Basic research
Asymmetric cell division during Bacillus subtilis sporulation

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

Probably the most controversial question of Bacillus subtilis cell division concerns the mechanism that ensures the precise location of the dividing septum in the center of the cell during vegetative growth but closer to one pole of the cell during sporulation. To date, the exact location where an asymmetric septum is formed is still unknown, and the accuracy with which this occurs has not been determined. In our work, we showed that the asymmetric septum is formed in 1/6 of the cell length measured from the cell pole and that the accuracy of its localization is comparable to the accuracy of the localization of the vegetative septum in the center of the cell. In this work, we also described the important role of SpoIIE, RefZ and MinCD proteins in determining the place where the asymmetric septum is formed. Nevertheless, we can only hypothesize how the cell finds the site of asymmetric division with high precision.


Projects:

1. VEGA 2/0007/17 - Mechanizmy asymetrického bunkového delenia počas sporulácie Bacillus subtilis (Mechanisms of asymmetric cell division during sporulation of Bacillus subtilis)
2. APVV-14-0181 - Vytváranie 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:

1. VALENČÍKOVÁ, R.- KRASCSENITSOVÁ, E. - LABAJOVÁ, N. - MAKROCZYOVÁ, J. - BARÁK, I. Clostridial DivIVA and MinD interact in the absence of MinJ. In Anaerobe, 2018, vol. 50, p. 22-31. (2017: 2.742 - IF, Q2 - JCR, 1.144 - SJR, Q2 - SJR)
2. MUCHOVÁ, K. - CHROMÍKOVÁ, Z. - VALENČÍKOVÁ, R. - BARÁK, I. Interaction of the Morphogenic Protein RodZ with the Bacillus subtilis Min System. In Frontiers in Microbiology, 2018, vol. 8, p. 1-10. (2017: 4.019 - IF, Q2 - JCR, 1.699 - SJR, Q1 - SJR)
3. BARÁK, I. - MUCHOVÁ, K. The positioning of the asymmetric septum during sporulation in Bacillus subtilis. In PLoS ONE, 2018, vol. 13, no. e0201979. (2017: 2.766 - IF, Q1 - JCR, 1.164 - SJR, Q1 - SJR)
4. WIEDORN, M.O. - OBERTHÜR, D. - BEAN, R. - SCHUBERT, R. - WERNER, N. - BARÁK, I. Megahertz serial crystallography. In Nature Communications, 2018, vol. 9, no. 4025. (2017: 12.353 - IF, Q1 - JCR, 6.582 - SJR, Q1 - SJR)
   

 

Basic research
Genome characterization of the bacterial strain Streptomyces lavendulae subsp. lavendulae in relation to the production of new biologically active substances.

Authors: Kormanec, J., Nováková, R.,Homerová, D., Fecková, Ľ., Řežuchová, B., Csolleiová, D., Bekeová, C., Mingyár, E., Ševčíková, B.

The sequences of some genomes of Streptomycetes sp., one of the most important producer of biologically active substances, revealed that their genomes contain dozens of gene clusters for various secondary metabolites, most of which are silent under laboratory conditions. Their activation can bring new effective biologically active substances. For this purpose, we determined the total genomic sequence of our model strain S. aureofaciens CCM3239 in cooperation with our co-workers. Its genome has a linear chromosome of 8,691,831 bp and a linear plasmid pSA3239 of 241,081 bp. We identified 8,101 genes in the sequence. By analyzing the sequence, we found that it is not the  S. aureofaciens strain as previously thought but the strain of S. lavendulae subsp. lavendulae CCM 3239. In its genome, we identified 26 gene clusters for various secondary metabolites, including  cluster for the polyketide antibiotic auricin, we have characterized in our previous work. The blue pigment indigoidine, and the aminoglycoside antibiotic streptothricin were among the other identified secondary metabolites.


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:

1. MINGYAR, E.- NOVÁKOVÁ, R.- KNIRSCHOVÁ, R.- FECKOVÁ, Ľ. - BEKEOVÁ, C. - KORMANEC, J. Unusual features of the large linear plasmid pSA3239 from Streptomyces aureofaciens CCM 3239. In Gene, 2018, vol. 642, no. 5, p. 313-323. (2017: 2.498 - IF, Q3 - JCR, 1.019 - SJR, Q1 - SJR)
2. BUSCHE, T. - NOVÁKOVÁ, R. - AL'DILAIMI, A. - HOMEROVÁ, D. - FECKOVÁ, Ľ. - REŽUCHOVÁ, B. - MINGYAR, E. - CSÖLLEIOVÁ, D. - BEKEOVÁ, C. - WINKLER, A. - ŠEVČÍKOVÁ, B. - KALINOWSKI, J. - KORMANEC, J. - RUCKERT, C. Complete genome sequence of Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly “Streptomyces aureofaciens CCM 3239”), a producer of the Angucycline-Type antibiotic Auricin. In Genome Announcements, 2018, vol. 6, no. 9, no. e00103-18. (2017: 0.553 - SJR, Q3 - SJR)

Applied research
Protecting our cultural heritage: implementing high-throughput sequencing to analyze the degrading microbiome and the use of bacterial enzymes in implementing biorestoration procedures.

