Cellular role and mode of action of two pivotal bacterial protein-kinases – PiBaKi

Our methodological approach is based on different expertise and technologies to identify protein-kinases targets, to study in vitro the biochemical properties of their substrates and partners and to construct mutant strains needed for the in vivo characterization of the role of phosphorylation.

We have already identified some substrates and partners of the two protein-kinases studied and initiated the characterization of the phenotypic features of strains deficient for the expression of these two protein-kinases.

While continuing substrates identification of the two protein-kinases, ours aims are to decipher the role of the phosphorylation of targets identified.

The project has started 30 months ago. Our first data have already been published in high-ranked international journals (Nature, Plos genetics, Molecular microbiology and Journal of Biological chemistry)
Our current work is extremely encouraging and should lead to outstanding outcomes.

Protein phosphorylation at serine, threonine and tyrosine is a major post-translational modification involved in the regulation of many fundamental mechanisms of eukaryotic cells. By contrast, in bacteria, the existence of serine/threonine- and tyrosine-kinases has been controversial for a long time. However, over the last two decades, an increasing amount of studies have clearly established that serine, threonine and tyrosine phosphorylation is involved in the regulation of a wide variety of biological processes in the bacterial cell ranging from biofilm formation, virulence, regulation of central and secondary metabolisms, competence to regulation of developmental processes. Bacteria produce eukaryotic-like kinases, but they also molded new enzymes sharing no resemblance with eukaryotic protein-kinases and being unique in exploiting other nucleotide-binding motifs. PiBaKi (Pivotal Bacterial Kinases) is a project focused on investigating the functional and cellular role of two conserved bacterial protein-kinases: one is homologous to eukaryotic protein-kinases and the second contains a Walker-A motif typically found in many ATPases/GTPases, and is specific to the bacterial kingdom. This study will be performed in two different bacteria, the ovococcal bacterial pathogen Streptococcus pneumoniae, and the rod-shape soil bacterium Bacillus subtilis. B. subtilis and S. pneumoniae both encode for a unique eukaryotic-like protein-kinase with PASTA (for Penicillin-binding protein And Ser/Thr protein kinase Associated) motifs, PrkC and StkP, respectively. While StkP would be involved in cell division, PrkC would signal bacteria to exit dormancy in response to peptidoglycan fragments released by the cells in the extracellular medium. However, the underlying regulatory mechanisms by which they exert their function remain elusive. Our recent work confirms that StkP is crucial for S. pneumoniae cell division and further showed that it participates in septum assembly and closure, cell shape and spatial localization of peptidoglycan biosynthesis sites through the phosphorylation of division proteins. We also observed that PrkC would be involved in cell shape determination but it would also affect ribosome biogenesis through the phosphorylation of ribosome-associated GTPases. PrkC would thus have a complex function. On the other hand, a new family of protein-kinases with a unique ATP-binding fold and widely spread in the bacterial kingdom has just been identified by two partners of this consortium. The cellular function of this protein, named YdiB and Spn1761 in B. subtilis and S. pneumonaie respectively, is beginning to emerge as a major player in oxidative stress but is very likely involved in other cellular processes as well. Our recent work with the S. pneumoniae protein-kinases StkP and Spn1761 and with their B. subtilis counterparts PrkC and YdiB offers thus interesting and promising hypotheses to decipher new processes for the regulation of bacterial physiology. The project is established by four research groups with a clear leadership status in the domain. Our strategies will strongly rely on specific competencies and preliminary results obtained by each partner. We will use the synergy created between our teams to apply molecular biology, genetic, biochemistry and microscopy approaches to identify StkP, PrkC, YdiB and Spn1761 partners and substrates and to investigate the regulatory mechanisms underlying these interactions and phosphorylations. From a fundamental point of view, this project will contribute to a better understanding of the inner workings of a bacterial cell. In applied terms, our study is expected to open new avenues towards specific inhibition of these bacterial protein-kinases and thus pave the way towards new antibacterial drugs.