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Projects / Programmes source: ARIS

DNA sampling II: a method for identification of directly or indirectly bound proteins at specific loci on bacterial chromosomes

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   

Code Science Field
P320  Natural sciences and mathematics  Nucleic acids, protein synthesis 

Code Science Field
1.06  Natural Sciences  Biological sciences 
Keywords
Bacteria, Pathogenic bacteria, Infection, Gene regulation, Gene expression, Transcription factor, Antibiotic, DNA sampling
Evaluation (rules)
source: COBISS
Researchers (20)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  16104  PhD Apolonija Bedina Zavec  Biotechnology  Researcher  2017 - 2020  153 
2.  24290  PhD Matej Butala  Biochemistry and molecular biology  Head  2017 - 2020  236 
3.  23399  PhD Tomaž Curk  Computer science and informatics  Researcher  2017 - 2020  253 
4.  25974  PhD Cene Gostinčar  Biotechnology  Researcher  2017 - 2020  338 
5.  32099  PhD Maja Grundner  Biochemistry and molecular biology  Researcher  2017 - 2020  35 
6.  53283  Maja Hostnik  Biochemistry and molecular biology  Researcher  2019 - 2020  18 
7.  52386  Eva Kočar  Biochemistry and molecular biology  Technical associate  2019 - 2020  29 
8.  18749  PhD Rok Kostanjšek  Biology  Researcher  2017 - 2020  473 
9.  00412  PhD Igor Križaj  Biochemistry and molecular biology  Researcher  2017 - 2020  729 
10.  53699  Amela Kujović  Biochemistry and molecular biology  Technical associate  2019 - 2020  25 
11.  17276  Jelka Lenarčič    Technical associate  2017 - 2020 
12.  18802  PhD Adrijana Leonardi  Biochemistry and molecular biology  Researcher  2017 - 2020  157 
13.  06994  PhD Peter Maček  Biochemistry and molecular biology  Researcher  2017 - 2018  523 
14.  35371  PhD Maruša Novak  Biotechnology  Researcher  2017 - 2018  34 
15.  35372  PhD Davor Obradović  Natural sciences and mathematics  Researcher  2017  14 
16.  51231  PhD Anja Pavlin  Biochemistry and molecular biology  Junior researcher  2018 - 2020  26 
17.  12048  PhD Marjetka Podobnik  Biochemistry and molecular biology  Researcher  2017 - 2020  319 
18.  15328  PhD Kristina Sepčić  Biochemistry and molecular biology  Researcher  2017 - 2020  731 
19.  15600  MSc Maja Šimaga    Technical associate  2019 - 2020 
20.  06905  PhD Tom Turk  Biochemistry and molecular biology  Researcher  2017 - 2020  620 
Organisations (4)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  21,316 
2.  0106  Jožef Stefan Institute  Ljubljana  5051606000  91,035 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,817 
4.  1539  University of Ljubljana, Faculty of Computer and Information Science  Ljubljana  1627023  16,298 
Abstract
Gene expression starts with regulatory proteins binding to DNA at the promoters. The ability of bacteria to tune their transcriptional programes (e.g. virulence or antibiotic resistance genes) in response to specific signals and changing environments plays a determining role in defining their pathogenic potential. In this process, the ability of proteins to target and bind to specific DNA sequences among a vast excess of nonspecific DNA is a fundamental property. In many cases different transcription factors, responding to different signals, bind at the same promoter. Thus, determining which transcription factors are specifically bound to selected promoter is of great importance in order to understand how multiple environmental signals are integrated to coordinate gene expression. This is particularly important to understand induction of virulence factors in pathogenic bacteria. Furthermore, molecules can be designed to specifically target transcription factors to inhibit gene expression in pathogens without affecting the resident commensals. A challenge remains to develop a reliable method to decode protein complexes residing on any specific genomic loci directly from bacteria. We developed a »DNA sampling« protocol that enables rapid isolation of specific DNA fragments with bound proteins in E. coli cells, which is to our knowledge the first example of identification of bacterial DNA-binding proteins in vivo. We have identified few shortcomings upon running the protocol. Therefore, the goal of this project is to modify the DNA sampling protocol into a reliable, affordable and more efficient method which will be applicable to diverse bacteria. We will improve the method to identify in the common or enterotoxigenic Escherichia coli strains or in the opprotunistic pathogen Pseudomonas aeruginosa each single transcription factor at selected promoters in determined conditions in vivo. We will achieve by either implementing the formaldehyde crosslinking and de-crosslinking steps into the DNA sampling protocol or by trapping the liberated, target nucleoprotein complexes in minicells. The latter approach does not rely on the use of chemical cross-linking agents, and will hence represent the closest example of in vivo transcription complexes isolated to date. DNA-associated proteins will be identified by mass spectrometry and the methylation profile of the target DNA assayed by methylation-sensitive restriction enzymes. Results obtained by the enhanced protocol will be validated computationally, in vitro and in vivo.
Significance for science
