Enzimspecifitás megváltoztatása in vitro evolúciós stratégiával = Alteration of enzyme specificity by a strategy based on in vitro evolution

A kutatás célja előző - hasonló című - pályázatunk, amelyben is vitro mesterséges evolúcióval előállítottunk egy megváltozott specifitású DNS-modifikációs metiláz enzimet, kiegészítése és lezárása volt. Elért eredményeink a következőkben foglalhatók össze: 1. Előállítottuk és részlegesen jellemeztük két újabb, szintén megváltozott specifitású mutánsát a korábban vizsgált M.SinI enzimnek. 2. Előállítottuk és részlegesen jellemeztük az M.SinI enzimnek és egyik megváltozott specifitású mutánsának az N-terminális régió első 65 aminosavától megfosztott, rövidített változatát, amelyek működőképesek voltak. 3. Újszerű, az irodalomban egyedülálló, kisérleti rendszert dolgoztunk ki az M.SinI enzim kívánt irányú specifitásváltozásának in vivo detektálására és jellemzésére. | This was the extension and continuation of the work done during our previous OTKA project (with the same title), in which we constructed, by using in vitro directed evolution techniques, a mutant DNA-modification enzyme with changed recognition specificity. The results of the present work could be summarized as follows: 1. By applying the same methods, we constructed two different new mutant variants of the previously studied M.SinI enzyme, with similarly changed recognition specificities, and partially characterized them. 2. We constructed and partially characterized truncated versions of the wild-type and one mutant variant of the M.SinI enzyme, without the N-terminal first 65 amino acids. These truncated enzymes preserved their enzymatic activity. 3. We developed an entirely new experimental system, for the in vivo detection and semiquantitative measurement of the changed recognition specificity of the studied M.SinI enzyme

Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs

<p>Abstract</p> <p>Background</p> <p>Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.</p> <p>Results</p> <p>By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome <it>E. coli </it>strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.</p> <p>Conclusions</p> <p>By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free <it>E. coli </it>strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.</p

Göran Wahlenberg úttörő tematikus térképe a Magas-Tátra vidékéről (1813–14)

Göran Wahlenberg (1780–1851) svéd orvos-botanikus és természetkutató 1813-ban végzett növényföldrajzi terepkutatásokat a Magas-Tátra és környéke hegyvidékén a Magyar Királyságban. Eredményeit 1814-ben Göttingenben publikálta a „Flora Carpatorum Principalium” című, latin nyelvű kötetében, melyhez két kartográfiai szempontból fontos ábrázolást, egy tematikus térképet és egy növényföldrajzi metszetet is mellékelt. Könyve és térképei az európai tudományos körökben is ismertté tették a terület földrajzi jellemzőit és botanikai értékeit, ugyanakkor a svéd kutató nemzetközi tudományos karrierje szempontjából is rendkívüli jelentőséggel bírtak. Tanulmányunkban a kartográfia mint formálódó tudományág felvilágosodás kori kialakulását folyamatként értelmezzük. Ebben a keretben értelmezzük Wahlenberg tematikus térképét, amely egyszerre növényföldrajzi és hipszometrikus ábrázolás a Magas-Tátra vidékéről. Az 1814-es térkép a kutató által mért magassági öveket szintvonallal és eltérő színekkel különíti el, így az első rétegszínezéses térkép a világon. A természetes vegetációzónák határvonalai a szárazföldi területen alkalmazott magasságvonalak prototípusainak tekinthetőek, mivel barometrikus magasságmérés alapján készültek. Tanulmányunk feltárja a térkép forrásait, névanyagának jellemzőit, kitér a tudós magasságmérési metódusára. Wahlenberg térképének és könyvének nemzetközi hatása kimutatható a 19. századi kortárs földrajzban és térképészetben Humboldttól Berghausig

Exploring the fitness benefits of genome reduction in Escherichia coli by a selection-driven approach

