Basic research
Structure and function of Mgm101 from C. parapsilosis

Authors: Pevala, V., Kutejová, E., Kunová, N., Bellová, J., Bauer, J.A.,

The yeast C. parapsilosis has a linear mitochondrial genome. Several proteins are involved in its stabilization and replication. In our work, we studied the protein CpMgm101 and showed that this protein is positioned at two localization sites, it is found in the mitochondria and in the nucleus. CpMgm101 is associated with the mitochondrial nucleoid and binds to a wide range of DNA structures. Based on SAXS measurements, the protein forms a C-shaped trimer. Using electron microscopy, we showed that Mgm101 forms circular structures at the protruding telomeric ends of mitochondrial DNA. The DNA binding activities of CpMgm101 together with the recombination properties suggest that Mgm101 is involved in various processes associated with the replication of the mitochondrial genome and the stabilization of its telomeres.



Projects:

1. VEGA 2/0113/14 ATP-závislé proteázy a homeostáza mitochondrií (ATP-dependent proteases and mitochondrial homeostasis)
2. VEGA 2/0122/11 Proteínové komplexy mitochondriálneho nukleoidu (Mitochondrial nucleoid protein complexes)
3. APVV-0123-10 Molekulárna architektúra, dynamika a evolúcia chromozómov v mitochondriách kvasiniek (Molecular architecture, dynamics and evolution of chromosomes in yeast mitochondria)
4. MVTS-1520 INSTRUCT

Publications:
PEVALA, V. - TRUBAN, D. - BAUER, J. - KOŠTAN, J. - KUNOVÁ, N. - BELLOVÁ, J. - BRANDSTETTER, M. - MARINI, V. - KREJČÍ, L. - TOMÁŠKA, L. - NOSEK, J. - KUTEJOVÁ, E. The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome. In Nucleic acids research, 2016, vol. 44, no. 5, p. 2227-2239. (2015: 9.202 - IF, Q1 - JCR, 7.358 - SJR, Q1 - SJR)
 
 

Basic research
Asymmetric cell division in Bacillus sp. and Clostrida sp.

Authors: Barák, I., Muchová, K., Chromíková, Z., Krascsenitsová, E., Labajová, N., Makroczyová, J.

Cell division of Escherichia coli and Bacillus subtilis belongs to the most intensively studied processes of bacterial cell division. In addition to the vegetative, centrally localized division septum, B. subtilis is also able to form an asymmetrically localized septum. However, it is not yet known how this division site is recognized by the division apparatus. The polar cell division septum formation is the first clear morphological feature of B. subtilis sporulation. SpoIIE is so far only sporulation-specific protein identified whose deletion or mutations lead to changes in the ultrastructure of the asymmetric septum. In our work, we showed that this protein is in direct contact with the RodZ protein and in this way with the entire cytoskeletal apparatus of the cell.

An important part of the regulation of vegetative and sporulation division is the Min system. In our other work, we have shown for the first time that the Min system from Clostridium difficile oscillates when expressed in B. subtilis cells. Clostridial Min system oscillation probably allows the creation of a concentration gradient of the cell division inhibitor, similar to that in E. coli.

Projects:

1. VEGA-2/0009/13 Štruktúra a funkcia proteínov zúčastňujúcich sa regulácie základných bunkových procesov Bacillus subtilis (Structure and function of proteins involved in the regulation of basic cellular processes of Bacillus subtilis)
2. APVV-14-0181Vytváranie proteínových komplexov počas asymetrického bunkového delenia v sporulujúcich bunkách Bacillus subtilis (Formation of protein complexes during asymmetric cell division in sporulating Bacillus subtilis cells)

Publications:
MAKROCZYOVÁ, J. - JAMROŠKOVIČ, J. - KRASCSENITSOVÁ, E. - LABAJOVÁ, N. - BARÁK, I. Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis. In MicrobiologyOpen, 2016, vol. 5, p. 387-401. (2015: 2.148 - IF, Q3 - JCR, 1.212 - SJR, Q2 - SJR)
MUCHOVÁ, K. - CHROMÍKOVÁ, Z. - BRADSHAW, N. - WILKINSON, A.J. - BARÁK, I. Morphogenic protein rodZ interacts with sporulation specific spoIIE in bacillus subtilis. In PLoS ONE, 2016, vol. 11, art. no. e0159076. (2015: 3.057 - IF, Q1 - JCR, 1.427 - SJR, Q1 - SJR)

Basic research
Complex mechanism of regulation of auricin antibiotic biosynthetic genes in Streptomyces aureofaciens CCM3239

Authors: Kormanec, J., Mingyár, E., Nováková, R., Bekeová, C., Fecková, Ľ.

