7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Prof. Dr.

Long as I can see the light – Control of cyanobacterial phototaxis orientation by the Tax1 system

Microbial Cell Biology 

In order to seek out optimal photosynthetic conditions, cyanobacteria exhibit phototaxis, navigating either towards or away from a light source via polar activation of type IV pili (T4P). The Tax1 system is a chemotaxis-like signal transduction pathway that governs a switch in cell polarity, which is crucial for positive phototaxis in Synechocystis sp. PCC 6803. The system consists of a blue/green light sensor, PixJ, which controls the histidine kinase PixL and two CheY-like response regulators, PixG and PixH. However, the molecular mechanisms by which Tax1 regulates polar pilus assembly and thereby phototactic orientation are poorly understood. Here, we investigated the phosphotransfer between PixL and its cognate response regulators in vitro and analyzed the localization and function of wild-type and phosphorylation-deficient PixG and PixH during phototactic motility. We demonstrated that both PixG and PixH are phosphorylated by PixL but have different roles in the regulation of phototaxis. Only phosphorylated PixG interacts with the T4P motor protein PilB1 and localizes to the leading cell pole under directional light, thereby promoting positive phototaxis. In contrast, PixH acts as a phosphate sink, reducing PixG phosphorylation and inhibiting positive phototaxis. In addition, we showed that the C-terminal receiver domain of PixL is essential for positive phototaxis, and modulates the kinase activity of PixL. Our findings reveal the molecular basis of positive phototaxis regulation by the Tax1 system and provide insights into the division of labor between PatA-type and CheY-like response regulators in cyanobacterial chemotaxis-like systems. Furthermore, these findings highlight similarities in the regulation of movement direction during twitching motility in phototactic and chemotactic bacteria.

Oral Presentation

PD Dr.

Insights into the Flagellar Motility System in Magnetotactic Bacteria

Magnetotactic bacteria (MTB) possess the remarkable ability to navigate by utilizing the geomagnetic field. This magnetic navigation arises from a combination of passive magnetic alignment, mediated by intracellular chains of magnetic organelles (magnetosomes), and active motility driven by flagella, associated with chemotactic responses, particularly aerotaxis. Magneto-aerotaxis is believed to facilitate orientation within aquatic habitats towards preferred growth-favoring oxygen concentrations. Despite MTB exhibiting motility patterns that are highly distinct from well-studied non-magnetotactic model bacteria (including a directionally biased North- or South-seeking motility towards the poles of the geomagnetic field), the underlying molecular mechanisms have remained largely unstudied. To gain insights into these mechanisms, I implemented high-speed live-cell imaging of fluorescently labeled flagella in the genetically tractable alphaproteobacterium Magnetospirillum gryphiswaldense, initially reported to possess one flagellum at each of its cell poles. Here, I propose that flagellation in M. gryphiswaldense is not as strictly bipolar as reported previously. Moreover, I will demonstrate how different configurations of the flagella control the movement and directional reversals of M. gryphiswaldense, including the wrapping and pushing of individual flagella. In the near future, visualizing rotating flagella in M. gryphiswaldense will facilitate further investigations into the complex motility behavior in MTB and allow us to unravel the interplay between aerotactic sensing, polar flagellation, and directionally biased motility in magnetic fields. Additionally, a comprehensive understanding of the biophysical aspects governing flagellar motility in magnetospirilla could potentially find application in the construction of bio-inspired microrobots guided and steered by magnetic forces.

Factors controlling in vivo phosphorylation of ReoM, an important player in control of peptidoglycan biosynthesis

Listeria monocytogenes, a Gr+ bacterium, 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 and identify potential pharmaceutical targets, we are studying a regulatory pathway that controls PG production in L. monocytogenes and related Gr+ bacteria1,2. 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. Artificial 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.1Wamp et al. (2020) Elife, 9, e560482Wamp et al. (2022) PLoS Pathogens, 18(3), e1010406

Cyanobacterial CurT proteins are involved in cell division.