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

Several protocols, based on the Illumina high-throughput sequencing (HGT) platform, have been optimized to detect and identify the microbiome responsible for the deterioration of our cultural heritage. The developed HGT approaches were able to analyze environmental (air samples, the surface of the glass surface of the sarcophagus) as well as historical objects (the surface of books, the mummified body, the internal organs of the mummy). The HGT workflow included: direct extraction of DNA or RNA from samples; PCR amplification of DNA or cDNA using specific bacterial and fungal genes; creation and analysis of Illumina libraries; bioinformatic evaluation of sequencing data. The extracellular enzyme mixture (EEM) from the bacterium Exiguobacterium undae, which was produced by cultivation in animal glia, was characterized. Proteomic analysis of EEM showed that bacillolysin and neutral metalloendopeptidase are mainly responsible for its proteolytic activity. This EEM was able to remove two types of animal glue from different types of material surfaces. These results suggest that EEM could serve as a potential new tool for the restoration of historical objects.


Projects:
APVV-15-0528 – Modifikované polyméry z obnoviteľných zdrojov a ich degradácia (Modified polymers from renewable sources and their degradation)VEGA 2/0061/17 – Inovatívne stratégie dezinfekcie: vplyv esenciálnych olejov na mikroflóru a materiály objektov kultúrneho dedičstva (Innovative disinfection strategies: the impact of essential oils on microflora and heritage materials)
Publications:

1. KRAKOVÁ, L. - ŠOLTYS, K. - PUŠKÁROVÁ, A. - BUČKOVÁ, M. - JESZEOVÁ, L. - KUCHARÍK, M. - BUDIŠ, J. - OROVČÍK, Ľ. - SZEMES, T. - PANGALLO, D. The microbiomes of a XVIII century mummy from the castle of Krasna Horka (Slovakia) and its surrounding environment. In Environmental microbiology, 2018, vol. 20, iss. 9, p. 3294-3308. (2017: 4.974 - IF, Q1 - JCR, 2.209 - SJR, Q1 - SJR)
2. JESZEOVÁ, L. - BAUEROVÁ-HLINKOVÁ, V. - BARÁTH, P. - PUŠKÁROVÁ, A. - BUČKOVÁ, M. - KRAKOVÁ, L. - PANGALLO, D. Biochemical and proteomic characterization of the extracellular enzymatic preparate of Exiguobacterium undae, suitable for efficient animal glue removal. In Applied Microbiology and Biotechnology, 2018, vol. 102, p. 6525-6536. (2017: 3.340 - IF, Q2 - JCR, 1.182 - SJR, Q1 - SJR)
3. KRAKOVÁ, L. - ŠOLTÝS, K. - OTLEWSKA, A. - PIETRZAK, K. - PURKRTOVÁ, S. - SAVICKÁ, D. - PUŠKÁROVÁ, A. - BUČKOVÁ, M. - SZEMES, T. - BUDIŠ, J. - DEMNEROVA, K. - GUTAROWSKA, B. - PANGALLO, D. Comparison of methods for identification of microbial communities in book collections: Culture-dependent (sequencing and MALDI-TOF MS) and culture-independent (Illumina MiSeq). In International Biodeterioration & Biodegradation, 2018, vol. 131, p. 51-59. (2017: 3.562 - IF, Q1 - JCR, 1.086 - SJR, Q1 - SJR)

Applied research
The effect of secondary structure of proteins on their quantification by the Bradford method

Authors: Baliová, M., Jurský, F.

Neurotransmitter transporters contain sequentially diverged N-terminal parts showing considerable flexibility and the character of so-called intrinsically disordered proteins. We have shown that these parts of proteins show an unstable absorption spectrum with Coomassie Brilliant Blue G250 dye, which makes it impossible to determine their concentration by the Bradford method. By mutating several glycines to alanines with the subsequent determination of the secondary structure using circular dichroism, the proportion of the helix structure in the GlyT1aN16 protein increased above 50%, which subsequently led to the stabilization of the absorption spectrum of the protein complex with Coomassie dye. A similar effect was previously observed with the addition of more than 50% of the ordered BSA protein. The results indicate that the Bradford method modified by us could be used for the detection of proteins containing more than 50% fraction of protein disorder.


