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Evolution of RAYT/REP system in Stenotrophomonas
Staněk, Tadeáš ; Nunvář, Jaroslav (advisor) ; Zahradník, Jiří (referee)
REP (repetitive extragenic palindromes) are short DNA motifs found in the intergenic regions of many bacteria, typically in the hundreds of copies per genome. Unable to mobilize on their own, REP are propagated by RAYT (REP-associated tyrosine transposase). Stenotrophomonas are G- bacteria with a broad ecological niche, belonging to the Gammaproteobacteria. RAYT/REP systems are widespread in them, with over 132,000 REP found in 102 strains. Several classes of REP coexist in the genomes, the presence of associated RAYT is essential for high REP abundance. REP are localised in "REP loci", a series of intergenic regions with high evolutionary dynamics - the number, sequence, and orientation of REP vary at a given locus. In addition to genomic islands, REP are absent from the replication termination site, a region showing non-standard evolutionary dynamics (variable number and order of genes), probably related to chromosome segregation. Stenotrophomonas are insertion sequences (IS) rich. 35 unique IS were identified and their insertion target was verified. Ten IS insert into REP, and four other target YPAL (Yersinia palindromic elements), DNA motifs so far known only in the genus Yersinia. A total of 14 out of 35 IS in Stenotrophomonas target intergenic motifs, while their copy number accounts for 75%...
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Characterization of membrane pores formed by newly discovered colicin FY from Yersinia frederiksenii
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Nunvář, Jaroslav (referee)
Colicins are toxic exocellular proteins used by Gram-negative bacteria for interspecies and intraspecies competition. The colicin FY is a pore-forming protein which was recently discovered at Masaryk university. Kolicin FY is produced by strain Yersinia frederiksenii Y27601 and is active against other strains of genus Yersinia. By comparison of aminoacid sequences of C-terminal domains of selected colicins it was proved, that colicin FY is closely related to colicin Ib (Bosák et al., 2012). In this work I was trying to create brief and integrated summary about the group of colicins from the perspective of an outer membrane traslocation mechanism, overcoming the periplasmic space up to isertion of C-terminal colicin domain into the inner membrane phospholipid bilayer. Other aim of my work was to generally summarize pore properties of known colicins and compare them with recently measured characteristics of colicin FY. Keywords: colicin, Yersinia, planar lipid membranes, membrane pore
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Bacterial REP elements: origins, variability and application.
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Pačes, Jan (referee) ; Melter, Oto (referee)
4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...
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Next generation sequencing in clinical virology: method optimization and it's use for samples with unknown infectious agent
Poláčková, Kateřina ; Kramná, Lenka (advisor) ; Nunvář, Jaroslav (referee)
The use of the MinION sequencer (Oxford Nanopore) was tested on samples prepared to simulate infectious samples. The tested procedure is to simulate work with a sample with an unknown pathogen. Therefore, a metagenomic approach was chosen. Three kits were tested: Rapid Barcoding Sequencing, PCR Barcoding and Premium whole genome amplification. Each kit differed in duration, difficulty to prepare and in amplification of nucleic acids. In total it was chosen eight viruses with different genome lengths and with varying types of the genome (5,6 - 152 kb, ss/ds RNA, dsDNA). Ten samples were prepared to simulate different types of infection (respiratory, gastrointestinal tract and urine), and one sample contained pure water as a negative control. Before preparation of the library with Oxford Nanopore's kits, DNase/RNase treatment was used. The viral RNA was transcribed into DNA and in chosen samples were amplificated to reach a higher concentration of nucleic acids. Rapid barcoding sequencing kit detected all selected viruses with the highest number of viral reads (4403) with a length between 100 and 250 nt and quality coverage of viral genomes. PCR Barcoding kit detected five out of eight viruses, and the number of identified reads with a length of 100-200 nt distinctly decreased. Premium whole genome...
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Bacterial REP elements
Staněk, Tadeáš ; Nunvář, Jaroslav (advisor) ; Zahradník, Jiří (referee)
Insertion sequences are the smallest type of mobile genetic element. They are formed from specific repetitive elements, between which gene for transposition is placed. That is an enzyme used for their genome propagation. REP elements are short repetitive sequences abundantly represented in many bacterial classes. They are placed in noncoding regions of genome. They have a palindromic structure, thanks to that their sides are complementary. ssRNA and ssDNA forms of REP can form a stem-loop structure. In the host genome, there are hundreds of these elements. REPs are usually grouped in higher structural units: REPIN, which has structure REP-spacer-REP, or BIME with iREP-spacer-iREP structure. These structures are associated with RAYT, tyrosine nuclease related to IS200/IS605 transposase family. The RAYT is a domesticated enzyme propagating REP elements through the genome. The presence of REP elements has an influence on transcription and translation of neighbouring genes. In addition, REPs interact with proteins important for cell physiology. This thesis is focused on the dissemination of REP elements, their characteristics, function in cells, and cooperation with RAYT nucleases. This thesis also summarizes the findings of RAYT and their similarities and differences from other families of...
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Bacterial REP elements: origins, variability and application.
Nunvář, Jaroslav
4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...
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Comparative structural analysis of Borreliella spp. virulent factors focusing on their surface topology conservation
Zdrha, Alois ; Drbal, Karel (advisor) ; Nunvář, Jaroslav (referee)
Gram-negative bacteria of genus Borreliella cause the nowadays spreading illness Lyme borreliosis. However, their classification as gram-negative bacteria is rather mislea- ding because they differ in their complex genome, pathogenic adaptation to their hosts and composition of their outer membrane. One species of borreliella can harbour up to 23 variable plasmids and one conserved chromosome and they still lack many crucial proteins needed for synthesis of key compounds. Hence, they have to obtain these compounds from the host. However, in order for borreliella to utilise the metabolic processes of the host, it has to first survive its immune response. Therefore borreliella contains many virulent factors including highly variable surface lipoproteins. The variability is a major obstacle to overcome when using their surface epitopes for detection and vaccine development because most of the dominant antigenic epitopes of borreliella are usually parts of the most variable regions of the lipoproteins. Nowadays, we can use many different algorithms which determine evolutionary conserved epitopes based on analysis of sequences of given lipoprotein, in order to find suitable targets for antibodies. In general, conserved epitopes are more suitable for vaccines, whereas more variable epitopes are better...
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The role of the AdeABC efflux system in resistance of Acinetobacter baumannii to aminoglycosides
Kladivová, Lucie ; Nemec, Alexandr (advisor) ; Nunvář, Jaroslav (referee)
Acinetobacter baumannii is an important nosocomial pathogen characterized by the ability to acquire and develop complex resistance to antimicrobial agents. This capability is caused by eflux systems removing molecules of antibiotics from bacterial intracellular space. AdeABC is an RND-type chromosomal eflux system specific for A. baumannii which has a broad substrate spectrum. In this work, we focused on functional analysis of AdeABC to define its role in the resistance development to aminoglycosides in genetically different strains. We studied a set of 15 epidemiologically and genotypically well characterized strains of A. baumannii which were fully susceptible to aminoglycosides and other antibiotics primarily effective against this species. We determined genotyp of AdeABC for these strains and performed a selection for resistant variants in the presence of netilmicin. Using real-time qRT-PCR we compared the expression of the transporter gene adeB in originally sensitive strains and selected variants. The obtained results confirmed that the increased expression of AdeABC significantly reduces susceptibility to aminoglycosides and other antibiotics. The results also suggest that the efflux system provides a significant selective advantage for nosocomial strains of A. baumannii.
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Bacterial REP elements: origins, variability and application.
Nunvář, Jaroslav
4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...
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