Current dogma suggests that transcription regulation in bacteria mainly occurs with proteins bound directly to DNA targets. However, within the project group we strongly believe a complex combination of DNA-bound transcription factors and to the DNA indirectly bound protein co-factors, are acting at one same promoter. We are lacking reliable methods for the isolation and identification of promoter DNA-transcription machinery complexes directly from cells, enabling one to monitor how this proteins change over time and in response to external stimuli. Methods to identify specific DNA-binding proteins in bacteria use crude cell extracts and immobilized DNA fragments to reconstitute protein-DNA fragments, and attempt to mimic the conditions inside the cell. Our protocol concerns with revealing and understanding the jet discerned basic principles of gene regulation directly in bacteria. Success of the project is guaranteed as the proposed innovative concept for capturing nucleoprotein complexes via two complementary approaches is based on the already validated DNA sampling protocol. Thus, efficiently probing the dynamics of transcription factors at specific DNA sequences in response to diverse stimuli directly in bacteria. Such powerful new approach will have great potential for the study of regulation of gene expression in bacteria (e.g. for toxin, antibiotic resistance genes) and it will be of use to any molecular biology laboratory studying bacterial transcription. In principle, the newly developed DNA sampling II method could be used to investigate any DNA segment and it will be applicable to diverse bacteria. This will provide us to address fundamental questions about how bacteria sense environmental stress and regulate gene transcription in order to survive in hostile conditions, as for instance during antibiotic therapy. Hence, it suggests a strong link to the complementary ChIP-based methods. In addition, a novel method using minicells does not rely on the use of chemical cross-linking agents, and hence will represent the closest example of in vivo transcription complexes isolated to date. Such approach will also enable us to examine epigenetic landscape of the target DNA. Furthermore, the novel method linked to electron microscopy on the isolated complexes from the minicells, would enable insight into the architecture of the transcription machinery, hence the DNA topology and volumetric measurements to determine the stoichiometry of proteins bound to the target DNA loci. In addition, immobilizing isolated promoter complexes, between DNA, RNA polymerase and transcription factors, on the surface of the SPR chip it would be possible to follow the initiation of transcription in real-time, by following the synthesis of short transcript upon addition of nucleotides. Clearly, the DNA sampling II protocol is a much needed molecular toolbox that can with development of more sensitive detection methods and the application of novel imaging technologies may eventually be developed further and to be applicable even on a whole genome scale.
Significance for the country
The development of drug-resistant bacteria is an unavoidable outcome of widespread antibacterial chemotherapy of infectious diseases. World Health Organisation recently called for an urgent and concerted action: »Combat drug resistance: no action today means no cure tomorrow.« We need to bring new classes of antibiotics to market to prevent people from dying from infections that have been treatable for decades, until resistance rendered our drugs ineffective. Drug discovery programs directed against proteins considered essential for in vivo bacterial viability have yielded few new therapeutic classes of antibiotics, therefore treatment options for combating bacteria resistant to multiple drugs are narrowing. One of the EU going on action plans set few years ago was reinforcement of research and development of innovative means to combat antimicrobial resistance. The studies proposed in this work fit into the key research areas of the European commission FP7 and Horizon 2020 for research and innovation pograms on antimicrobial drug resistance. Notably, results obtained by our approach are also highly complementary to the Innovative Medicines Initiative (IMI) NewDrugs4BadBugs program, a partnership between European Commission and the European pharmaceutical industry, which focuses especially on pushing novel types of antimicrobial drugs to the market. By illuminating dark matter of gene regulatory mechanisms, by uncovering hidden complexities of transcription factors associated with specific loci on bacterial chromosomes, we can obtain major aspects into bacterial biology such as pathogenesis and antibiotic resistance development. We will illustrate the enhanced protocol on virulence-associated promoters in enterotoxigenic E. coli, responsible for significant portion of pediatric deaths, and in opportunistic pathogen P. aeruginosa, a leading cause of hospital-acquired infections. The DNA sampling II protocol will enable to identify pathogen-specific virulence transcription regulators and enable the design and production of next generation, safe antimicrobial agents or novel, fast diagnostic tools to identify pathogens.  Thus, a long term goal of this project is to transfer knowledge from academics to industry. These long-term activities are out of the scope of the proposed project, but they might lead to a range of commercialization options, including private-public partnerships, IP drug licensing, and spin-off companies.
Most important scientific results Final report
Most important socioeconomically and culturally relevant results Interim report, final report
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