Andreas Diepold

DNA-PAINT enables super-resolution imaging with high multiplexing capacity, yet it is rarely used in nanoscale bacteriology. A likely reason is the lack of straightforward protocols and of systematic characterization of imager-docking strand pairs inside intact bacterial cells, whose envelope and dense cytoplasm differ markedly from both the DNA origami in vitro systems used to develop them, and eukaryotic cells. In this work, we establish a screening platform based on RNA-FISH, in which we extend the 16S rRNA-directed EUB338 probe with docking sequences of choice. This allows imager-docking pairs to be benchmarked directly inside Escherichia coli cells and characterized for parameters such as specificity, binding kinetics, and localization precision. We screened 12 different DNA-PAINT sequences and used an optimized set to perform four-target 3D Exchange-PAINT in single E. coli cells. We further applied our strategy to image three effector mRNAs in the Gram-negative pathogen Yersinia enterocolitica and quantified RNA clusters and their 3D distribution. Our work provides a framework and practical guidelines for high-quality 3D DNA-PAINT imaging in Gram-negative bacteria and should facilitate its broader adoption in nanoscale bacteriology.

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces have potential for biotechnological applications. However, while a limited set of genetic tools exist, the lack of secretion assays and fine-tuning of secretion constrain both fundamental studies and bioengineering of B. bacteriovorus . Here, we present a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. Building on functional native and synthetic promoters from the Anderson library with varying expression levels of fluorescent reporter protein mScarletI3, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

Major bacterial pathogens manipulate eukaryotic target cells by injecting effector proteins through type III secretion systems (T3SS). Recent in situ observations revealed that these large molecular machines, often referred to as injectisomes, are remarkably dynamic and adaptive entities, with the cytosolic T3SS components forming a mobile network that recruits effectors to the export machinery. In contrast to these soluble components, the transmembrane rings anchoring the injectisome are stably associated – with one exception. Using functional assays, live cell microscopy, and photobleaching experiments, we found that SctD, which constitutes the inner membrane ring of the T3SS, exchanges subunits in secreting injectisomes in Yersinia enterocolitica . To elucidate the biological significance of this unexpected dynamic behavior of a key structural component, we investigated its role in the assembly and function of the T3SS. Using engineered SctD variants whose exchange rate can be modulated, we found that exchange supports the integration of export apparatus components into assembled membrane rings and efficient secretion of effectors. Our findings uncover a new aspect of the molecular function and regulation of the T3SS, which may apply to other secretion systems and molecular machines.

Pseudomonas aeruginosa, a versatile Gram-negative opportunistic pathogen, relies on multiple virulence mechanisms, including a Type III Secretion System (T3SS) and several Type VI Secretion Systems (T6SS), to establish infections. The bacterial universal second messenger cyclic di-guanylate (c-di-GMP) orchestrates the lifestyle transitions of Pseudomonas aeruginosa between motile and biofilm-associated states and influences the expression of virulence traits. While it is clear that these systems are interconnected, their precise interaction on the single-cell level has remained unclear. In this study, we use single-cell analysis to dissect the role of c-di-GMP in the heterogeneity of virulence factors in P. aeruginosa populations. Our results confirm earlier findings that on the population level, high c-di-GMP levels lead to increased formation and activity of the H1-T6SS, while negatively influencing formation and activity of the T3SS. On the single-cell level, we further characterize the virulence crosstalk within P. aeruginosa populations by presenting a cooperative relationship among T3SS and flagellum and antagonistic relationships between presence of the H1-T6SS and the T3SS as well as the flagellum. Overall, this c-di-GMP-orchestrated heterogeneity and crosstalk of virulence systems suggest a strategy to optimize survival and pathogenicity under varying environmental conditions in the framework of the motile-sessile lifestyle transition.

The T3SS injectisome is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. While in some bacterial species, T3SS-negative bacteria benefit from the activity of their T3SS-positive siblings, the T3SS model organism Yersinia enterocolitica was thought to uniformly express and assemble injectisomes. In this study, we found that Yersinia actively suppress T3SS expression, assembly and activity at higher cell densities, such as inside microcolonies. This effect is highly specific to the T3SS, reversible, and distinct from stationary phase adaptation. It is conferred by the main T3SS transcription factor VirF, which is downregulated at higher densities and whose in trans expression restores T3SS activity. The concomitant downregulation of the VirF-dependent adhesin YadA led to a drastic reduction in bacterial cell adhesion. We propose that this active suppression of T3SS secretion and cell attachment at higher local bacterial densities promotes a switch during Yersinia infection from a T3SS-active colonization stage to a bacterial replication and dissemination phase.

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces hold potential for biotechnological applications. However, the limited availability of versatile genetic tools and secretion assays constrain both fundamental studies and bioengineering of B. bacteriovorus . Here, we developed a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. We investigated functional native and synthetic promoters from the Anderson library with varying expression levels and demonstrated their efficacy in driving expression of the fluorescent reporter protein mScarletI3 at both the population and single-cell level. Additionally, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

The type III secretion system (T3SS) is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. Type III secretion and bacterial physiology are known to be tightly interconnected and influence each other. Most notably, secreting cells undergo growth arrest in the T3SS model organisms Yersinia , Salmonella and Shigella . The molecular basis of this phenotype, referred to as secretion-associated growth inhibition, is debated. In Yersinia , T3SS genes are encoded extra-chromosomally in the plasmid of Yersinia virulence (pYV), whose copy number increases upon induction of T3SS secretion. In this study, we characterize the link between T3SS activity and subcellular organization by localizing and quantifying the pYV and chromosomal DNA in Yersinia enterocolitica . We find that activation of secretion not only increases the number of pYV plasmids per bacterium, but that the plasmids also move towards the membrane and poles. This relocalization is not caused by transertion (coupled transcription, translation and translocation) of effectors, but part of a broader DNA rearrangement, leading to a distinct relocalization of chromosomal DNA to mid-cell. We hypothesize that these striking DNA rearrangements occurring during secretion are a main factor in the secretion-associated growth inhibition of pathogenic bacteria.

Gram-negative bacteria can use type III secretion systems to inject effector proteins into eukaryotic target cells. Most effectors are co-expressed with specific chaperone proteins that are required for the secretion of their cognate effector. Although chaperones share characteristics across species, no common mechanism of action has been identified. In particular, it remains unclear, if and how chaperones target effectors to the type III secretion injectisome. In this study, we analyzed the interaction network, cellular localization, mobility, and function of SycH and SycE, two Yersinia enterocolitica T3SS chaperones, in live bacteria. While both chaperones strongly interacted with their cognate effectors, SycH additionally bound two negative regulators, YscM1/2, whereas SycE consistently showed weak interactions and proximity to various other effectors. In contrast, the chaperones did not specifically interact with the injectisome or the cytosolic T3SS components that were recently found to shuttle effectors to the injectisome. Mobility measurements and single particle tracking support these findings. Taken together, our results indicate a handover of the effector YopH from its chaperone SycH to the shuttle complexes in the bacterial cytosol and raise the possibility that a similar mechanism applies to other effector/chaperone pairs .