The angucycline antibiotic auricin is modified by aminodeoxyhexose D-forosamine. We identified and characterized genes encoding biosynthetic proteins responsible for D-forosamine biosynthesis. We also characterized their regulation. Up to two glycosyltransferases are uniquely responsible for attaching D-forosamine to auricin. By characterizing the regulation of these modifier genes, we found that while the biosynthetic genes for polyketide aglycone construction are regulated through the pathway-specific regulators Aur1P and Aur1R, the modifier genes responsible for the final steps of D-forosamine biosynthesis and its attachment are differentially regulated through two pathway-specific regulators SARP, Aur1PR3 and Aur1PR4 families. These genes are also under the differential control of Aur1P and Aur1R regulators. These results revealed a unique mechanism of antibiotic biosynthetic gene regulation at two levels. The genes for the polyketide aglycone core are under the control of Aur1P and Aur1R, and the modifying genes under the control of Aur1PR3 and Aur1PR4.


Projects:
APVV-15-0410 - Syntetická biológia pre produkciu nových biologicky aktívnych látok u streptomycét (Synthetic biology for the production of new biologically active substances in Streptomycetes)
Publications:
BEKEOVÁ, C. - REHÁKOVÁ, A. - FECKOVÁ, Ľ. - VLČKOVÁ, S. - NOVÁKOVÁ, R. - MINGYAR, E. - KORMANEC, J. Characterisation of the genes involved in the biosynthesis and attachment of the aminodeoxysuga. In Applied Microbiology and Biotechnology, 2016, vol. 100, p 3177-3195. (2015: 3.376 - IF, Q2 - JCR, 1.256 - SJR, Q1 - SJR)

Basic research
High diversity of peroxidase and catalase genes discovered in the genome of the filamentous soil fungus Chaetomium cochliodes with significant biotechnological potential

Authors: Zámocký, M., Kamlárová, A., Chovanová, K.

Using the NGS method, we completely sequenced the genome of the filamentous fungus Chaetomium cochliodes, which is known for the production of the antibiotic chetomin and many other secondary metabolites. Our sequencing analyzes clearly show that this mesophilic fungus contains genes for thermostable proteins from its direct evolutionary ancestors, which were thermostable Ascomycete fungi. The genome of C. cohliodes (34.7 Mbp in 6036 contigs) contains up to 20 different genes for evolutionarily divergent families of peroxidases and catalases, which remove reactive oxygen compounds from the cell. In two cases, we managed to prove that these antioxidant genes were transferred to the fungal genome from bacteria by the mechanism of horizontal gene transfer (HGT). Using the real-time PCR method, we discovered a different way of expression of individual hydroperoxidase genes and cloned the most important of them for the purpose of preparing recombinant antioxidants and biocatalyzers for various polymerization reactions.


Projects:

1. VEGA 2/0021/14 - Fylogenomický a fyziologický výskum reakcií na oxidačný stres v termofilných a mezofilných mikroorganizmoch (Phylogenomic and physiological research of reactions to oxidative stress in thermophilic and mesophilic microorganisms)
2. APVV 14/0375 – Syntetická biológia a produkcia peroxidáz de novo (Synthetic biology and de novo production of peroxidases)

Publications:

1. ZÁMOCKÝ, M. - TAFER, H. - CHOVANOVÁ, K. - LOPANDIC, K. - KAMLÁROVÁ, A. - OBINGER, C. Genome sequence of the filamentous soil fungus Chaetomium cochliodes reveals abundance of genes for heme enzymes from all peroxidase and catalase superfamilies. In BMC Genomics, 2016, vol. 17, p. 763. (2015: 3.867 - IF, Q1 - JCR, 2.348 - SJR, Q1 - SJR)
2. CHOVANOVÁ, K. - ZÁMOCKÝ, M. Detection of the antibacterial effect of Chaetomium cochliodes Palliser CCM F-232 based on agar plugs and unprocessed fungal substances from cultivation media. In Biologia, 2016, vol. 71, no. 11, p. 1204—1211. (2015: 0.719 - IF, Q4 - JCR, 0.329 - SJR, Q3 - SJR)

Applied research
Methodological survey to determine the microbial community degrading the surface of albumin photographs and the application of biocides to biofilms of the catacombs in Rome

Authors: Pangallo, D., Kraková, L., Puškárová, A., Bučková, M.

