Biology subjects

Turgay, K.

Publications and source records attributed to Turgay, K..

7 recordsLinked to original sources

bioRxiv2025

Targeting the translation machinery: The RNase Y specificity factor Y-complex coordinates ribosome degradation

Limiting the synthesis and activity of ribosomes is crucial for adaptation to stresses, such as heat or nutrient starvation. In Bacillus subtilis, this can be achieved through the coordinated action of the alarmones (p)ppGpp and the transcription factor Spx. Here, we performed a genetic screen to uncover novel factors contributing to the heat shock response of B. subtilis. We identified the Y-complex, which confers specificity to the endonuclease RNase Y, as a critical player during stress conditions, such as heat or transition into stationary phase. This protein complex is required for processing diverse RNAs, notably the maturation of mRNAs encoding proteins involved in translation and metabolism. We further demonstrate that the Y-complex and RNase Y initiate the degradation of rRNAs of mature ribosomes, lowering their abundance. We propose that the Y-complex is a regulatory hub that modulates gene expression, adjusts protein synthesis and resource allocation.

microbiology↗

bioRxiv2024

Soft X-ray tomography reveals variations in B.subtilis biofilm structure upon tasA deletion

Bacterial biofilms are complex communities of cells within a self-produced extracellular matrix. They play crucial roles in healthcare, nutrition, agriculture and environmental research, yet an analysis of their elaborate 3D architecture remains challenging. Understanding mechanisms of biofilm formation, particularly the effects of chemical, physical, and genetic influences or modifications, is crucial but requires structural information at subcellular resolution to enable a community-level analysis of biofilms. In this work, we developed a "biofilm-in-capillary" growth method compatible with full-rotation soft X-ray tomography, providing high-resolution 3D imaging of bacterial cells and their surrounding extracellular matrix during biofilm formation, without drying or fixation steps. This approach offers 50 nm isotropic spatial resolution, rapid imaging time, and quantitative native analysis of biofilm structure. We demonstrate the potential of our method using Bacillus subtilis biofilms, detecting coherent alignment and chaining of wild-type cells while they are travelling towards the oxygen-rich capillary tip region. In stark contrast, the genetic knock-out {Delta}tasA shows a loss of cellular orientation, including changes in the extracellular matrix in volume and chemical density. Notably, we show that the addition of TasA protein to a culture of a {Delta}tasA strain restores the extracellular matrix density and leads to a compaction of cell assemblies, yet no chaining is observed as for the wildtype. Our approach to imaging biofilms is scalable and transferable, opening new avenues for examining biofilm structure and function across various species, including mixed biofilms, and observing 3D reorganization in response to genetic and environmental factors.

microbiology↗

bioRxiv2024

A novel ClpC adaptor protein that functions in the developing Bacillus subtilis spore

Bacterial protein degradation machinery consists of chaperone-protease complexes that play vital roles in bacterial growth and development and have sparked interest as novel antimicrobial targets. ClpC-ClpP (ClpCP) is one such chaperone-protease, recruited by adaptors to specific functions in the model bacterium Bacillus subtilis and other Gram-positive bacteria including the pathogens Staphylococcus aureus and Mycobacterium tuberculosis. Here we have identified a new ClpCP adaptor protein, MdfA ("Metabolic differentiation factor A", formerly YjbA), in a genetic screen for factors that help drive B. subtilis toward metabolic dormancy during spore formation. A knockout of mdfA stimulates gene expression in the developing spore, while aberrant expression of mdfA during vegetative growth is toxic. MdfA binds directly to ClpC to induce its oligomerization and ATPase activity, and this interaction is required for the in vivo effects of mdfA. Finally, a co-crystal structure reveals that MdfA binds to the ClpC N-terminal domain at a location analogous to that on the M. tuberculosis ClpC1 protein where bactericidal cyclic peptides bind. Altogether our data and that of an accompanying study by Riley, Lyda and colleagues support a model in which MdfA induces ClpCP-mediated degradation of metabolic enzymes in the developing spore, helping drive it toward metabolic dormancy.

microbiology↗

bioRxiv2022

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

Initiation of bacterial DNA replication takes place at the origin of replication, a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response. Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be a valuable tool for biotechnological applications. ImportanceEven though bacteria are constantly replicating under laboratory conditions, natural environments expose them to various stresses like lack of nutrients, high salinity, and pH changes, which can keep them in non-replicating states. Non-replicating states can allow bacteria to become less sensitive or tolerant to antibiotics (persisters), remain inactive in specific niches for an extended period (dormancy), and adapt to some hostile ecosystems. Non-replicating states have been studied due to the possibility of repurposing energy to produce additional metabolites or proteins. Using CRISPRi targeting bacterial replication initiation sequences, we successfully arrested the replication of B. subtilis. We observed that non-replicating cells continued growing but not dividing, and the initial arrest did not induce global stress conditions such as SOS or stringent response. Notably, these cells continued their metabolic activity and translation. This study provides comprehensive insights into the physiological response of replication initiation blockage in B. subtilis.

