Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Prof. Dr.

Molecular basis for the targeting process of injectisome substrate proteins in Salmonella enterica

Prokaryotic cell biology

A major virulence factor of many pathogenic gram-negative bacteria, including Salmonella enterica Typhimurium, is the injectisome, a needle-like structure that injects toxins into host cells during infection. The injectisome employs a type-III secretion system (T3SS) for the export of toxin proteins. Before initiating toxin secretion, it secretes its own structural components through a highly controlled self-assembly process. However, because T3SS substrates do not contain a typical cleavable secretion signal in the aminoterminal region of the peptide sequence [1], it remains unknown how these substrates are selected from thousands of cytoplasmic proteins and specifically targeted to the T3SS. This project explores the hypothesis that substrate gene transcription and translation occur in close proximity to the T3SS, enabling efficient recognition and secretion. This concept is inspired a process known as transertion in which transcription, translation, and membrane insertion of proteins are spatially coordinated. Recent findings suggest that Vibrio parahaemolyticus utilizes transertion for T3SS substrate secretion [2], but its role in Salmonella remains unclear. To test this hypothesis, co-localization analyses of chromosomal substrate gene loci and the T3SS, as well as spatial studies on membrane proximity will be conducted. In summary, this project will provide critical insights into the type III secretion process, shedding light on the assembly mechanism of the injectisome and the secretion of toxins during infection. Ultimately, it will elucidate the potential role of local transcription and translation of substrate genes in these processes.[1] Michiels T, Wattiau P, Brasseur R, Ruysschaert JM, Cornelis G (1990). Secretion of Yop proteins by Yersiniae. Infect Immun 58(9), 2840–2849 [2] Kaval KG, Chimalapati S, Siegel SD, Nalleli G, Jananee J, Ankur BD, Kim O (2023). Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion. Nat Commun 14, 1178

Poster

PD Dr.

The bacterial predator Myxococcus xanthus uses two LcrH-like chaperones with unprecedented substrates to lyse prey cells

In soil environments, microorganisms constantly compete for limited nutrients. To secure their resources, selected bacterial species act as predators, i.e. they kill other bacteria to consume their biomass. An important model to study bacterial predation is Myxococcus xanthus, which uses a broad prey spectrum and possesses a distinct predation machinery. M. xanthus kills its prey in direct cell-cell contact using two protein secretion systems [1]. A Tad-like "Kil" system seems to mediate prey killing, and an atypical type 3 secretion system (T3SS*) is responsible for prey lysis [2]. Compared to other T3SS, the T3SS* in M. xanthus lacks a needle and other outer membrane components, yet it is fully functional.To address the question of whether and how the T3SS* might secrete effector proteins across the M. xanthus cell envelope to the prey cell, we initially investigate the role of putative chaperones. These chaperones presumably bind specific substrates to recruit them for secretion. Phenotypic analysis showed that two out of four LcrH-like chaperones within the T3SS* gene cluster are required for T3SS*-dependent prey lysis. However, homologues to known T3SS effectors were not found in the M. xanthus genome. We are currently looking into candidate effectors for each chaperone by screening deletion mutants. First interactions between chaperone-effector pairs were identified using in vitro pulldown assays. To localize these T3SS* components upon prey contact, we perform fluorescence microscopy and aim to unravel their role within the predation machinery.References: Arend, K. I. Schmidt, J. J., Bentler, T., Lüchtefeld, C., Eggerichs, D., Hexamer, H. M., & Kaimer, C., (2021). Myxococcus xanthus predation of Gram-positive or Gram-negative bacteria is mediated by different bacteriolytic mechanisms. Applied and Environmental Microbiology 87(5). Thiery, S., Turowski, P., Berlemann, J. E., Kaimer, C., (2022) The predatory soil bacterium Myxococcus xanthus combines a Tad- and an atypical type 3-like protein secretion system to kill bacterial cells. Cell Reports, 40, 1-29.This work is funded by Deutsche Forschungsgemeinschaft DFG.

