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Production of galactosidases by yeasts of genus Cryptococcus growing on lactose medium
Pavlatovská, Barbora ; Molnárová,, Jana (referee) ; Stratilová, Eva (advisor)
This work focuses on the induction of galactosidases by yeasts of genus Cryptococcus growing on lactose medium and relations between this production and growth of yeasts. At the end biotyping using mass spectrometry is described. All tested and relative yeasts, hydrolytic enzymes -, -galactosidase and used analytical methods as UV-VIS spectrophotometry and mass spectometry, especially MALDI-TOF are described in the theoretical part. The experimental part consists of description of instrumentations, preparation of necessary solutions, cultivation of microorganisms, measurement of enzymatic activity spectrophotometrically and biotyping of yeasts by mass spectrometry. Each of the 18 tested yeast strains, including species Cr. canescens (CCY 17-3-13), Cr. flavescens (CCY 17-3-6, CCY 17-3-15, CCY 17-3-29, CCY 17-3-31, CCY 17-3-33, CCY 17-3-38), Cr. flavus (CCY 17-3-5), Cr. laurentii (CCY 17-3-2, CCY 17-3-9, CCY 17-3-17, CCY 17-3-24), Cr. magnus (CCY 17-4-39, CCY 17-4-40), Cr. saitoi (CCY 17-3-18), Cr. victoriae (CCY 17-3-26) and Bulleromyces albus (CCY17-3-35, CCY 17-3-37) from the Culture Collection of Yeasts, Institute of Chemistry of SAS (CCY), was cultured for 96 hours in a liquid medium with lactose. During cultivation, the quantity of cells was determined as well as enzyme activities of - and -galactosidase in the medium and on the cell surface. Lactose medium was shown not to be suitable culture medium for all Cryptococcus strains, because some of them grew on it very slowly (CCY 17-3-29, CCY 17-3-5, CCY 17-3-26, CCY 17-3-35), or showed a long adaptation phase (CCY 17-3-2, CCY 17-3-6). A comparison of the growth and surface -galactosidase production curves showed that the link between lactose medium and production of this enzyme does not exist in generally. The results did not confirm neither the expected impact of this enzyme on the growth of strains nor the anticipated induction of -galactosidase by lactose. For instance, the fastest growth on lactose showed the strain Cr. canescens CCY 17-3-13, which exhibited a very low activity of this enzyme on its surface. Relatively increased production of this enzyme was observed on the surfaces of the type strain Cr. laurentii CCY 17-3-2, strain Cr. flavescens CCY 17-3-31 and Cr. flavus CCY 17-3-5. The production of -galactosidase by capsular yeasts was strain-dependent with the exception of the members of Cr. flavescens species. Because of this, the expected general influence of this enzyme on the re-building of the protective cryptococcal capsule can be excluded. Only Cr. flavescens strains can be generally considered as producers of lactose-inducible surface -galactosidase. In other cases, the production of this enzyme matters on single strain and not on species, as can be seen in the case of type strain Cr. laurentii and the other Cr. laurentii strains. Similar induction by lactose medium was also observed with non-cryptococcus yeasts and fungi and therefore the link with the capsules can be also excluded. In connection with the selected culture medium, the second aim of this work was to test the suitability of lactose medium for genus Cryptococcus biotyping by mass spectrometry. Normalized spectra showed very low score between strains of the same species and as a consequence, close related strains were included in the different evolutionary branches using Biotyper programme. These findings led to the conclusion that lactose medium is an unsuitable medium.
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Biotyping of ascomycetous yeasts
Jurnečková, Alena ; Dudášová,, Hana (referee) ; Stratilová, Eva (advisor)
In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.
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Effect of medium composition on the mass spectra of the yeast species of Cryptococcus laurentii and Cryptococcus flavescens
Ledvina, Vojtěch ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.
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Biotyping of ascomycetous yeasts
Jurnečková, Alena ; Dudášová,, Hana (referee) ; Stratilová, Eva (advisor)
In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.
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Effect of medium composition on the mass spectra of the yeast species of Cryptococcus laurentii and Cryptococcus flavescens
Ledvina, Vojtěch ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.
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Production of galactosidases by yeasts of genus Cryptococcus growing on lactose medium
Pavlatovská, Barbora ; Molnárová,, Jana (referee) ; Stratilová, Eva (advisor)
This work focuses on the induction of galactosidases by yeasts of genus Cryptococcus growing on lactose medium and relations between this production and growth of yeasts. At the end biotyping using mass spectrometry is described. All tested and relative yeasts, hydrolytic enzymes -, -galactosidase and used analytical methods as UV-VIS spectrophotometry and mass spectometry, especially MALDI-TOF are described in the theoretical part. The experimental part consists of description of instrumentations, preparation of necessary solutions, cultivation of microorganisms, measurement of enzymatic activity spectrophotometrically and biotyping of yeasts by mass spectrometry. Each of the 18 tested yeast strains, including species Cr. canescens (CCY 17-3-13), Cr. flavescens (CCY 17-3-6, CCY 17-3-15, CCY 17-3-29, CCY 17-3-31, CCY 17-3-33, CCY 17-3-38), Cr. flavus (CCY 17-3-5), Cr. laurentii (CCY 17-3-2, CCY 17-3-9, CCY 17-3-17, CCY 17-3-24), Cr. magnus (CCY 17-4-39, CCY 17-4-40), Cr. saitoi (CCY 17-3-18), Cr. victoriae (CCY 17-3-26) and Bulleromyces albus (CCY17-3-35, CCY 17-3-37) from the Culture Collection of Yeasts, Institute of Chemistry of SAS (CCY), was cultured for 96 hours in a liquid medium with lactose. During cultivation, the quantity of cells was determined as well as enzyme activities of - and -galactosidase in the medium and on the cell surface. Lactose medium was shown not to be suitable culture medium for all Cryptococcus strains, because some of them grew on it very slowly (CCY 17-3-29, CCY 17-3-5, CCY 17-3-26, CCY 17-3-35), or showed a long adaptation phase (CCY 17-3-2, CCY 17-3-6). A comparison of the growth and surface -galactosidase production curves showed that the link between lactose medium and production of this enzyme does not exist in generally. The results did not confirm neither the expected impact of this enzyme on the growth of strains nor the anticipated induction of -galactosidase by lactose. For instance, the fastest growth on lactose showed the strain Cr. canescens CCY 17-3-13, which exhibited a very low activity of this enzyme on its surface. Relatively increased production of this enzyme was observed on the surfaces of the type strain Cr. laurentii CCY 17-3-2, strain Cr. flavescens CCY 17-3-31 and Cr. flavus CCY 17-3-5. The production of -galactosidase by capsular yeasts was strain-dependent with the exception of the members of Cr. flavescens species. Because of this, the expected general influence of this enzyme on the re-building of the protective cryptococcal capsule can be excluded. Only Cr. flavescens strains can be generally considered as producers of lactose-inducible surface -galactosidase. In other cases, the production of this enzyme matters on single strain and not on species, as can be seen in the case of type strain Cr. laurentii and the other Cr. laurentii strains. Similar induction by lactose medium was also observed with non-cryptococcus yeasts and fungi and therefore the link with the capsules can be also excluded. In connection with the selected culture medium, the second aim of this work was to test the suitability of lactose medium for genus Cryptococcus biotyping by mass spectrometry. Normalized spectra showed very low score between strains of the same species and as a consequence, close related strains were included in the different evolutionary branches using Biotyper programme. These findings led to the conclusion that lactose medium is an unsuitable medium.
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