Projects:
VEGA 2/0064/17 - Úloha kalpainom vytváraných degrónov v regulácii transportérov neurotransmiterov (The role of calpain-produced degrons in the regulation of neurotransmitter transporters)
Publications:
BALIOVÁ, M. - JURSKÝ, F. Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250. In Electrophoresis, 2018, vol. 39, p. 1357-1360. (2017: 2.569 - IF, Q2 - JCR, 0.745 - SJR, Q2 - SJR)
 

International research projects
Lactoferrin as a natural plasminogen inhibitor

Authors: Gutekova, M., Petrovčíková, E., Kutejova, E., Leksa, V.

Lactoferrin is found in human breast milk but it is also present in tears, saliva or urine. Research by the Slovak Academy of Sciences and the Medical University of Vienna indicates that by blocking plasminogen, lactoferrin blocks the invasion of tumor cells and the activation of plasminogen by the Borrelia bacterium, which is the causative agent of infectious Lyme disease. Our results significantly contribute to the understanding of the many antimicrobial, antitumor and immunomodulatory activities of lactoferrin and underline its utility as a potential therapeutic tool.


Projects:

1. VEGA 2/0020/17 – Ľudský mliečny bioaktívny glykoproteín laktoferín ako regulátor homeostázy (Human milk bioactive glycoprotein lactoferrin as a regulator of homeostasis)
2. APVV-16-0452 – Regulácia pericelulárnej proteolýzy: od molekulárnych mechanizmov k novým subsetom imunitných buniek a terapeutickým nástrojom (Regulation of pericellular proteolysis: from molecular mechanisms to new subsets of immune cells and therapeutic tools)
3. FWF - Austrian Science Fund P22908 - The Integral Regulatory Role of (CD222) in T cell activation

Publications:

1. ZWIRZITZ, A. - REITER, M. - ŠKRABANA, R. - OHRADANOVA-REPIC, A. - MAJDIC, O. - GUTEKOVÁ, M. - CEHLÁR, O.- PETROVČÍKOVÁ, E. - KUTEJOVÁ, E. - STANEK, G. - STOCKINGER, H. - LEKSA, V. Lactoferrin is a natural inhibitor of plasminogen activation. In Journal of Biological Chemistry, 2018, vol. 293, p. 8600-8613. (2017: 4.011 - IF, Q2 - JCR, 2.672 - SJR)
2. VIČÍKOVÁ, K. - PETROVČÍKOVÁ, E. - MANKA, P. - DRACH, J. - STOCKINGER, H. - LEKSA, V. Serum and urinary levels of CD222 in cancer: origin and diagnostic value. In Neoplasma, 2018, vol. 65, p. 762-768. (2017: 1.696 - IF, Q4 - JCR, 0.639 - SJR, Q3 - SJR)
3. OHRADANOVA-REPIC, A. - MACHACEK, C. - CHARVET, C. - LAGER, F. - LE ROUX, D. - PLATZER, R. - LEKSA, V. - MITULOVIC, G. - BURKARD, T.R. - ZLABINGER, G. - FISCHER, M.B. - FEUILLET, V. - RENAULT, G. - BLÜML, S. - BENKO, M. - SUCHANEK, M. - HUPPA, J.B. - MATSUYAMA, T. - CAVACO-PAULO, A. - BISMUTH, G. - STOCKINGER, H. Extracellular purine metabolism is the switchboard of immunosuppressive macrophages and a novel target to treat diseases with macrophage imbalances. In Frontiers in Immunology, 2018, vol. 9, no. 852. (2017: 5.511 - IF, Q1 - JCR, 2.803 - SJR, Q1 - SJR)
4. PETROVČÍKOVÁ, E. - VIČÍKOVÁ, K. - LEKSA, V. Extracellular vesicles – biogenesis, composition, function, uptake and therapeutic applications. In Biologia, 2018, vol. 73, p. 437-448. (2017: 0.696 - IF, Q4 - JCR, 0.299 - SJR, Q3 - SJR)
5. LEKSA, V. - SCHILLER, H.B. - STOCKINGER, H. Biotin-chasing assay to evaluate uPAR stability and cleavage on the surface of cells. In Proteases and Cancer : methods and protocols. - New York : Humana Press, 2018, p. 39-47
   

 

International research projects
Heterological production of the antitumor agent mitramycin in a genetically modified host strain Streptomyces lividans

Authors: Nováková, R., Homerová, D., Knirschová, R., Fecková, Ľ., Řežuchová, B., Ševčíková, B., Kormanec, J.

