|
|
Analysis of promoters of genes encoding sigma factors in pathogenic Corynebacteria using Corynebacterium glutamicum model
Kučera, Tomáš ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The aim of this thesis was to idetify potential promoters of genes encoding sigma factors in pathogenic corynebacteria using the model bacterium Corynebacterium glutamicum and to assess the effect of amino acid sequences of sigma proteins on promoter recognition. Sigma factors are essential for transcription and regulation of gene expression and play a key role in controlling promoter activity. Understanding the regulation of genes encoding sigma factors is esential because they contribute to virulence and pathogenicity and regulate the host immune response. In this study, the promoter regions of genes encoding sigma factors in Corynebacterium ulcerans and Corynebacterium diphtheriae were analyzed. First, potential promoter sequences in the genomes of C. ulcerans and C. diphtheriae were identified based on knowledge of consensus sequences of confirmed promoters in the model organism. The already tested two-plasmid system established in C. glutamicum was used to evaluate the activity of the proposed promoter regions. A potentional promoter region was inserted into the pEPR1 vector upstream of the gfpuv reporter gene, and sigma factor genes were inserted downstream of the inducible promoter in the pEC-XT99A vector. Using fluorescence, the activity of the reporter gene in response to induced sigma...
|
|
|
The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav ; Nešvera, Jan (advisor) ; Branny, Pavel (referee) ; Lichá, Irena (referee)
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
|
|
|
The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid- producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
|
|
|
Overlaps of sigma factors regulons of RNA polymerase in Corynebacterium glutamicum
Zíková, Jaroslava ; Pátek, Miroslav (advisor) ; Sudzinová, Petra (referee)
Sigma factor (σ) is a part of the RNA polymerase enzyme complex. This complex (referred to as a holoenzyme) ensures the recognition of the consensus promoter sequences of the individual genes and the initiation of transcription. Seven sigma factors were found in Corynebacterium glutamicum. The genome of this bacterium encodes one primary factor σA and another six alternative sigma factors: σB , σC , σD , σE , σH a σM . These alternative sigma factors are expressed in response to changes in the internal and external environment and ensure the adaption of the bacterium to growth conditions. They are also one of many ways to regulate gene expression at the transcriptional level. In specific cases, the regulation of gene expression is caused by alternative sigma factors that recognize corresponding dual (recognized alternatively by two sigma factors) or overlapping promoters. Thus, the genes controlled by these promoters are classified into overlapping regulons. Key words: Corynebacterium glutamicum, sigma factor, RNA polymerase, transcription, promoter, regulons, RNA-seq, in vitro transcription, in vivo two-plasmid system
|
|
|
Function of stress sigma factors of RNA polymerase SigD, SigE, SigH and SigM in transcription regulation network of Corynebacterium glutamicum
Dostálová, Hana ; Pátek, Miroslav (advisor) ; Bobek, Jan (referee) ; Halgašová, Nora (referee)
Grampositive bacterium Corynebacterium glutamicum is an important industrial producer of amido acids and other metabolites. Its genome encodes 7 sigma (σ) subunits of RNA polymerase: primary factor σA , primary-like σB and five alternative sigma factors, σC , σD , σE , σH and σM (sigma factors with extracytoplasmic function). This study is focused on revealing so far unknown regulons of stress sigma factors or closer description of regulons whose genes are controled by σD , σE , σH and σM . These factors were partially described for their activity during surface (σD and σE ), heat (σE , σH and σM ) and oxidative (σH and σM ) stress response. We assumed that the genes of each regulon are transcribed from promoters of a single class. For the purpose of detailed promoter analysis, it was necessary to develop methods which can quickly and reliably assign sigma factor to particular promoters and, thus, respective genes. For this purpose, a combination of in vivo (two-plasmid system) and in vitro (in vitro transcription) techniques was developed that allow to specify this assignment. We identified 9 σH /σE - promiscuous promoters (PamtR, Pcg0378, Pcg1121, Pcg3309, Pcg3344, PclgR, PdnaJ, PdnaK and PsigB), 7 σD /σH - promiscuous promoters (Pcg0607, Pcg2047, Pcmt2, PfadD2, Plpd, PlppS and PrsdA) a 9 σH /σM...
|
|
|
Hybrid sigma factors of RNA polymerase in Corynebacterium glutamicum
Blumenstein, Jan ; Štěpánek, Václav (advisor) ; Krásný, Libor (referee)
Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...
|
|
|
The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid- producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
|
|
|
Regulatory network controlled by sigma factors of RNA polymerase in Corynebacterium glutamicum
Kučera, Tomáš ; Pátek, Miroslav (advisor) ; Roučová, Kristina (referee)
An important feature of bacteria is an ability to adapt to changing environment by regulating gene expression. Level of gene expression and its right timing depends mainly on activation or repression of the gene by transcriptional regulators and recognition of the respective promoter by the sigma factor which is a subunit of RNA polymerase. Transcription regulators with sigma factors and other control elements, form a complex regulatory network. The regulatory network in Corynebacterium glutamicum is one of the best studied networks among gram-positive bacteria owing to genome sequencing and application of a number of techniques at the genome level. There has been a lot of success in understanding the roles of individual regulators and interactions between regulators in response to changes in environment. This work summarizes currently known knowledge of mutual relationships between sigma factors, the influence of sigma factors on transcriptional regulators and their cooperative effect on the initiation of transcription. In the thesis, a regulatory network of sigma factors in C. glutamicum and a regulatory cascade in response to the stress situation is schematically created. Key words: sigma factor (FS), Corynebacterium glutamicum, transription regulator (TR), transcription, regulation
|
|
|
Characteristics of expression vectors for Corynebacterium glutamicum and their use for studies of sigma factors of RNA polymerase
Dvořáková, Pavla ; Pátek, Miroslav (advisor) ; Konopásek, Ivo (referee)
The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...
|
|
|
The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav ; Nešvera, Jan (advisor) ; Branny, Pavel (referee) ; Lichá, Irena (referee)
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
|
guest ::