Artificial simplification of bacterial genomes is thought to have the potential to yield cells with reduced complexity, enhanced genetic stability, and improved cellular economy. Of these goals, economical gains, supposedly due to the elimination of superfluous genetic material, and manifested in elevated growth parameters in selected niches, have not yet been convincingly achieved. This failure might stem from limitations of the targeted genome reduction approach that assumes full knowledge of gene functions and interactions, and allows only a limited number of reduction trajectories to interrogate. To explore the potential fitness benefits of genome reduction, we generated successive random deletions in E. coli by a novel, selection-driven, iterative streamlining process. The approach allows the exploration of multiple streamlining trajectories, and growth periods inherent in the procedure ensure selection of the fittest variants of the population. By generating single- and multiple-deletion strains and reconstructing the deletions in the parental genetic background, we showed that favourable deletions can be obtained and accumulated by the procedure. The most reduced multiple-deletion strain, obtained in five deletion cycles (2.5% genome reduction), outcompeted the wild-type, and showed elevated biomass yield. The spectrum of advantageous deletions, however, affecting only a few genomic regions, appears to be limited

Potent Chimeric Antimicrobial Derivatives of the Medicago truncatula NCR247 Symbiotic Peptide

In Rhizobium-legume symbiosis, the bacteria are converted into nitrogen-fixing bacteroids. In many legume species, differentiation of the endosymbiotic bacteria is irreversible, culminating in definitive loss of their cell division ability. This terminal differentiation is mediated by plant peptides produced in the symbiotic cells. In Medicago truncatula more than similar to 700 nodule-specific cysteine-rich (NCR) peptides are involved in this process. We have shown previously that NCR247 and NCR335 have strong antimicrobial activity on various pathogenic bacteria and identified interaction of NCR247 with many bacterial proteins, including FtsZ and several ribosomal proteins, which prevent bacterial cell division and protein synthesis. In this study we designed and synthetized various derivatives of NCR247, including shorter fragments and various chimeric derivatives. The antimicrobial activity of these peptides was tested on the ESKAPE bacteria; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli as a member of Enterobacteriaceae and in addition Listeria monocytogenes and Salmonella enterica. The 12 amino acid long C-terminal half of NCR247, NCR247C partially retained the antimicrobial activity and preserved the multitarget interactions with partners of NCR247. Nevertheless NCR247C became ineffective on S. aureus, P. aeruginosa, and L. monocytogenes. The chimeric derivatives obtained by fusion of NCR247C with other peptide fragments and particularly with a truncated mastoparan sequence significantly increased bactericidal activity and altered the antimicrobial spectrum. The minimal bactericidal concentration of the most potent derivatives was 1.6 mu M, which is remarkably lower than that of most classical antibiotics. The killing activity of the NCR247-based chimeric peptides was practically instant. Importantly, these peptides had no hemolytic activity or cytotoxicity on human cells. The properties of these NCR derivatives make them promising antimicrobials for clinical use

A prospective, longitudinal monocentric study on laser Doppler imaging of microcirculation: comparison with macrovascular pathophysiology and effect of adalimumab treatment in early rheumatoid arthritis

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Evaluation of bloodstream infections during chemotherapy-induced febrile neutropenia in patients with malignant hematological diseases: single center experience

From year to year, it is important to get an overview of the occurrence of causative agents in febrile neutropenic patients to determine the empiric treatment. Thus our aims were to evaluate a four-year period regarding the prevalence of bloodstream infections and the most important causative agents. During this period, 1,361 patients were treated in our hematology ward because of various hematological disorders. 812 febrile episodes were recorded in 469 patients. At that time, 3,714 blood culture (BC) bottles were sent for microbiological investigations, 759 of them gave positive signal. From the majority of positive blood culture bottles (67.1%), Gram-positive bacteria, mainly coagulase-negative staphylococci (CNS), were grown. Gram-negative bacteria were isolated from 32.9% of the positive blood culture bottles, in these cases the leading pathogen was Escherichia coli. The high prevalence of CNS was attributed to mainly contamination, while lower positivity rate for Gram-negative bacteria was associated with the use of broad-spectrum empiric antibiotic treatment

In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants▿

The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease