Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We found that ancient (~ a thousand years old) hemp fiber associated microorganisms and/or enzymes awoken from lake sediment are capable of degradation of complex plant polysaccharides and we mimicked the process of hemp retting at a laboratory scale using ancient biomaterial.
• We have analyzed microbial content of honeybee intestine as well as fungi and pollens.

Opis badań

• Translatomics

Protein biosynthesis is a fundamental process in every living cell. Translation is performed by the ribosome and associated factors. Besides the set of conserved and essential translation factors including initiation, elongation, release and recycling factors, evolution selected for a range of proteins which can help preserve and sustain translational machinery during stress conditions including starvation, stationary phase or antibiotic stress. Regulation of gene expression at the translational level is allows for a rapid and transient response to a variety of stimuli or environmental changes, allowing for adaptation of cell growth. Such adaptation would manifest not in the transcriptome composition change, but rather in the changed rate at which mRNA is engaged by the ribosome leading to altered translatome –which results in differing protein levels. Many translation related factors involved in the response to non-optimal growth conditions are well conserved. For decades ribosomes were considered homogeneous macromolecules carrying an unchangeable set of ribosomal RNAs and proteins and the role of the ribosomes in gene expression regulation was neglected. Over time, experimental evidence suggested that ribosomes may take a role of regulatory elements. Due to high-complexity of translational machinery, the ribosomal adaptation could be achieved by different means including varying stoichiometry of ribosomal proteins, presence of paralogous of ribosomal proteins, post-translational modifications of r-proteins or variations in rRNA sequences. Moreover, specialized translation factors are known to help protein biosynthesis machinery to respond to aberrations of bacterial growth conditions. For example, Elongation factor EF-P helps to translate consecutive polyproline stretches. The ribosome functional adaptation of the translational machinery may represent the need for immediate implementation of information about the environmental changes, providing the cell with rapid reaction system related to the usage of a specific pool of mRNAs.

In our lab, we deploy the sporulation process in Bacillus subtilis bacteria as a model to study specialized ribosomes. B. subtilis is an endospore forming bacterium. Once the cell commits to sporulation, it stops to divide and enters hours-long, tractable process to become a spore, which gives an excellent model to study the involvement of translational apparatus in the regulation of sporulation. In this project, we are looking for factors which may determine specialization of the ribosomes during sporulation. We have selected protein factors which we predict may participate in translation regulation. Deletion strains for each factor and in some cases also double and triple knock-out (KO) strains were prepared and characterized phenotypically. For further analyses we chose deletion strains showing no growth defects in optimal, nutrient-rich conditions but at the same time exhibiting strong defects during  severe stress like starvation, extreme temperatures, exposure to detergents, antibiotics or salt. Such phenotype indicates that the investigated protein factor is necessary for specialization of the bacterium. This approach allowed us to prioritize the importance of factors for bacterial growth and survival.

We use a combination of Next Generation Sequencing based analyzes of transcriptome (RNAseq) and translatome (Ribosome profiling, RIBOseq) to identify genes regulated by the action of the investigated factors, combined with genetics, molecular biology, biochemistry and bioinformatical analyses, to explain the molecular role of these factors in gene expression regulation. Together with Dr. Jean-Paul Armache, we will determine the atomic model of the factors bound to the ribosome using Cryo-EM. Knowledge gained in this project will not only broaden  our understanding of translation per se but also, it will give valuable insights into regulatory networks of stress response mechanisms in bacteria.

• Metagenomics

In addition, in our lab, we carry on a project investigating the environmental samples – lake sediments collected at various depths. In this project, we aim to explore the metagenomes obtained from different sediment samples corresponding to different times in history. We are particularly interested in two aspects of information hidden in the extracted DNA: antibiotic resistance genes (ARGs) and their distribution throughout the centuries, as well as their correlation with the environmental factors including climate changes and the lake trophic status. Thanks to next generation shotgun sequencing of the environmental DNA extracted from different depths, we intend to investigate and analyze: (I) metagenome composition – which will inform us about the trophic status of the lake and serve as a proxy to establish the environmental changes across past 2000 years, and (II) microbiome composition – we are especially interested in antibiotic resistance profile, i.e. the resistome, and how it evolved over time. By expanding the traditional biophysical analyses of lake sediments with the metagenome and microbiome analyses from the individual layers of sediment, we will be able to build a more exhaustive picture of the interplay between the environmental conditions and the establishment, maintenance and dissemination of ARGs in the environment.

