Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We have described the translatome of the spore in Bacillus subtilis: Translation and translational machinery are temporally and spatially organised in B. subtilis cells during sporulation.
• Asymmetric septum plays an important role in the spatial organisation of the ribosomes and serves as a hub in the cell’s developmental control, including translation regulation.
• Translocation of the ribosomes into the spore requires peptidoglycan rearrangements by SpoIIDMP.
• The forespore “inherits” the mother cell’s ribosomes.
• The SpoIIIAH-SpoIIQ complex is responsible for maintaining the turgor of the spore and prevents spore deflation resulting from chromosome back-translocation from the spore to the mother cell.

Opis badań

Protein biosynthesis is a fundamental process in every living cell and is performed by the ribosome and associated factors. 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. Regulation of gene expression at the translational level allows for a rapid and transient response to a variety of environmental stimuli without the need to change the transcriptome. Rather, what changes is the rate at which mRNA is engaged by the ribosome leading to the altered translatome and, as a result, differing protein levels. Due to the high-complexity of the translational machinery, the ribosomal adaptation could be achieved by different means including varying stoichiometry of ribosomal proteins, paralogs and post-translational modifications of the r-proteins or variations in rRNA sequences. Specialized translation factors are also known to help the protein biosynthetic machinery to respond to aberrant growth conditions.

In our lab, we employ the sporulation process in Bacillus subtilis, an endospore forming bacterium, as a model to study translation regulation. Upon starvation, the bacterial cell develops a survival form – a spore – in a process of asymmetric cell division. This process is tightly regulated on the transcriptional level, however, little is known about how translation is involved in the regulation of this process. Once the cell commits to sporulation, it stops dividing and enters an hours-long, tractable process to become a spore, which is an excellent model in which to study the involvement of the translational apparatus in the regulation of cellular development. In our research we look for factors which regulate translation during sporulation, e.g. factors determining ribosomal specialization. We use state-of-the-art approaches including Next Generation sequencing methods such as RNA-seq and ribosome profiling, supported by in-depth bioinformatical analyses. We also apply fluorescent microscopy techniques, including super-resolution structured illumination microscopy (SIM) imaging and click-chemistry mediated biochemical assays. In our toolkit we also have various genetic, molecular biology and biochemistry based assays.

In our research we have described the sequential changes in the translatome of B. subtilis during sporulation. Translation and translational machinery are temporally and spatially organised in B. subtilis during the process of sporulation, which is especially important during asymmetric septation. During sporulation, translation undergoes two silencing events, first prior to the asymmetric septation and then at the end of sporulation, in preparation for mother cell autolysis. At the first translation silencing event, the ribosomes are transiently arrested and are translating at very low levels. This translational halt lasts until the two asymmetric compartments (spore and the mother cell) are formed and transcription is separated based on dedicated sigma factors. Interestingly, the ribosomes do not dissociate from the mRNA, but instead are paused for a while on the mRNA template awaiting their new subcellular destination. We have also discovered that ribosomes are transported into the spore after the chromosome is fully translocated and during peptidoglycan rearrangement at the asymmetric septum. The peptidoglycan of the asymmetric septum is partially degraded with the aid of the SpoIIDMP protein complex, allowing for membrane migration and new peptidoglycan synthesis and thus, for successful spore engulfment. In our research we showed that the asymmetric septum plays an important role in the spatial organisation of the ribosomes and serves as a hub in the cell’s developmental control, including translation regulation. The process of ribosome translocation into the spore is thus dependent on the peptidoglycan rearrangement of the asymmetric septum and, without it, the spores do not mature.

We have also investigated the role of three zinc independent paralogs of ribosomal proteins during sporulation in B. subtilis. The triple deletion strain showed no growth difficulties in optimal, nutrient-rich conditions but at the same time exhibited defects during cellular stress – sporulation caused by nutrient limitation. Such phenotype indicates that the investigated protein factors are necessary for specialization of the bacterial cell. Indeed, although expressed at low to moderate levels, the three investigated proteins make up a subpopulation of ribosomes which is important for timely and effective sporulation. The lack of all three proteins results in delayed sporulation and reduced germination efficiency. This is most probably a result of dysregulated translation of key sporulation and metabolism related genes and especially, of the delayed and inefficient translation silencing occurring prior to asymmetric septation, as compared to WT.

In our lab, we are also investigating specialized translation factors associated with the biosynthesis and action of antibiotics produced by Myxococcus xanthus. The interplay between translation and antibiotic biosynthesis is an attractive research subject. Biosynthesis of antimicrobials targeting translation is, with a doubt, imposing a burden on the bacterial translational machinery. Thus, by investigating the putative antibiotic biosynthetic gene cluster encoded in the M. xanthus genome, as well as expression of the paralogs of translation factors, we would like to describe how and when antibiotic biosynthesis is switched-on in bacteria and how the cell is protected from self-intoxication. Such research could not only help to provide new insights into the action of the translational machinery, but also to characterize putative novel determinants of antibiotic resistance.

In the future, we plan to study the translatome of the Psychrophilic bacteria isolated in the Antarctic.

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

• Iwańska, O., Latoch, P., Kovalenko, M., Lichocka, M., Hołówka, J., Serwa, R., Grzybowska, A., Zakrzewska-Czerwińska, J., & Starosta, A. L. (2025). Ribosomes translocation into the spore of Bacillus subtilis is highly organised and requires peptidoglycan rearrangements. Nature communications, 16(1), 354. https://doi.org/10.1038/s41467-024-55196-9.
• Iwańska, O., Latoch, P., Kopik, N., Kovalenko, M., Lichocka, M., Serwa, R., & Starosta, A. L. (2024). Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation. Nature communications, 15(1), 7188. https://doi.org/10.1038/s41467-024-51654-6.
• Nowacka, M., Latoch, P., Izert, M. A., Karolak, N. K., Tomecki, R., Koper, M., Tudek, A., Starosta, A. L., & Górna, M. W. (2022). A cap 0-dependent mRNA capture method to analyze the yeast transcriptome. Nucleic acids research, 50(22), e132. https://doi.org/10.1093/nar/gkac903.
• Iwańska, O., Latoch, P., Suchora, M., Pidek, I. A., Huber, M., Bubak, I., Kopik, N., Kovalenko, M., Gąsiorowski, M., Armache, J. P., & Starosta, A. L. (2022). Lake microbiome and trophy fluctuations of the ancient hemp rettery. Scientific reports, 12(1), 8846. https://doi.org/10.1038/s41598-022-12761-w.
• Kopik, N., Chrobak, O., Latoch, P., Kovalenko, M., & Starosta, A. L. (2021). RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing. Journal of visualized experiments : JoVE, (174), 10.3791/62544. https://doi.org/10.3791/62544.

Współpraca

• Jean-Paul Armache, Penn State University, USA. https://www.armachelab.psu.edu
• Anna Sroka-Bartnicka, Medical University in Lublin, Poland.
• Maria Górna, Structural Biology Group, Biological and Chemical Research Centre, University of Warsaw, Poland. https://gorna.uw.edu.pl/en/team/maria-gorna
• Przemysław Gródnik, Małopolskie Centrum Biotechnologii, Uniwersytet Jagielloński, Kraków, Poland.

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.