Basic research
A unique complex mechanism of regulation of the antibiotic auricin by the global gamma-butyrolactone receptor system in Streptomyces aureofaciens CCM3239

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

We have identified the sagA and sagR genes encoding the gamma-butyrolactone (GBL) system in S. aureofaciens CCM3239. By characterizing the mutants of these genes, we proved the role of this GBL system in the global regulation of secondary metabolism, in the regulation of auricine and indigoidine production. We characterized the molecular mechanism of this regulation. The receptor protein SagR binds to the promoters of the genes for the auricin-specific regulators Aur1P and Aur1R in the exponential phase of growth, while this inhibitory effect is unblocked by accumulated GBL at the entry of the stationary phase of growth. We discovered a unique feedback mechanism of regulation of the global regulatory SagA/SagR GBL genes by the pathway-specific auricin regulator Aur1R, which blocked the transcription of these genes after entering the stationary phase of growth. These results revealed a unique complex mechanism of auricin regulation at two levels. The task of this complex system is to precisely ensure the expression of auricin biosynthetic genes only at the stage of the stationary phase of growth and at the same time to block the expression of global GBL regulators at this stage, when their function is no longer required.


Projects:

APVV-0203-11 - Molekulárne mechanizmy biosyntézy, regulácie a horizontálneho prenosu génov zodpovedných za produkciu biologicky aktívnych látok u streptomycét

Publications:

1.Mingyar, E., Feckova, L., Novakova, R., Bekeova, C., Kormanec, J.: A ?-butyrolactone autoregulator-receptor system involved in the regulation of auricin production in Streptomyces aureofaciens CCM 3239. Applied Microbiology and Biotechnology 99 (2015) 309-325. [IF: 3,337]

2. Knirschova, R., Novakova, R., Mingyar, E., Bekeova, C., Homerova, D., Kormanec, J.: Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces. Journal of Microbiological Methods 113 (2015) 1-3. [IF: 2,026]

3. Medema MH at al. : Minimum information about a biosynthetic gene cluster. Nature Chemical Biology 11 (2015) 625-631. [IF: 12,996]

Basic research
Study and characterization of replication proteins gp41 and gp43 of bacteriophage BFK20

Authors: G. Bukovská, N. Halgašová, B. Šoltészová, V. Pevala, E. Kutejová

Within the framework of the study of the composition of phage BFK20 replisome, using the two-hybrid bacterial system BACTH, we identified several new protein-protein interactions. We demonstrated a strong interaction of the gp41 protein from phage BFK20 (a helicase from the SFII family) with the host protein DnaG (primase), moderately strong with DnaZX and DnaN, and weak with SSB and Dnaδ. We also confirmed the interaction of gp41 and DnaG by in vitro methods using recombinant proteins gp41HC and DnaGN. We have characterized the gp43 replication protein of phage BFK20, a multifunctional protein with a primase-polymerase and helicase domain. The isolated recombinant protein gp43-1 contained both domains and gp43C contained only the helicase domain. We tested their oligomeric state using SEC-MALS analysis and enzyme activity. The gp43-1 protein forms functional hexamers SAV 7 Activity Report and exhibits strong ssDNA-dependent NTPase activity and helicase activity. The gp43C protein showed weak NTPase and no helicase activity. We assume that the gp43 protein belongs to the replicative helicases of the SF4 family and requires the presence of both domains for activity.
Projects:

VEGA 2/0122/14 - Replizóm korynefága BFK20 - štúdium fágových replikačných proteínov.

Publications:

1. Solteszova, B., Halgasova, N., Bukovska, G. Interaction between phage BFK20 helicase gp41 and its host Brevibacterium flavum primase DnaG. (2015) Virus Res. 196: 150-156. [IF2014 2.324]

2. Halgasova, N., Solteszova, B., Pevala, V., Kostan, J., Kutejova, E., Bukovska, G. A RepA-like protein from bacteriophage BFK20 is a multifunctional protein with primase, polymerase, NTPase and helicase activities. (2015) Virus Res. 210: 178-187. [IF2014 2.324]

Basic research
Characterization of the potential binding pocket for benzophenanthridine alkaloids on the glycine transporter GlyT1

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

Benzophenanthridine alkaloids chelerythrine and sanginarine are naturally occurring plant substances, exhibiting a broad-spectrum effect on bacteria, as well as mammalian cells. They are mainly investigated for their antitumor effects. Currently, however, their new receptors are still being discovered, enabling a better understanding of the complex mechanism of their effect. Recently, we managed to identify the glycine transporter GlyT1 as a new receptor for these alkaloids. In the latest work, we identified two structurally adjacent amino acid residues in the GlyT1 molecule, located in the cytoplasmic part of the transporter. Molecular docking of alkaloids into this space allowed us to design their potential alkaloid binding pocket. Mutational analysis as well as reduction of sanginarine pointed to the important role of aromatic staking interactions. The findings can be used to design new inhibitors based on benzophenanthridines. GlyT1 inhibitors are important mainly as anti-psychotics and agents against neuropathic pain.