microbiology↗

bioRxiv2022

Protein arginine phosphorylation and de-phosphorylation facilitate protein homeostasis by an AAA+ chaperone protease system

We could demonstrate that the AAA+ unfoldase ClpC together with the protein arginine kinase and adaptor protein McsB, its activator McsA and the phosphatase YwlE form a unique chaperone system. Here, the McsA-activated McsB phosphorylates and targets aggregated substrate proteins for extraction and unfolding by ClpC. Sub-stoichiometric amounts of the YwlE phosphatase enhanced the ClpC/McsB/McsA mediated disaggregation and facilitated the de-phosphorylation of the arginine-phosphorylated substrate protein extruded by ClpC, allowing its subsequent refolding. Interestingly, the successfully refolded protein escaped degradation by the loosely associated ClpP protease. This unique chaperone system is thereby able to disaggregate and refold aggregated proteins but can also remove severely damaged protein aggregates by degradation.

microbiology↗

bioRxiv2019

The alarmone (p)ppGpp is part of the heat shock response of Bacillus subtilis

Here, B. subtilis was used as a model organism to investigate how cells respond and adapt to proteotoxic stress conditions. Our experiments suggested that the stringent response, caused by raised levels of the (p)ppGpp alarmone, plays a role during thermotolerance development and the heat shock response. Accordingly, our experiments revealed a rapid increase of cellular (p)ppGpp levels upon heat shock as well as salt- and oxidative stress. Strains lacking (p)ppGpp exhibited increased stress sensitivity, while raised (p)ppGpp levels conferred increased stress tolerance to heat- and oxidative stress. During thermotolerance development, stress response genes were highly up-regulated together with a concurrent transcriptional down-regulation of the rRNA, which was influenced by the second messenger (p)ppGpp and the transcription factor Spx. Remarkably, we observed that (p)ppGpp appeared to control the cellular translational capacity and that during heat stress the raised cellular levels of the alarmone were able to curb the rate of protein synthesis. Furthermore, (p)ppGpp controls the heat-induced expression of Hpf and thus the formation of translationally inactive 100S disomes. These results indicate that B. subtilis cells respond to heat-mediated protein unfolding and aggregation, not only by raising the cellular repair capacity, but also by decreasing translation involving (p)ppGpp mediated stringent response to concurrently reduce the protein load for the cellular protein quality control system.\n\nAuthor SummaryHere we demonstrate that the bacterial stringent response, which is known to slow down translation upon sensing nutrient starvation, is also intricately involved in the stress response of B. subtilis cells. The second messengers (p)ppGpp act as pleiotropic regulators during the adaptation to heat stress. (p)ppGpp slows down translation and is also involved in the transcriptional down-regulation of the translation machinery, together with the transcriptional stress regulator Spx. The stress-induced elevation of cellular (p)ppGpp levels confers increased stress tolerance and facilitates an improved protein homeostasis by reducing the load on the protein quality control system.

microbiology↗

bioRxiv2019

Xenogeneic regulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

SPO1 phage infection of Bacillus subtilis results in a comprehensive remodelling of processes leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins for the specific shut down of various host processes including transcription, DNA synthesis and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module we identified eight gene products which attenuated B. subtilis growth. Out of the eight gene products that attenuated bacterial growth, a 25 kDa protein, called Gp53, was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Results reveal that Gp53 functions like a phage encoded adaptor protein and thereby appears to alter the substrate specificity of the ClpCP protease to modulate the proteome of the infected cell to benefit efficient SPO1 phage progeny development. It seems that Gp53 represents a novel strategy used by phages to acquire their bacterial prey.\n\nSignificance statementViruses of bacteria (phages) represent the most abundant living entities on the planet, and many aspects of our fundamental knowledge of phage-bacteria relationships remain elusive. Many phages encode specialised small proteins, which modulate essential physiological processes in bacteria in order to convert the bacterial cell into a factory for phage progeny production - ultimately leading to the demise of the bacterial cell. We describe the identification of several antibacterial proteins produced by a prototypical phage that infects Bacillus subtilis and describe how one such protein subverts the protein control system of its host to benefit phage progeny development. The results have broad implications for our understanding of phage-bacteria relationships and the therapeutic application of phages and their gene products.

microbiology↗