Cyanobacteria are in large characterized by their capacity for aerobic photolithoautotrophic growth. The latter is driven by oxygenic photosynthesis whose molecular machinery resides within a specialized intracellular membrane system termed &quot;thylakoid&quot; in all but the most early-branching cyanobacterial species. Thylakoid membranes have undergone severe morphological changes during cyanobacterial evolution and the transition into endosymbiotic plastids. Here, the CurT/CURT1-like transmembrane proteins constitute a key determinant of thylakoid architecture in both cyanobacteria and the green lineage of algae and land plants. The evolutionary origin of this protein family, however, remains highly elusive, with the appearance of &quot;fully-formed&quot; CurT-like proteins roughly coinciding with the evolution of thylakoids themselves.Microscopic analyses reveal a number of hitherto undescribed defects is cell division and thylakoid partitioning in curT-depleted mutants of Synechocystis sp. PCC 6803 which may provide the first relevant cue towards the origin of these fascinating proteins. CurT-depleted Synechocystis sp. PCC 6803 cells show impaired Z-ring formation, while a filamentous phenotype can be observed in Synechococcus sp. PCC 7942 curT mutants, all of which suggests an (original) involvement of CurT in cell division. Through phylogenetic analyses we identified a candidate for a distant sister clade to cyanobacterial/plastid CurT/CURT1 within the inherently thylakoid-less Firmicutes phylum. With Firmicutes and cyanobacteria being closely related phyla with overlapping cell division features, CurT/CURT1-like proteins having been originally obtained through horizontal gene transfer and stemming from a non-photosynthetic context becomes conceivable. Initially aiming at unravelling a tentative secondary function of CurT/CURT1 in cyanobacteria, we are now confident to have uncovered the evolutionary origins of these prominent photosynthesis-associated proteins in Gram-positive-like cell division.

Membrane binding triggers MinD ATPase activity in Bacillus subtilis

Bacterial cell division relies on a complex protein network, and since most rod-shaped bacteria divide at midcell, correct placement of the division apparatus is crucial. One of the most studied division-site selection systems is the Min system, best understood in Escherichia coli, where defects lead to aberrant cell division and thus minicells. The active component MinC is localized through MinD, a Walker-type ATPase that dimerizes and interacts with the membrane upon ATP binding. The third protein MinE stimulates MinD ATPase activity, breaking the dimer and releasing it from the membrane, leading to a pole-to-pole oscillation. In contrast, Bacillus subtilis localizes MinD through MinJ and DivIVA to regions of negative curvature without oscillation.Previously, we described a role of the Min system in B. subtilis in divisome disassembly (1) and high MinD dynamics to allow translocation to new septa (2). Here, we aimed to investigate how MinD membrane interaction is controlled and how ATPase activity is triggered in B. subtilis.We employed in-vitro and in-vivo methods, including biochemical analysis of purified MinD and ATPase mutants as well as single-particle tracking (SPT) of MinD fusions.Our findings suggest that MinD ATPase activity is solely stimulated by lipid-membrane interaction, while a membrane-binding mutant (I260E) is inactive. Bio-layer interferometry (BLI) with immobilized liposomes confirmed binding of monomeric MinD mutants (G12V, K16A) independent of ATP or dimerization, while a trapped-dimer mutant (D40A) showed reduced dissociation. SPT affirmed these findings, indicating similar shifts in mobility and dwelling times.In summary, these results support the hypothesis that frequent MinD membrane association and rapid dissociation upon dimerization generates a sharp protein gradient in B. subtilis, without the requirement for a functional MinE homolog. Quick cycling of MinD in dense areas aided by local MinJ recruitment establishes a dynamic gradient, ensuring correct Min component localization throughout the cell cycle.van Baarle S, Bramkamp M. 2010. PLoS One 5:e9850.Feddersen H, Wurthner L, Frey E, Bramkamp M. 2021. mBio 12.

Biogenesis of magnetic organelles

The biogenesis of membranous cell organelles relies on extensive membrane remodeling and precise spatio-temporal targeting of proteins. While these processes are well characterized in eukaryotes, their molecular-level understanding in prokaryotes, which typically form organelles of lower complexity, still remains limited.Here we analyzed the biogenesis of magnetosomes, membrane-enclosed magnetite nanoparticles used for magnetic navigation, as a model system for prokaryotic organelle formation. Using phenotypic assessments, proteome profiling, protein interaction studies, and fluorescence microscopy in Magnetospirillum gryphiswaldense, we identified three so-far uncharacterized proteins of the HOTT protein family that are crucial for the late stages of magnetosome membrane biogenesis. Since we also identified a remote homology between the HOTT protein family and chloroplast protein translocation systems, our findings suggest a novel mechanism of organelle biogenesis in prokaryotes. Moreover, analyses of subcellular localization patterns of GFP-labelled HOTT proteins in different mutant backgrounds are not consistent with the currently accepted model of magnetosome biogenesis. Thus, we propose a new spatiotemporal model for magnetosome biogenesis.

Oral Session
Microbial Cell Biology

Termin

Chair

Thema

Programm

Oral SessionMicrobial Cell Biology

Termin

Chair

Thema

Programm

Oral Session
Microbial Cell Biology