and differentiation on inert microbeads- morphology and proteome study
Tesařová, Eva ; Weiser, Jaroslav (advisor) ; Lichá, Irena (referee)
Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...
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Diversity of the soil microbial community and its functional aspects in man-influenced environments
CHROŇÁKOVÁ, Alica
Diversity of the soil microbial community and its functional aspects were investigated in man-influenced environments, such as colliery spoil heaps in post mining sites and upland pasture used for outdoor cattle husbandry. The study was based on the cultivation of bacteria and streptomycetes as well as culture-independent approaches. Cultivated bacteria and streptomycetes were characterized by phenotypic and genotypic means. The culture-independent approaches were based on an analysis of environmental DNA in terms of both qualitative and quantitative parameters.
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Struktura a funkce tmRNA u streptomycet
Bobek, Jan ; Palečková, Petra ; Mikulík, Karel
The functional trans-translation system is important to allow survival of antibiotic producing microorganism under unfavourable conditions. The ssrA genes from S. coelicolor, S. collinus, S. aureofaciens and S. griseus were amplified and subjected to sequence analysis. To investigate the role of tmRNA in streptomycetes we have examined the level of tmRNA during the growth and differentiation under various physiological conditions
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Aktivace translačního systému v průběhu buněčné diferenciace streptomycet
Bobek, Jan ; Palečková, Petra ; Vohradský, Jiří ; Mikulík, Karel
Synchronously germinating spores of S. granaticolor were used to study the activation of translational system and expression of proteins. Proteomic approach was used to gain more information on expression of proteins during germination on two types of media differing in ability of cells to produce antibiotic granaticin. The requirement of functional trans-translational system for reactivation of translation in S. aureofaciens exposed to unfavourable conditions was examined. Abundance of tmRNA is connected with changes in the rate of translation and with the accumulation of tetracycline. Our data indicate that trans-translation could be involved in self-defence against antibiotics synthesized
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