In order to find suitable procedures for the preservation of objects of our cultural heritage, we have developed different strategies including analyzes of microbial communities of albumen photographs and several approaches capable of analyzing material properties. This study is one of the few that analyzes albumen photographs from the turn of the 19th and the beginning of the 20th century. The chemical composition of albumin photographic samples was evaluated using FTIR spectroscopy (Fourier transform infrared spectroscopy) and X-ray fluorescence (XRF). These two non-invasive techniques revealed the complex nature of albumen photographs, which are composed of a mixture of proteins, cellulose and salt. Microbial analysis was performed using culture-dependent (culture in different media) and culture-independent approaches and SEM analysis. The isolated microorganisms were tested in order to monitor their lipolytic, proteolytic, cellulolytic, as well as catalase and peroxidase abilities.

The effects of biocidal biofilm treatment were tested in the Domitilla catacombs in Rome (Italy). The aim of this research was to compare the changes in the microbial community in one site of the Domitilla catacombs (CD15), before and after a biocide treatment (a mixture of quaternary ammonium compounds and octylisothiazolone, OIT), over a period of one month. A multi-step approach was used based on microscopy, culture method and molecular technique (f-ITS and 16S rDNA), phenotypic and genetic analysis of culturable microbial populations. Our results highlighted that the biocide treatment had insufficient effect against cyanobacteria, while the bacterial population increased quantitatively but changed radically in terms of diversity. In fact, some bacteria multiplied at the expense of organic matter released from dead microorganisms. Furthermore, our data describe how microbial interactions can have different responses depending on favorable conditions for one type of microorganism in relation to others. This study illustrates the real risk of applying a biocidal treatment to microbial communities and the need to subject the treatment to monitoring and re-evaluation.

Projects:
VEGA 2/0103/14 - Ochrana našich spomienok: štúdium biodeteriorácie fotografických a kinematografických materiálov (Protection of our memories: study of biodeterioration of photographic and cinematographic materials)
Publications:

1. PUŠKÁROVÁ, A. - BUČKOVÁ, M. - HABALOVÁ, B. - KRAKOVÁ, L. - MAKOVÁ, A. - PANGALLO, D. Microbial communities affecting albumen photography heritage: a methodological survey. In Scientific Reports, 2016, vol. 6, no. 20810 p. 1-14. (2015: 5.228 - IF, Q1 - JCR, 2.034 - SJR, Q1 - SJR)
2. URZI, C. - DE LEO, F. - KRAKOVÁ, L. - PANGALLO, D. - BRUNO, L. Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome. In Science of the Total Environment, 2016, vol. 572, p. 252–262. (2015: 3.976 - IF, Q1 - JCR, 1.653 - SJR, Q1 - SJR)

Applied research
Use of Coomassie Brilliant Blue G250 as a sensor for protein flexibility and disorder detection

Authors: Juhasová, A., Baliová, M., Jurský, F.

Coomassie Brilliant Blue interacts with proteins, and despite the dependence of this interaction on the amino acid composition, its interaction with individual proteins is relatively stable and is used as a standard for the quantitative determination of proteins called the Bradford Assay. In our work, we managed to find proteins, N-terminal sections of glycine transporters GlyT1 and GlyT2, showing an atypical dynamic interaction with Coomassie Brilliant Blue G250. This method of interaction made it impossible to determine them using the Bradford method, since after their initial interaction with the dye, the absorption maximum at 595 nm used by Bradford gradually decreased within 15 minutes and a new absorption maximum at 750 nm was formed. Circular dichroism of the N-terminal sections showed that it is the so-called disordered proteins, which indicates that the observed anomalies are probably caused by an unstructured character with high flexibility. This process was suppressed by fusion of the N-terminal regions with GST as well as by the presence of GST or BSA in the assay solution, suggesting that the exposure of similar domains in proteins may be masked by the presence of other ordered protein domains. However, after subcloning and recombinant overexpression of protein domains, spectral analysis of their complexes with Coomassie Brilliant Blue can provide a relatively affordable alternative method for screening protein domains with a potentially disordered and flexible character.


