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Significance of protein phosphorylation for bacterial cell
Gregorová, Michaela ; Branny, Pavel (advisor) ; Lišková, Petra (referee)
Phosphorylation - most common post-translational modification has an important role in many cellular processes of bacteria. Bacteria contain enzymes that are in charge of adding phosphoryl group (kinases) or enzymes with reciprocal activity (phosphatases). Reversible phosphorylation and dephosphorylation of proteins are fundamental for signal transduction from the environment to the cell. These modifications can affect enzymatic activity, protein stability, localization as well as interaction with another protein. Due to the complexity of these phosphorylation networks, bacterial cells are capable to adapt very effectively to changing environmental conditions.
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Molecular mechanisms of bacterial persistence to antibiotics
Jirsová, Anežka ; Lichá, Irena (advisor) ; Branny, Pavel (referee)
The ability to persist is inherent in the vast majority of bacterial species. Persisters represent a small heterogenous fraction of the bacterial population that can tolerate antibiotics. Unlike resistant cells, which are genetically distinct from the rest of the antibiotic-sensitive population, persister cells form a genetically indistinguishable subpopulation. Persisters arise either as a result of stress caused by adverse environmental conditions, or persister subpopulation arise as a result of stochastic induction independent of the presence of stressful conditions. There are various mechanisms by which bacterial cells have been shown to induce a persistent state. Toxin-antitoxin systems and their interaction with stringent response effectors play an important role during the development of persistence. The persistent state is also affected by changes in proton-motive force (PMF) and the fluctuations in the gene expression level of energy generating enzymes of the Krebs cycle (TCA). In this thesis, persister cells are characterized and the current knowledge about the molecular mechanisms leading to the induction of persistence in bacteria is summarized. Key words: bacteria, persistence, antibiotics, stringent response, PMF, TCA
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Intestinal metabolism of bilirubin
Jirásková, Alena ; Branny, Pavel (advisor) ; Španová, Alena (referee) ; Pátek, Miroslav (referee)
CONCLUSIONS In this study we focused on the process of bilirubin reducfion catalyzed by an anaerobic intestinal bacterium C' peýingen.s. We aimed to undertake analysis of bile pigments metabolized by C. perfringens and their respective reduction products and to identify gene(s) encoding protein(s) involved in metabolism of bilirubin. Analysis of bile pigments metabolized by C. perfringens and their respective reduction products 1) The C. perfringens strain BRI isolated from neonatal stools reduces a variety of different bile pigments indicating that this broad substrate speciÍicity could be an effective tool for disposal of electrons produced in catabolic pÍocesseswithin thesebacteria. 2) The examined strain reduces UCB only to the level of urobilinogen. Other bacterial sfoains and species, absent in neonates, are presumed to be essential for catabolism to the level ofstercobilinogen. Identification of gene(s)involved in bilirubin metabolism 1) The C. perfringens strain BRl is resistant to the transformation of plasmid DNA mediatedby electroporation and thereforeit is not a candidate suitable for transposonmutagenesis. A transformab|e C. peýingens P90.2,2. strain was found to reduce bilirubin. Rapid and simple method suitable for electroporation of this strain was developed providing transformation...
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Dimerization of Streptococcus pneumoniae eukaryotic-type SerThr protein kinase and characterization of its substrate, phosphoglucosamine mutase GlmM
Pallová, Petra ; Branny, Pavel (advisor) ; Janata, Jiří (referee) ; Španová, Alena (referee)
104 7 Závěr 7.1 Proteinkinasa StkP Připravili jsme kmen S. pneumoniae KDTM-his, který ve svém genomu kóduje epitopem značenou kinázovou doménu proteinkinasy StkP ukotvenou do membrány pomocí transmembránové domény. Imunologickou detekcí s monoklonální protilátkou proti histidinové kotvě jsme potvrdili lokalizaci proteinu v membránové frakci S. pneumoniae. V in vitro kinázových reakcích jsme prokázali, že se jedná o plně funkční protein s autofosforylační aktivitou. Pomocí in vivo reportérového systému jsme zjistili, že transmembránová doména a extracelulární doména proteinkinasy StkP tvoří stabilní dimery. Dimerizace proteinkinasy StkP byla následně potvrzena pomocí nativní elektroforézy. Je tedy velmi pravděpodobné, že proteinkinasa StkP se in vivo vyskytuje ve formě dimeru a dimerizace je nutným předpokladem její autofosforylační aktivity. Na základě těchto výsledků jsme vyslovili hypotézu, že protein kódující kinázovou doménu postrádající transmembránovou doménu není funkční, neboť není schopen dimerizace a podléhá degradaci. 7.2 Fosfoglukosaminmutasa GlmM V in vitro kinázové reakci na bezbuněčných lyzátech S. pneumoniae Cp1015, ∆stkP a ∆phpP-stkP v přítomnosti rekombinantní proteinkinasy StkP jsme prokázali fosforylaci proteinu o molekulové hmotnosti odpovídající fosfoglukosaminmutase GlmM....
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Molecular dynamics of proteins interacting with substrate or ligand: mitochondrial processing peptidase and FixL oxygen sensor
Dvořáková Holá, Klára ; Janata, Jiří (advisor) ; Váchová, Libuše (referee) ; Branny, Pavel (referee)
I I Ovenvrew I I I II I I I I I I I I I The presenteddoctoralthesis includesfive publishedscientificarticlesand one manuscriptpreparedfor submission.All describestudieson threeproteinmodels.The four papersnumbered(1)- (4)inthelistofpublications,sharea commonobjectivee.i.observingand describingfunctionalproteindynamicsandconformationchangeinducedby ligandor substrate binding,andrepresentthemainresultofmyPhDwork. Thepapers(1)and(4)offerresultsof a projectfrommyhomelaboratoryattheInstitute ofMicrobiologyAS CR, LaboratoryforBiologyofSecondaryMetabolism,underthesupervisionof JiřÍ Janata, PhD. The projectis focusedon the protein-proteindynamicsinteractionof mitochondrialprocessingpeptidase(MPP) fromSaccharomycescerevisiaewithits preproteins substrates. The papers(2)and(3)describeresultsof a project,inwhichI haveparticipatedduring myMarieCuriefellowshipattheEcolePolytechnique(PalaiseauCedex,France),Laboratoryfor OpticsandBiosciences,in2004.Theprojectconcernsresearchon proteinstructuraldynamicsof theheme-basedoxygensensorFixLfromBradyrhizobiumjaponicum,inwhichoxygenbindingto the heme sensor domain inducesconformationchange,which regulatesthe activityof neighboringkinasedomain. ln bothprojects,analogyin methodicalapproach,i.e.seriesof molecularbiologyand...
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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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Control of cell division of Streptococcus pneumoniae by unique signaling pathway
Kubincová, Hana ; Branny, Pavel (advisor) ; Fišer, Radovan (referee)
Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...
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