Genetická analýza N.meningitidis multilokusovou sekvencí určí povahu populací působících invazivní onemocnění se zřetelem na vysoce virulentní varianty, odpovědné ve středoevropském regionu za onemocnění se zvýšeně závažným průběhem a smrtností.; Genetic analysis of N. meningitidis populations by multilocus gene sequencing that describe highly virulent clones specific for Central European region and responsible for invasive disease of unusually serious course and high fatality rate.

• MeSH
• faktory virulence MeSH
• meningokoková meningitida MeSH
• Neisseria meningitidis genetika   MeSH
• sekvenční analýza MeSH
• sepse MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• infekční lékařství
• biologie
• epidemiologie
• mikrobiologie, lékařská mikrobiologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

A new separation and quantification method using ultra high-performance liquid chromatography (UHPLC) with UV detection was developed for the detection of sibiromycin in fermentation broth of Streptosporangium sibiricum. The solid phase extraction method based on cation-exchange was employed to pre-concentrate and purify fermentation broth containing sibiromycin prior to UHPLC analysis. The whole assay was validated and showed a linear range of detector response for the quantification of sibiromycin in a concentration from 3.9 to 250.0 μg mL⁻¹, with correlation coefficient of 0.999 and recoveries ranging from 71.66±3.55% to 74.76±5.18%. Method limit of quantification of the assay was determined as 0.18 μg mL⁻¹ and was verified with resulting RSD of 9.6% and accuracy of 97.6%. The developed assay was used to determine the sibiromycin production in 12 different fermentation broths. Moreover, several natural sibiromycin analogues/derivatives were described with pilot characterization using off-line mass spectrometry: the previously described dihydro-sibiromycin (DH-sibiromycin) and tentative bis-glycosyl forms of sibiromycin and its dihydro-analogue.

• MeSH
• Actinomycetales metabolismus   MeSH
• aminoglykosidy analýza   chemie   metabolismus   MeSH
• extrakce na pevné fázi MeSH
• fermentace MeSH
• hmotnostní spektrometrie metody   MeSH
• kalibrace MeSH
• kultivační média speciální chemie   MeSH
• lineární modely MeSH
• molekulární struktura MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• stabilita léku MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE OF WORK: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.

• MeSH
• aminohydrolasy genetika   metabolismus   MeSH
• Aspergillus niger enzymologie   genetika   MeSH
• Escherichia coli genetika   MeSH
• exprese genu MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genom fungální MeSH
• Gibberella enzymologie   genetika   MeSH
• klonování DNA MeSH
• Neurospora crassa enzymologie   genetika   MeSH
• nitrily metabolismus   MeSH
• organické látky metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

• MeSH
• aminohydrolasy biosyntéza   genetika   izolace a purifikace   metabolismus   MeSH
• Aspergillus niger enzymologie   genetika   MeSH
• bakteriální proteiny genetika   izolace a purifikace   metabolismus   MeSH
• klonování DNA metody   MeSH
• komplementární DNA MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• radiační rozptyl MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky MeSH
• stabilita enzymů MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• odvolaná publikace MeSH
• práce podpořená grantem MeSH

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.

• MeSH
• acetolaktátsynthasa genetika   chemie   metabolismus   MeSH
• aktivace enzymů MeSH
• alosterická regulace imunologie   MeSH
• bakteriální proteiny genetika   chemie   metabolismus   MeSH
• bodová mutace MeSH
• katalytická doména imunologie   MeSH
• konformace proteinů MeSH
• missense mutace MeSH
• molekulární modely MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• Streptomyces enzymologie   genetika   MeSH
• substituce aminokyselin MeSH
• valin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH

Transfer-messenger RNA (tmRNA, 10Sa RNA, ssrA) is bacterial RNA having both tRNA and mRNA properties and playing an essential role in recycling of 70S ribosomes that are stalled on defective mRNA. The trans-translational system is thought to play a crucial role in bacterial survival under adverse conditions. Streptomycetes are Gram-positive soil bacteria exposed to various physical and chemical stresses that activate specialized responses such as synthesis of antibiotics and morphological differentiation. Comparative sequence analysis of ssrA genes of streptomycetes revealed the most significant differences in the central parts of tag-reading frames, in the stop codons and in the 15-34 nucleotide sequences following stop codons. A major challenge in understanding the interactions that control the function of tmRNA is the definition of protein interactions. Proteins from various phases of development of Streptomyces aureofaciens associated with tmRNA were analyzed. Using affinity chromatography on tmRNA-Sepharose and photo cross-linking experiments with [(32)P]labeled tmRNA seven proteins, the beta and beta'-subunits of DNA dependent RNA polymerase, polyribonucleotide nucleotidyltransferase (PNPase), ribosomal protein SS1, ATP-binding cassette transporters, elongation factor Tu, and SmpB were identified among the proteins associated with tmRNA of S. aureofaciens. We examined the functional role of ribosomal protein SS1 in a defined in vitro trans-translation system. Our data show that the protein SS1 that structurally differs from S1 of Escherichia coli is required for translation of the tmRNA tag-reading frame.

• MeSH
• bakteriální RNA genetika   metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• financování organizované MeSH
• messenger RNA metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• proteiny vázající RNA metabolismus   MeSH
• proteosyntéza MeSH
• retardační test MeSH
• ribonukleoproteiny metabolismus   MeSH
• ribozomální proteiny metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• spory bakteriální metabolismus   MeSH
• Streptomyces coelicolor genetika   růst a vývoj   MeSH

tmRNA and protein SmpB are the main components required for rescue of stalled ribosomes incapable of properly elongating or terminating the polypeptide chain. We examined the tmRNA level and protein synthesis in Streptomyces aureofaciens, S. griseus and S. collinus synthesizing tetracycline, streptomycin and kirromycin, respectively, during various stress conditions. Downshift in temperature caused a decrease in protein synthesis but the level of tmRNA increased. Shift up in temperature induced decay of tmRNA in all strains and in S. collinus led to stimulation and in S. aureofaciens and S. griseus to inhibition of protein synthesis. At high NaCl concentrations protein synthesis was inhibited and tmRNA decayed. Shift in pH from 7.0 to 5.0 had no pronounced effect on the tmRNA level while upshift to pH 9.0 in S. collinus and S. aureofaciens caused inhibition of protein synthesis and decay of tmRNA in S. collinus. In contrast, protein synthesis and tmRNA level increased in S. griseus at the alkaline pH. Our data show that tmRNA abundance is important for survival of streptomycetes under certain unfavorable conditions.

• MeSH
• bakteriální RNA analýza   genetika   MeSH
• elektroforéza v polyakrylamidovém gelu metody   využití   MeSH
• finanční podpora výzkumu jako téma MeSH
• hybridizace genetická genetika   MeSH
• northern blotting metody   využití   MeSH
• proteosyntéza genetika   MeSH
• reakce na tepelný šok genetika   MeSH
• Streptomyces aureofaciens genetika   metabolismus   MeSH
• Streptomyces griseus genetika   metabolismus   MeSH

• MeSH
• chloramfenikol biosyntéza   MeSH
• financování vládou MeSH
• messenger RNA biosyntéza   genetika   MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• proteosyntéza genetika   MeSH
• RNA transferová biosyntéza   genetika   MeSH
• Streptomyces aureofaciens genetika   izolace a purifikace   růst a vývoj   MeSH
• techniky in vitro MeSH
• tetracyklin MeSH