Projects:
VEGA  2/0086/13 - PDZ interakcie GABA transportéra GAT1 (PDZ interactions of GABA transporter GAT1)
Publications:
JUHÁSOVÁ, A. - BALIOVÁ, M. - JURSKÝ, F. A dynamic interaction of coomassie dye with the glycine transporters N-termini. In Protein Journal, 2016, vol. 35, p. 371–378. (2015: 1.029 - IF, Q4 - JCR, 0.411 - SJR, Q3 - SJR)

International research projects
The role of the N-terminal domain of human Lon protease in stabilizing the structure and maintaining activity

Authors: Kutejová, E., Pevala, V., Kunová, N., Ondrovičová, G., Bauer, J., Bellová, J., Ambro, Ľ.

Lon protease is a very important part of mitochondria. It participates in maintaining homeostasis and stabilizing mitochondrial DNA. An altered level of this protease leads to serious diseases, such as myopathy and cancer. In collaboration with the 1st Medical Faculty of the Charles University in Prague, we have shown by cryo-electron microscopy that the human Lon protease has a unique structure. Its proteolytic and ATPase domains (AP domain) form a hexameric barrel, while the N-terminal domain forms a trimer of a dimer. In the presence of the non-hydrolyzable analog of ATP (AMP-PNP), the AP domain forms a closed structure together with the N-terminal domain. ADP binding results in a change in the structure of both the AP domain and the N-terminal domain. We found that loss of 156 amino acids from the N-terminus results in structural instability and loss of enzyme activity. The N-terminal domain is therefore essential both for the Lon protease structure itself and for its activity.



Projects:

1. VEGA 2/0113/14 ATP-závislé proteázy a homeostáza mitochondrií (ATP-dependent proteases and mitochondrial homeostasis)
2. APVV-0123-10 Molekulárna architektúra, dynamika a evolúcia chromozómov v mitochondriách kvasiniek (Molecular architecture, dynamics and evolution of chromosomes in yeast mitochondria)
3. CZ.1.05/1.1.00/02.0109 Project BIOCEV

Publications:
KEREICHE, S. - KOVÁČIK, L. - BEDNÁR, J. - PEVALA, V. - KUNOVÁ, N. - ONDROVIČOVÁ, G. - BAUER, J. - AMBRO, Ľ. - BELLOVÁ, J. - KUTEJOVÁ, E. - RAŠKA, I. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease. In Scientific Reports, 2016, vol. 6, no. 33631, p. 1-10. (2015: 5.228 - IF, Q1 - JCR, 2.034 - SJR, Q1 - SJR)

International research projects
Variability of dipicolinic acid and calcium in Clostridial strains

Authors: Barák, I., Jamroškovič, J., Chromíková, Z.

Bacterial spores are extremely resistant to the effects of various environmental conditions such as high temperature, drought and chemical oxidizing agents. Spores are enveloped by several layers of proteins and peptidoglycan, which envelop the center of the cell with DNA high in calcium and dipicolinic acid (DPA). Within the framework of this project, we succeeded in using new spectroscopic and electron microscopic methods to show the heterogeneity of DPA and calcium content in different strains of Clostridium, but also to show heterogeneity within the same population of cells.


Projects:
Scopes – Swiss National Foundation (IZ73Z0_152527 / 1) - Úloha homeostázy kovov, ich redukcia a sporulácia v rezistencii Gram-pozitívnych baktériách (The role of metal homeostasis, reduction and sporulation in resistance of Gram-positive bacteria)
Publications:
JAMROŠKOVIČ, J. - CHROMÍKOVÁ, Z. - LIST, C. - BÁRTOVÁ, B. - BARÁK, I. - BERNIER-LATMANI, R. Variability in DPA and Calcium Content in the Spores of Clostridium Species. In Frontiers in Microbiology, 2016, vol. 7, p. 1791. (2015: 4.165 - IF, Q1 - JCR, 1.869 - SJR, Q1 - SJR)