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Preparation and characterization of LmbX protein involved in lincomycin biosynthesis
Jiráčková, Petra ; Janata, Jiří (advisor) ; Janderová, Blanka (referee)
Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...
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Characterization of gycin-rich loop of mitochondrial processing peptidase alpha subunit from Saccharomyces cerevisiae
Chytrá, Dana ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which belongs to M16B subfamily of metaloendopeptidases. A universal function of this enzyme is in recognition and cleavage of great number of mitochondrial preprotein presequences, which differ in length and amino acid sequence. MPP consists of catalytical β-MPP and probably recognizing α-MPP. The most conservative region in α-MPP is GRL - glycine-rich loop. Its function is supposed in primary interaction with preprotein presequence. It is possible to study conformational change of GRL after binding the substrate by fluorescence experiments. In this diploma thesis the constructs coding the α-MPP with the single reporter tryptophan residue in the position 289 or 299 were prepared using site-directed mutagenesis. These forms of α-MPP were produced in E. coli BL21(DE3)+pGroESL. Activities of MPP dimer containing α-MPP with the single tryptophan residue in the reporter position were compared with MPP from wild type of S. cerevisiae. Used substrate was yeast malate dehydrogenase precursor with fused presequence (pMDH) from three organisms (yeast, mouse and melon). These presequences differ in their length. Activities of MPP dimer containing α-MPP with the single reporter tryptophan residue in the position 289 were about 70 % while...
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Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters
Koběrská, Markéta ; Janata, Jiří (advisor) ; Lichá, Irena (referee) ; Kormanec, Ján (referee)
Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters Markéta Koběrská PhD thesis 2010 The introductory part of the thesis presents isolation and sequencing of lincomycin gene cluster from type strain Streptomyces lincolnensis ATCC 25466. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin industrial strain Streptomyces lincolnensis 78-11. Analysis of the cluster flanking regions revealed its localization within the genome of S. lincolnensis ATCC 25466. The cluster-bearing cosmid was integrated into the chromosome of lincomycin non-producing strains Streptomyces coelicolor CH 999 and Streptomyces coelicolor M 145. The modified strains heterologously produced lincomycin, but the level dropped to approximately 1-3% of the production in S. lincolnensis ATCC 25466. The exact sequence of lincomycin gene cluster from the type strain allowed isolation and sequence analysis of the gene cluster of structurally related celesticetin. The analysis revealed 24 putative genes, 18 of them homologous with the genes participating in lincomycin biosynthesis. Four celesticetin specific genes are encoding enzymes involved in the salicylate biosynthesis and attachment, one is coding for...
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Genetic characterization of tetracycline resistance in selected soil isolates of bacteria.
Hucková, Tereza ; Lichá, Irena (advisor) ; Janata, Jiří (referee)
GENETIC CHARACTERIZATION OF TETRACYCLINE RESISTANCE IN SELECTED SOIL ISOLATES OF BACTERIA Abstract Antibiotic resistance is becoming increasingly widespread among bacterial organisms. To respond to the situation it is necessary to understand in detail all the mechanisms of resistance as well as expansion possibilities of resistance genes in the environment. In this study we attempted to identify tetracycline resistance determinants in selected soil gram-positive and gram-negative isolates. The isolates originate from unfertilized soil and from soil fertilized with tetracycline-contaminated manure. We tested the soil isolates for the presence of twenty three selected tetracycline resistance determinants and presence of tetracycline resistance determinants in DNA libraries. We identified one of the tetracycline resistance determinants tet(K) in Staphylococcus sp. A DNA fragment was amplified with primers for tet(M) determinant in Arthrobacter sp., but its presence was not confirmed by the sequence analysis. None of the tested tetracycline resistance determinants were present in the genera Chryseobacterium and Stenotrophomonas. However, we managed to prove the ongoing horizontal gene transfer between the genus Stenotrophomonas and the genus Chryseobacterium. The transferred sequences encoded hybrid protein and...
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The Shared Steps of Lincomycine and Pyrrolo-benzodiazepine Biosynthetic Pathways.
Jiráčková, Petra ; Fišer, Radovan (referee) ; Janata, Jiří (advisor)
Lincomycin and its semi-synthetic derivate clindamycin are therapeutically used antibiotics inhibiting protein synthesis in sensitive bacteria. Their structure is composed of an amino-sugar and an amino-acid moiety, which are linked with an amide bond. Some time ago the sequence of lincomycin-production gene cluster was established and functions of the most proteins coded from gene cluster were proposed on the basis of a comparison with known proteins and gene-inactivations. For a long time it was clear that the amino-acid moiety of linkomycin (propylproline) is related to a dihydropyrrol moiety of pyrrolo[1,4]benzodiazepine (PBD). Both of these moieties are derived from L-tyrosine and they undergo the same biosynthetic pathway at first. But other biosynthetic steps divide. It is believed that a transfer of genes from the PBD-production gene cluster to the lincomycin-production gene cluster could modify the structure of propylproline and improve traits of lincomycin e.g. increase antimalaric effects. The most effective antibiotics are alkyl derivatives, their chemical synthesis is very complicated, but they could be prepared via genetic engineering as so-called hybrid antibiotics.
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