Projects:

VEGA 2/0084/13 - Inhibícia transportérov neurotransmiterov benzofenantridínovými alkaloidmi.

Publications:

Jursky, F., Baliova, M., Juhasova, A.: Structural insights into the benzophenanthridines binding to human glycine transporter GlyT1. Eur. J. Pharmacol. 765 (2015) 1-6. [IF2014 2.532]

Applied research
Different survey strategies applied to determine the microbial community degrading the surface of a fresco and an epoxy sculpture

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

Culture-dependent and culture-independent approaches were used to analyze the bacterial community of the surface of the fresco of the Roman catacombs and the eukaryotic and prokaryotic microflora of the surface of the epoxy statue of St. Cyril and Methodius (Devín). DNA and RNA were extracted directly from two areas of the fresco. In addition, DNA was extracted from a mixture of bacteria incubated in Petri dishes containing R2A and B4 media. The results demonstrated that a complex bacterial community (mainly composed of filamentous Actinobacteria, Firmicutes and Proteobacteria) colonizes two different white biofilms and their detection, quantitative and qualitative, could be revealed thanks to a combination of different approaches, as each method provided different information that was only partially overlapped. A microflora of fungi and bacteria was isolated from the epoxy sculpture, which was subsequently clustered using fluorescent ITS, identified by sequencing the ITS region and the 16S rRNA gene section, and tested for lipolytic abilities by three types of agar tests. Various clone libraries of bacteria, cyanobacteria, fungi and algae have been constructed. The results indicated the presence on the surface of the sculpture of interesting and diverse microbial communities composed of stone-dwelling representatives, algal photobionts, cyanobacteria, "black yeast" and plant pathogenic fungi. The survey provided new information about the potential microflora inhabiting the epoxy substrate.
Projects:

Publications:

1. Pangallo, D., Buckova, M., Krakova, L., Puskarova, A., Sakova, N., Grivalský, T., Chovanova, K., Zemankova, M. Biodeterioration of epoxy resin: a microbial survey through culture-independent and culture-dependent approaches. (2015) Environ. Microbiol 17: 462-479. [IF 6.201]

2. Krakova, L., De Leo, F., Bruno, L., Pangallo, D., Urzí, C. Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies. (2015) Environ. Microbiol 17: 1738-1752. [IF 6.201]

International research projects
Study of the self-aggregating properties of morphogenetic proteins of the Bacillus subtilis spore coat

Authors: D. Krajčíková, I. Barák

Spores, dormant cell forms of the bacterium Bacillus subtilis, are characterized by exceptional resistance to adverse physical and chemical influences. This feature is largely due to the existence of a protective protein layer on their surface, called the spore coat. A group of morphogenetic proteins play a key role in its formation, which control the deposition of nearly 70 proteins on the surface of the spore in a precise temporal and spatial arrangement. Using various biochemical, biophysical and genetic methods, we found that morphogenetic proteins are in contact with each other. All our results indicate that morphogenetic proteins form the basic construction of the spore coat, on which other coat proteins are gradually added. Using the AFM technique (atomic force microscopy), we characterized the dynamics of their mutual contact in detail. At the same time, we studied the interaction of other proteins that control the formation of the outer layer of the spore envelope. Using cryo transmission electron microscopy, we determined the structure of some morphogenetic proteins, such as CotY and CotE. We found that envelope proteins have a remarkable ability to self-assemble into very complex but regular structures, which may have considerable potential for the development of new nano-biomaterials.


Projects:

1. VEGA 2/0131/14 - Spórový obal Bacillus subtilis – štúdium tvorby a samo-organizujúcich sa vlastností bielkovín spórového obalu.

2. APVV 14-0181 - Vytváranie proteínových komplexov počas asymetrického bunkového delenia v sporulujúcich bunkách Bacillus subtilis.

Publications:

1. Jiang S., Qiang W., Krajcikova D., Tang J., Tzokov S.B., Barak I., Bullough P.A. (2015) Diverse supramolecular structures formed by self-assembling proteins of the Bacillus subtilis spore coat. Mol. Microbiol. 97: 347-359. (CC-časopis, IF =5.1)

2. Liu H., D. Krajcikova, Z. Zhang, H. Wang, I. Barak and J. Tang (2015) Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single moleculeforce spectroscopy. J. Struct. Biol. (CC-časopis, IF =3.2)