YejL is involved in Gram-negative outer membrane biogenesis

The outer membrane of Gram-negative bacteria consists of two leaflets that differ in their composition: the inner leaflet contains mostly phospholipids (PL) whereas the outer leaflet is mostly composed of lipopolysaccharides (LPS). LPS provide a physical barrier against hydrophobic molecules like antibiotics and bile salts, therefore, the balance of the two major components is essential for viability. To maintain the optimal ratio of LPS and PL, their biosynthesis pathways are connected at multiple sites, resulting in a complex protein network (Möller et al., 2023). Since an overproduction of LPS leads to cell death, the first committed enzyme in the LPS pathway, LpxC, is posttranslationally degraded by the protease FtsH, which is facilitated by the adaptor protein LapB. The adaptor function of LapB is inhibited by the inner membrane protein YejM (LapC) upon lack of LPS, resulting in stabilisation of LpxC. Interestingly, the yejM gene is located downstream of yejL, which encodes for a so far uncharacterised protein. Unlike other proteins involved in the regulatory interplay, YejL is not essential. However, YejL is conserved among gammaproteobacteria, contains 75 amino acids and forms a dimer with a tight conformation.To investigate the function of YejL in the complex regulatory network, we performed protein-protein interaction studies using bacterial two-hybrid, pull down, microscale thermophoresis (MST) and far western dot blot analysis. We identified various proteins involved in outer membrane biosynthesis that might interact with YejL. The bacterial two-hybrid assay and MST showed that the first protein of the PL pathway, FabZ, as well as the first protein of the LPS pathway, LpxA, bind to YejL. In addition, MST and far western dot blot revealed an interaction between YejL and MreB which coordinates cell shape.Overall, our studies indicate that YejL is likely involved in the regulatory interplay of outer membrane biogenesis.

Functional analysis of CRISPR-Cas systems in antibiotic-producing actinomycetes

Many Actinomycetes are important producers of valuable secondary metabolites with potent properties like antimicrobial, anti-tumor or immunosuppressive activity. However, their genetic engineering is often prohibited by mechanisms that prevent the stable establishment of foreign DNA. CRISPR-Cas systems are known as adaptive prokaryotic immunity systems. They play an important role in the defence against foreign mobile genetic elements like plasmids or phages. Recently, also the involvement of CRISPR-Cas in other cellular processes including DNA-repair or cell-differentiation was described. But so far the knowledge about the function of CRISPR-Cas in Actinomycetes is still very limited.The goal of this project is the functional analysis of CRISPR-Cas systems in antibiotic-producing actinomycetes. This includes adaptive immunity as well as functions beyond immunity, such as a potential role of CRISPR-Cas in regulation of antibiotic production. For this, we use the industrial aminoglycoside antibiotic producer Streptoalloteichus tenebrarius as model strain. It is particularly interesting, as its genome encodes 33 predicted biosynthetic gene clusters and three CRISPR-Cas systems (Region 1-3).Bioinformatic sequence analysis on the origin of the spacers did not result in matches to known sequences from databases. Gene expression studies revealed the transcription of all cas-genes under standard laboratory conditions. The immunity function can be investigated with DNA-uptake assays based on transformation, conjugation and/or phage infection. To identify a phage that infects S. tenebrarius, an extensive screening of newly isolated phages as well as known actinomycetes phages was conducted. It was shown, that S. tenebrarius is not included in the host range of all tested phages.Since the genetic manipulation of S. tenebrarius is challenging, the CRISPR-Cas systems of S. tenebrarius were heterologously expressed in Escherichia coli and Streptomyces coelicolor. The DNA-Uptake assays performed with the heterologous hosts expressing CRISPR-Cas Region 1 did not reveal activity as adaptive immunity system so far.Furthermore, CRISPR-Cas deletion mutants in S. tenebrarius were generated. The deletion of CRISPR-Cas Region 1 led to a sporulation-deficient phenotype. This suggests an involvement of CRISPR-Cas in cell-differentiation processes in S. tenebrarius.

Effects of cell shape changes on Listeria monocytogenes in vitro virulence

Introduction: Listeria monocytogenes is a foodborne pathogen characterized by its capability to invade and replicate in multiple human cell types and to spread from cell to cell. In addition to known virulence factors, also the absence of the late cell division protein DivIVA leads to severe virulence defects (1, 2). Based on the characteristic cell-chaining phenotype of the L. monocytogenes divIVA mutant, the question arose how the cell shape affects in vitro virulence. Therefore, we here compare the virulence properties of coccoid and filamentous mreB (involved in lateral cell growth) and ezrA (involved in Z-ring formation) mutants to the cell-chaining phenotype of the divIVA mutant.Methods: IPTG-dependent mreB and ezrA depletion strains were constructed and the presence of the expected coccoid and filamentous phenotypes was verified during growth under standard laboratory conditions and in in vitro infection experiments. All cell morphotypes were tested for replication, invasion and cell-to-cell spread in various eukaryotic cell lines. Additionally, the expression of actin tails, required for movement and spreading during infection, was analysed by phalloidin staining.Results: The coccoid and filamentous growth of the mreB and ezrA depletion strains did neither impair the multiplication in infected macrophages nor did it influence the invasion in human hepatocytes, as opposed to the replication and invasion deficient divIVA mutant. On the other hand, filamentous growth of the ezrA depletion strain led to reduced plaque sizes compared to the wild type. No cell-to-cell spread could be observed for the coccoid growing mreB depletion strain as well as the divIVA mutant, which coincided with a reduced abundance or absence of actin filaments.Conclusions: Our findings indicate that the cell shape plays a minor role for efficient in vitro infection. The severe virulence defect of the divIVA mutant therefore seems to be associated with a DivIVA-dependent influence on other virulence factors or their release via the SecA2 secretion route (1).1 – Halbedel et al. (2012), Mol Micro, 83: 821-839.2 – Kaval et al. (2014), Mol Micro, 94: 637-654.

Identification of EcpK, a BY-pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus

Bacteria secrete a diverse array of polysaccharides that have important functions in many physiological processes and industrial applications. In the ubiquitous Wzx/Wzy-dependent pathway, polysaccharide biosynthesis is initiated by a phosphoglycosyltransferase transferring a sugar-1 phosphate to undecaprenyl phosphate. Next, glycosyltransferases add sugar units to synthesize the repeat unit, which is then "flipped" to the periplasm by a Wzx flippase. Subsequently, a Wzy polymerase in conjunction with a polysaccharide-co-polymerase (PCP) polymerizes repeat units. Finally, in Gram-negative bacteria, an OPX protein in conjunction with the PCP translocate the polysaccharide across the outer membrane. Thus, PCPs in Wzx/Wzy-dependent pathways in Gram-negative bacteria are at the nexus between polymerization and translocation of the polysaccharide. These PCPs belong to the 2a family and interact directly with the Wzy polymerase and the OPX protein. Generally, PCP-2a proteins are inner membrane proteins with an extended periplasmic domain, a C-terminal cytoplasmic bacterial tyrosine kinase (BYK) domain and form octameric assemblies. Interestingly, PCP-2a proteins lacking either the BYK domain entirely or motifs important for Tyr phosphorylation have been described. Here, we focus on one such protein, EpsV for exopolysaccharide (EPS) biosynthesis in Myxococcus xanthus. Computational structural biology confirmed that EpsV lacks the BYK domain entirely. Using phylogenomics and computational structural approaches, we identify EcpK as a standalone cytoplasmic BY-pseudokinase, lacking key residues required for tyrosine phosphorylation and show experimentally that EcpK is essential for EPS biosynthesis. Using mass-spectrometry based proteomics, bacterial-two-hybrid assays and computational analyses, we show that EcpK directly interacts with EpsV. Based on these findings, we suggest that EcpK functions as a scaffold protein and by direct protein-protein interactions, rather than by Tyr phosphorylation, facilitates EpsV activity. Notably, EcpK and EpsV orthologs are present in other bacteria, suggesting a broad conservation of this novel, non-canonical PCP-2a mechanism.

Prof.

Characterization of luciferase-fluorophore reporters for tracking of single molecules

Single-Molecule Microscopy is a powerful tool for studying molecular dynamics in living cells. Monitoring the kinetics of intracellular processes, single-molecule tracking (smt) grants a deeper understanding of behavioral states of biomolecules as these often correlate with diffusive behavior [1].Crucial for smt is a constant signal and specific labelling. While the latter poses a major problem with conventional dye labeling systems, the use of fluorescent proteins prevents non-specific binding and still achieves sufficient quantum yields [2]. Even more advantageous is the use of luciferases as a marker due to their long signal half-life. However, their quantum yield is typically too low for smt, as smt requires a high temporal resolution in the millisecond range [3].The combination of luciferases and FPs, often referred to as Nano-lanterns, unifies their advantages by exploiting Fluorescence Resonance Energy Transfer (FRET) to enhance the light output of the luciferase [4]. FRET describes the non-radiative energy transfer via resonance from a donor- to acceptor-fluorophore and depends upon the overlap of the emission and excitation spectra of the donor and acceptor molecules, as well as close proximity of the donor and acceptor and their respective orientation.In this work, we present our efforts to characterize different luciferase-fluorophore reporters for single-molecule tracking in living cells. Therefore, we established a stable read-out system employing the monomeric inner membrane protein LacY from E. coli as the biological test system. We chose LacY due to its slow diffusion properties [5], its monomeric structure, and the localization of its two termini on the cytoplasmic side of the membrane [6]. Regulating gene expression by a L-Rhamnose inducible promoter, this low expression system enables laser free high-resolution tracking of low abundant biomolecules. We tested and characterized different constructs in which fluorophores and luciferases were fused to the distinct termini of LacY.[1] DOI: 10.1146/annurev-anchem-091922-073057[2] DOI: 10.1007/s00216-016-9781-8[3] DOI: 10.1021/cb3002478[4] DOI: 10.1016/j.cbpa.2015.05.029[5] DOI: 10.1101/2024.11.05.622141[6] DOI: 10.1146/annurev.biophys.35.040405.102005

Cytosolic factors controlling in vivo phosphorylation of ReoM, an important player in control of Listeria monocytogenes peptidoglycan biosynthesis

Listeria monocytogenes is characterised by a thick cell wall consisting mainly of peptidoglycan (PG). The cell wall counteracts the high internal turgor pressure and ensures viability. To fully understand PG biosynthesis, we are studying a regulatory pathway that controls PG production in L. monocytogenes and related Gr+ bacteria1,2,3. This pathway controls the proteolytic degradation of MurA, the enzyme that catalyses the first committed step of PG biosynthesis. MurA is degraded by the protease ClpCP in the presence of unphosphorylated ReoM. However, when ReoM is phosphorylated by the PASTA-domain containing protein serine/threonine kinase PrkA, the interaction of ReoM with MurA is inhibited and MurA degradation is prevented. The phosphorylation can be reversed by the phosphatase PrpC. In this way, PrkA and PrpC are involved in the decision whether PG biosynthesis is switched on or off. We here have studied the phosphorylation profile of ReoM in living L. monocytogenes cells to clarify under which growth conditions ReoM is phosphorylated and thus PG biosynthesis activated, respectively. Our results show that ReoM is fully phosphorylated under most tested laboratory growth conditions. However, we also found factors that prevented the phosphorylation of ReoM as, for example, ReoM phosphorylation was reduced in a mutant lacking the divisome gene gpsB. Additionally, ReoM phosphorylation decreased in the late stationary growth phase and this decrease was dependent on the presence of the phosphatase PrpC. Furthermore, overexpression of MurA exerted a negative feedback on ReoM phosphorylation and this required a direct contact between ReoM and MurA, as well as MurA enzymatic activity and substrate binding. These findings indicate that MurA and PrkA compete for ReoM as their shared interaction partner and suggest that the involved proteins constitute a safety valve mechanism ensuring controlled degradation of excess MurA to align the MurA amounts and the rate of PG biosynthesis with nutrient supply and growth.1-Wamp et al. (2020). PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM. Elife, 9, e56048.2-Wamp et al. (2022). MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling. PLoS Pathogens, 18(3), e1010406.3-Rothe et al. (2024) Cytosolic factors controlling PASTA kinase-dependent ReoM phosphorylation. Mol Microbiol. 2024 Oct;122(4):514-533.

Novel Cag outer protein interactions of the Helicobacter pylori Cag Type 4 SecretionSystem and direct physical network of outer membrane proteins HopQ and HopZ withT4SS components

Background. Type IV secretion systems, for instance the CagT4SS of Helicobacter pylori, encode membrane-spanning protein complexes in Gram-negative bacteria which can transport various molecules. To elucidate mechanisms and new functionalities, we use the CagT4SS as a model, which is an important virulence factor in the human H. pylori infection [1,2,3]. Recently, structural information on the CagT4SS has been substantially improved by cryo-EM [4]. However, important structural and functional information is lacking, in particular on protein interactions between T4SS outer proteins [5], and of T4SS outer proteins with other proteins,Methods and Results. In the present study, we followed the hypothesis that H. pylori T4SS outer proteins may form an outer protein complex, together with other, non-CagT4SS proteins, which may also be essential for T4SS function under different conditions. Using interaction screens of H. pylori CagT4SS outer proteins, followed by biochemical and functional characterization, we have enhanced here our knowledge on functional protein-protein interactions of the CagT4SS outer proteins. This extends to newly identified physical interactions of outer membrane proteins HopQ and HopZ with CagT4SS proteins. We have further identified and quantitated direct physical interactions of T4SS outer proteins with HopZ and HopQ, which play an important role in T4SS function, and of both HopZ and HopQ with themselves and with host cell factors CEACAM and integrin. Furthermore, we determined an influence of pH on Hop-Cag interactions. Utilizing chromosomal tag insertions in H. pylori, we confirmed surface-exposed co-localization of HopQ with the T4SS in the absence or presence of human gastric epithelial cells using high-resolution microscopy. Functionally opposing roles of HopQ and HopZ for the T4SS were uncovered in pro-inflammatory human cell activation.Conclusions. In summary, we identified novel interactions between H. pylori outer membrane proteins and CagT4SS outer proteins and characterized them as functionally important for membrane transport processes. This will help to refine structural and functional details of the dynamic surface-exposed complex of the CagT4SS. 1. Andrzejewska, J., J Bacteriol 2006; doi: 10.1128/JB.00060-06. 2. Boenig, T., et al., Sci Rep 2016; doi: 10.1038/srep38101. 3. Pham, K.T., et al., Plos One 2012; doi: 10.1371/journal.pone.0035341. 4. Hu, B., et al., 2019; doi: 10.1128/JB.00060-06. 5. Bats, S.H., et al., Int J Med Microbiol 2018; doi: 10.1016/j.ijmm.2018.02.005.

Prokaryotic cell biology 2

Termin

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