Metodologia

• Ribosome profiling

RIBOseq enables direct monitoring of the exact position of the ribosome on transcript, i.e. the translatome. Unlike RNAseq (total mRNA sequencing), RIBOseq not only gives the information about the mRNA composition in the cell at a given time, but also provides the rate of translation of each mRNA, number of copies of protein synthesized, unveils stalling events, evaluates the character of small-RNAs or reveals cryptic open reading frames, which may lead to discovery of novel proteins and pathways. The method is based on the fact that during translation approximately 28-30 nucleotides of the mRNA are buried within the ribosomal small subunit. Upon treatment with a nuclease, such ribosome-protected mRNA fragments (footprints) can be characterized by deep sequencing. Each footprint indicates which mRNA was being translated and where the ribosome was located. Deep sequencing of ribosomal footprints provides density profiles of the ribosomes’ positions, enabling qualitative/quantitative measurements of translation across the whole genome.

Moreover, the use of tagged proteins or antibodies raised against a tested factor allows to apply this technique in a targeted way, in which factor-bound ribosomes are purified using affinity or immuno-purification methods – targeted RIBOseq.

• Bioinformatical analyses

We have optimized analysis workflows for sequencing data obtained in Next Generation Sequencing approaches, including RNAseq, RIBOseq, metagenomics (amplicon, shotgun sequencing). We established dedicated pipelines for different data types, however, allowing for a high degree of customizing in order to obtain most comprehensive and thorough analyses.

Wybrane publikacje

• Peil, L.*, Starosta, A. L.*, Virumae, K., Atkinson, G. C., Tenson, T., Remme, J., and Wilson, D. N. (2012). Lys34 of translation elongation factor EF-P is hydroxylated by YfcM. Nature Chemical Biology 8, 695-697.
• Ude, S., Lassak, J., Starosta, A. L., Kraxenberger, T., Wilson, D. N., and Jung, K. (2013) Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science 339, 82-85.
• Starosta, A. L., Lassak, J., Peil, L., Atkinson, G.C., Virumäe, K., Tenson, T., Remme, J., Jung, K., and Wilson, D.N.Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site. Nucleic Acids Research 42, 10711–10719
• Polikanov, Y.S.*, Starosta, A.L.*, Juette, M.F.*, Altman, R.B., Terry, D.S., Lu, W., Burnett, B.J., Dinos, G., Reynolds, K.A., Blanchard, S.C., Steitz, T.A., Wilson, D.N. (2015) Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A. Molecular Cell 2015 Jun 4;58(5):832-44. *contributed equally
• Jenner, L.*, Starosta, A. L.*, Terry, D. S., Mikolajka, A., Filonava, L., Yusupov, M., Blanchard, S. C., Wilson, D. N., and Yusupova, G. (2013) Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. PNAS 110, 3812-3816. *contributed equally

Współpraca

• Jean-Paul Armache, Penn State University, USA, https://www.armachelab.psu.edu/
• Marek Tchórzewski, Department of Molecular Biology, Faculty of Biology and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University.
• Dr hab. Anna Sroka-Bartnicka, Medical University in Lublin.
• Aneta Ptaszyńska, Department of Immunobiology, Faculty of Biology and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University.
• Maria Górna, Structural Biology Group, Biological and Chemical Research Centre, University of Warsaw https://gorna.uw.edu.pl/en/team/maria-gorna
• Jürgen Lassak, Biozentrum, Ludwig-Maximilians University of Munich, Germany

Nagrody i wyróżnienia

• Agata Starosta. Prime Minister Prize for an Outstanding Habilitation Achievement. 2020, Poland
• Agata Starosta. Fellowship for an Outstanding Young Scientists. 2018. Ministry of Science and Higher Education, Poland.