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Function of alternative sigma factors of RNA polymerase in stress response of Corynebacterium glutamicum
Dostálová, Hana ; Nešvera, Jan (advisor) ; Lichá, Irena (referee)
The aim of this thesis was to contribute to understanding of the functions of the alternative sigma factors of RNA polymerase, σH a σM , during stress response of C. glutamicum. The role of σH and σM in the transcription of the gene sigM encoding σM and the genes of the operon dnaK-grpE-dnaJ-hspR which encode proteins involved in heat-shock response of C. glutamicum was studied. The promoters of the tested genes were cloned into the "promoter-probe" vector pET2 and their activity was determined by the measuring of specific activity of the reporter enzyme chloramfenicol acetyltransferase. It was found, that the heat stress has a moderate positive effect on the activity of the promoter of the gene sigM (P-sigM), whereas no effect of the oxidative stress induced by diamide was found. It was proved, that deletion of the gene sigH or sigM itself does not lead to decrease of activity of the promoter P-sigM neither in standard conditions nor after heat-shock. On the other hand, complete abolition of the activity of the promoter P-sigM was observed in the strain C. glutamicum ∆sigH∆sigM. The promoter of the gene sigM is thus recognized in standard conditions and after heat stress is recognized by both sigma factors σH and σM , but not by the sigma factor σA . It was shown, that mutation in the -10 region...
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Characterization of bacterial genes encoding DbtC-like extradiol dioxygenase with bioremediation potential for aromatic compounds in locality Hradčany
Šnajdrová, Renata ; Bořek Dohalská, Lucie (advisor) ; Nešvera, Jan (referee)
Aromatic pollutants pose a serious environmental problem. Petroleum and its derivates belong to the most abundant contaminants in the Czech Republic and their sanation is a priority objective for improving the life quality of the population. Bioremediation is a technology taking advantage of the natural capacity of soil and water microbial community to degrade environmental pollutants. Deeper understanding and detailed knowledge on specialized bacterial species, pathways and genes is required for selection, optimization and application of targeted bioremediation approach and for monitoring of its results. Recent analysis of a metagenomic library constructed from highly contaminated soil of the former military air-base Hradčany has identified a novel group of catabolic genes encoding extradiol dioxygenase similar to DbtC of Burkholderia sp. DBT1. The DbtC-like enzymes are among the three priority groups of extradiol dioxygenases with biodegradation relevance for the locality. The present study of soil bacterial isolates and metagenomic fosmid clones harboring the genes of interest gained evidence about the dbtC-like genes as a part of highly mobile gene cassette. Transposon insertion mutagenesis identified the genes joined with the expression of the extradiol dioxygenase activity. The dbtC-like genes were...
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Role of the yxkO gene of Bacillus subtilis in responce to environmental stress.
Petrovová, Miroslava ; Lichá, Irena (advisor) ; Nešvera, Jan (referee)
ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.
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Characterization of Corynebacterium glutamicum promoters responding to increase of temperature
Zemanová, Martina ; Nešvera, Jan (advisor) ; Lichá, Irena (referee) ; Felsberg, Jurgen (referee)
My thesis is focused on characterizationof Corynebacteriumglutamicum promoters responding to increase of temperature.C. glutamicum is the most importantrepresentativeof a gram-positive,non-pathogenic,amino-acidproducing bacteria.Its strainsare industrialproducersof glutamic acid, lysine, threonine, tryptophaneand phenylalanine(Liebl et al., l99l). Amino acids producedby microbial fermentationare used in medicine(as infusionliquids or therapeutics), chemical industry (as detergentsand cosmeticscompounds),food industry (as additives), agriculture and stockbreeding (as growth-regulatoradditives to the animalsfeed)(Hermann,2003). Althoughknowledgeof organizationsof its genesinvolvedin biosynthesis of variousaminoacidsincreasedconsiderablyduringlastyears,still littleis known about molecularsignals controllinggene expression. Since regulationof gene expressionon the level of transcriptionplays crucial role, understandingthe structureand functionof transcriptioninitiationsignals(promoters)is of primary importance.Promoters, which can be easily switahed on and off during fermentationsby externalfactorsmay ensurethe controlledoverexpressionofthe chosengenes.Therefore,the C. glutamicumpromotersrespondingto increaseof temperaturehavebeenisolated. Two differentapproachesfor isolationof the promoterswere employed. First...
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