One of the main goals of the 7RP StrepSynth project was to prepare S. lividans RedStrep 1 strains with improved biotechnological properties for the production of biologically active substances. Based on several analyses, we deleted several relevant genes and gene clusters for secondary metabolites in the S. lividans genome using the efficient PCR bpsA deletion system developed by us. We prepared a collection of S. lividans RedStrep 1.0 to 1.8 strains with sequentially deleted gene clusters for interfering secondary metabolites act, red, cda, cpk, mel, hrdD gene for sigma factor and scbAR genes for gamma-butyrolactone system. In these strains, we demonstrated a dramatic increase in the heterologous production of the antitumor polyketide mithramycin A. This increase (up to 3 g/L medium) was six times higher than the overproducing mutant strain for mithramycin production, S. argillaceus. These results demonstrated the biotechnological impact of the RedStrep 1 strains prepared by us for the production of biologically active substances.


Projects:
7RP EU project StreSynth (č. 613877) a APVV-DO7RP-0038-12 – Rewiring the Streptomyces cell factory for cost-effective production of biomolecules
Publications:

1. NOVÁKOVÁ, R.- NÚÑEZ, L.E. - HOMEROVÁ, D. - KNIRSCHOVÁ, R. - FECKOVÁ, Ľ. - REŽUCHOVÁ, B. - ŠEVČÍKOVÁ, B. - MENÉNDEZ, N. - MORÍS, F. - CORTÉS, J. - KORMANEC, J. Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains. In Applied Microbiology and Biotechnology, 2018, vol. 102, p. 857–869. (2017: 3.340 - IF, Q2 - JCR, 1.182 - SJR, Q1 - SJR)
2. REŽUCHOVÁ, B. - HOMEROVÁ, D. - ŠEVČÍKOVÁ, B. - NUÑEZ, L.E. - NOVÁKOVÁ, R. - FECKOVÁ, Ľ. - ŠKULTÉTY, Ľ. - CORTÉS, J. - KORMANEC, J. An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA. In Applied Microbiology and Biotechnology, 2018, vol. 102, p. 10231-10244. (2017: 3.340 - IF, Q2 - JCR, 1.182 - SJR, Q1 - SJR)
3. HAMED, M.B. - VRANCKEN, K. - BILYK, B. - KOEPFF, J. - NOVÁKOVÁ, R. - VAN MELLAERT, L. - OLDIGES, M. - LUZHETSKYY, A. - KORMANEC, J. - ANNÉ, J. - KARAMANOU, S. - ECONOMOU, A. Monitoring protein secretion in streptomyces using fluorescent proteins. In Frontiers in Microbiology, 2018, vol. 9, no. 3019. (2017: 4.019 - IF, Q2 - JCR, 1.699 - SJR, Q1 - SJR)
   

 

International research projects
Characterization of the unique evolution of the starch-binding domain of laforin

Authors: Janeček, Š., Kuchtová, A.

Laforin is a glucan phosphatase that catalyzes the dephosphorylation of glycogen. Mutations in his gene lead to the so-called Lafor's type of epilepsy (fatal progressive myoclonic epilepsy), which is characterized by the presence of water-insoluble hyperphosphorylated polyglucosans, called Lafor's bodies. Human laforin consists of an N-terminal carbohydrate-binding module (so-called starch-binding domain) from the CBM20 family and a C-terminal catalytic domain with dual phosphatase specificity. Laforin is conserved in all vertebrates and in some basal metazoa and a small group of protozoa. In the present study, the evolutionary relationships between the starch-binding domains of laforin CBM20 were described with an emphasis on its newly identified hypothetical orthologs in the genus Trichinella (parasitic nematode), two sequence inserts were found in the CBM20 domain of laforin from parasitic coccidia, and it was found that hypothetical orthologs of laforin from some protozoa and algae contain more than one copy of the CBM20 domain.


Projects:
VEGA 2/0146/17 - Evolúcia amylolytických enzýmov (Evolution of amylolytic enzymes)
Publications:

1. KUCHTOVÁ, A. - GENTRY, M.S. - JANEČEK, Š. The unique evolution of the carbohydrate?binding module CBM20 in laforin. In FEBS Letters, 2018, vol. 592, no. 4, p. 586-598. (2017: 2.999 - IF, Q2 - JCR, 1.991 - SJR, Q1 - SJR)
2. ABBOTT, W. - ALBER, O. - BAYER, E. - BERRIN, J.G. - BORASTON, A. - JANEČEK, Š. Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes. In Glycobiology, 2018, vol. 28, p. 3-8. (2017: 3.664 - IF, Q2 - JCR, 1.493 - SJR, Q1 - SJR)