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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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Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a homologue of Escherichia coli protein YjjG
Vacková, Zuzana ; Lichá, Irena (referee) ; Branny, Pavel (advisor)
ANGLICKÝ ABSTRAKT Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a likely homolog of Escherichia coli protein YjjG. Bacterial cells are constantly exposed to innumerable toxic substances, either in their external environment or by by-products of their own metabolism. For these reasons, the bacterial cells evolved several mechanisms to cope with this challenge. These mechanisms are represented by: blocking the uptake, export by specific transporters as well as specific inactivation of these substance by enzymes. A particular group of these toxic substances are noncanonica nucleotides, which can directly inhibit bacterial cell DNA replication or can result in increased mutation rate. Enzymes recognizing these modified derivatives are known as "house-cleaning" nucleotide phsphateses, which can inactivate the potentially mutagenic nucleotides and prevent their incorporation into DNA and RNA. Some of the "house- cleaning" enzymes belong to a group of haloacid dehalogenase enzymes (haloacid dehalogenase-like hydrolase superfamily), which are found in many bacterial species. This thesis is focused on the function of hypothetical protein Spr1057 of Streptococcus pneumoniae with an unknown function. Sequence comparison revealed that Spr1057 has a significant...
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Effect of environmental stresses on mutability of Bacillus subtilis - role of mismatch-repair system
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Konopásek, Ivo (referee)
The everchanging nature of bacterial environment requires adaptation to emerging novel conditions. One proposed way of adaptation involves increased generation of genetic variability in response to harmful conditions - a phenomenon called adaptive mutagenesis. However, the details of mechanisms of adaptive mutagenesis, and even its very existence, are far from clear. Our goal was to subject the Gram-positive model bacterium Bacillus subtilis to variety of environmental stresses, examine the rate of mutagenesis occuring and compare it to unstressed conditions. Next we wondered if there was a role for mismatch-repair system (MMR), the major pathway for mutation avoidance, in these processes. To accomplish this, we constructed systems to monitor the expression of MMR components both on transcription and translation level. We also developed a mathematical model for precise mutation rate determination in order to quantify the intensity of mutagenic processes. The monitoring of MMR proteins translation failed due to high background endogenous fluorescence present in B. subtilis cells. However, we found out, using transcription reporter system, that the expression of MMR is not influenced by imposition of harsh hyperosmotic shock upon cells. The expression of MMR was also barely influenced by nutrient limitation...
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Study of persistence phenomenon at Staphylococcus aureus
Kubištová, Lucie ; Lichá, Irena (advisor) ; Melter, Oto (referee)
Staphylococcus aureus is human pathogen and is causative factor of many diseases with different character, from light infections of the skin to life-threatening sepsis. S. aureus often colonizes cystic fibrosis patient's lungs and causes long-standing pneumonia, which can cause death in these patients. Infection caused by S. aureus are, as most of the infections, treated with antibiotics. Failure in treatment is caused because of presence of resistant strains. It was shown, that treatment failure can be caused by different type of the antibiotic tolerance - by the persistence. Persisters are phenotypic variants of isogenic bacterial population, that is unaffected by antibiotic treatment even though they don't have genetics determinants of the resistance. The phenomenon of the persistence in bacteria is still relatively poorly understood. The aim of my thesis was to contribute to the characterization of the persistence in S. aureus. We have optimized a method for rapid identification of persisters' amount in the bacterial population after treatment with antibiotics using the method of measuring the killing curves. We have found out, that exposure to osmotic stress causes increase in number of persistence in the bacterial population by 1 - 2,5 order. Using quantitative PCR method we have analyzed the...
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