Single-cell analysis of bacterial extracellular filament regulation and assembly

Im Laufe der Evolution haben Bakterienarten äußerst vielfältige und ausgeklügelte extrazelluläre Strukturen entwickelt, die es ihnen ermöglichen, Substrate in ihre Umgebung abzugeben oder Wirtszellen während einer Invasion anzugreifen. Diese Sekretionssysteme sind an vielen bakteriellen Mechanismen wie Biofilmbildung, Zellmotilität, Virulenz oder Gentransfer und Verbreitung von Antibiotikaresistenzen beteiligt. Das Verständnis des Aufbaus und der Regulierung dieser Strukturen ist angesichts der zunehmenden Entwicklung multiresistenter Bakterien von entscheidender Bedeutung. Darüber hinaus geht der Aufbau solcher Strukturen unweigerlich auf Kosten wertvoller zellulärer Energieressourcen, was einen spannenden Parameter darstellt, um zu untersuchen, wie Bakterien den optimalen Mechanismus zum Ausgleich zwischen zellulären Mechanismen und Energieverbrauch wählen. Diese Arbeit konzentriert sich auf den Aufbau und die Regulierung bakterieller extrazellulärer Filamente, insbesondere des flagellaren Typ-III-Sekretionssystems (T3SS). Im ersten Kapitel werden Defekte in der Zellmorphologie aufgezeigt, die durch die Deletion des FlhE-Proteins während des Zusammenbaus der Flagellen verursacht werden, was die Bedeutung der Regulierung des Membranpotentials verdeutlicht. Das zweite Kapitel zeigt, dass der Assemblierungsmechanismus der Flagellen-Filamente, welcher dem Injektions-Diffusions-Modell entspricht, im Vergleich zu anderen Sekretionssystemen schneller ist und für die Energieerhaltung optimiert ist. Das dritte Kapitel untersucht die Rolle des Pilus beim Plasmid-Transfer, der mit einem Typ-IV-Sekretionssystem (T4SS) assoziiert ist, und liefert zusätzliche Hinweise darauf, dass der Pilus als Kanal für den Plasmid-DNA-Transfer dienen kann. Im letzten Kapitel wird ein neuer Biosensor zur Messung des Gehalts an bis-(3'-5')-zyklischem Diguanosinmonophosphat (c-di-GMP) entwickelt, einem entscheidenden Molekül in bakteriellen Mechanismen, die Zellmotilität und Biofilmbildung miteinander verbinden. Insgesamt bietet diese Arbeit Einblicke in die Regulation des flagellaren T3SS und des T4SS-Pilus, ein neues Werkzeug zur Untersuchung von c-di-GMP und Einblicke, wie Bakterien entscheidende Überlebensparameter und die Optimierung eines energiesparenden Aufbaus abwägen.Through evolution, bacterial species have developed highly diverse and sophisticated extracellular structures enabling them to secrete substrates in their environment or to target host cells during invasion. Those secretion systems are involved in many bacterial mechanisms such as biofilm formation, cell motility, virulence or gene transfer and antibiotic resistance dissemination. Understanding the assembly and regulation of these structures is crucial due to the increasing development of multi-drug resistant bacteria. Moreover, the assembly of such structures inevitably comes at the cost of valuable cellular energy resources, representing an exciting parameter to study how bacteria selected the optimal mechanism to balance cellular mechanisms and energy consumption. This thesis focuses on the assembly and regulation of bacterial extracellular filaments, notably the flagellar type III secretion system (T3SS) flagellum . The first chapter reveals cell morphology defects caused by the deletion of the FlhE protein during flagellum assembly, highlighting the importance of membrane potential regulation . Chapter two illustrates that the flagellar filament assembly mechanism, following the injection-diffusion model, is faster compared to other secretion systems and optimized for energy conservation. The third chapter investigates the role of the pilus in plasmid transfer, associated with a type IV secretion system (T4SS), and gives additional evidence that the pilus may also act as a channel for plasmid DNA transfer. The final chapter develops a new biosensor for measuring bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) levels, a crucial molecule in bacterial mechanisms linking cell motility and biofilm formation. Overall, this thesis provides insights into the regulation of the flagellar T3SS and the T4SS pilus, a new tool to study c-di-GMP, and how bacteria balance crucial survival parameters and energy-conserving assembly optimization

Hook‐basal‐body assembly state dictates substrate specificity of the flagellar type‐III secretion system

The assembly of the bacterial flagellum is orchestrated by the secretion of distinct early and late secretion substrates via the flagellar‐specific type‐III secretion system (fT3SS). However, how the fT3SS is able to distinguish between the different (early and late) substrate classes during flagellar assembly remains poorly understood. In this study, we investigated the substrate selectivity and specificity of the fT3SS of Salmonella enterica at different assembly stages. For this, we developed an experimental setup that allowed us to synchronize hook‐basal‐body assembly and to monitor early and late substrate secretion of fT3SSs operating in either early or late secretion mode, respectively. Our results demonstrate that the fT3SS features a remarkable specificity for only the substrates required at the respective assembly stage. No crosstalk of substrates was observed for fT3SSs operating in the opposing secretion mode. We further found that a substantial fraction of fT3SS surprisingly remained in early secretion mode. Our results thus suggest that the secretion substrate specificity switch of the fT3SS is unidirectional and irreversible. The developed secretion substrate reporter system further provides a platform for future investigations of the underlying molecular mechanisms of the elusive substrate recognition of the T3SS.H2020 European Research Council http://dx.doi.org/10.13039/100010663Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Humboldt‐Universität zu Berlin http://dx.doi.org/10.13039/501100006211Peer Reviewe

Control of membrane barrier during bacterial type-III protein secretion

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.Peer Reviewe

Protein Export via the Type III Secretion System of the Bacterial Flagellum

The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane.Peer Reviewe

Protein Export via the Type III Secretion System of the Bacterial Flagellum

The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.Peer Reviewe

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

ABSTRACTInitiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies