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

Prof. Dr.

The role of FimV in cellular processes in Pseudomonas putida

Prokaryotic cell biology

It is important for bacteria to be able to precisely coordinate intracellular processes such as the placement of motility structures, e.g. the flagellum or the pilus machinery. Therefore, polar landmark proteins such as HubP or FimV are widely distributed in different bacterial species and help them to control the spatiotemporal regulation of polar regions within the bacterial cell. While the HubP proteins of Vibrio or Shewanella are comparably well characterized and their involvement in various essential processes such as chromosome segregation or the correct placement of the chemotaxis machinery has been shown, the role of FimV in Pseudomonas putida remained unclear. To characterize the role of FimV in P. putida, we examined FimV with respect to cellular processes known to involve FimV or HubP in other species. Motility assays showed that deletion of FimV significantly reduces the spreading behavior of the bacteria, while it does not alter the flagellation pattern of the bacteria. We could also show that FimV is not essential for the correct placement of the chemotaxis system, whereas deletion of the polar marker FlhF delocalizes the chemotaxis protein CheA from the cell pole. Replacement of the entire HubP/FimV protein from Shewanella putrefaciens and Pseudomonas putida, respectively, showed that the spreading behavior is phenotypically similar to a deletion mutant. In contrast, the introduction of the C-terminal FimV domain of Pseudomonas into Shewanella HubP led to a restriction of the spreading behavior of Shewanella, whereas the introduction of the FimV domain of Shewanella into Pseudomonas slightly improved the spreading of the Pseudomonas bacteria. Taken together, the results show that the polar landmark proteins HubP and FimV are not necessarily functional orthologs but that their function is highly species-specific.

Poster

Chromosome maintenance in Escherichia coli – the role of SeqA in mismatch repair

To achieve faithful transfer of the genetic information from one generation to the next, bacteria have evolved different chromosome maintenance systems. In Escherichia coli two such chromosome maintenance systems are based on the DNA sequence motif GATC, which is methylated at the N6 position of the adenines by the DNA adenine methyltransferase (Dam). During DNA replication, unmodified nucleotides are incorporated into the newly synthesized DNA strand, which results in a hemi-methylation of the GATC motifs for a short period of time after the passing of the replication fork. These hemi-methylated GATC motifs are targeted by MutH, a component of the mismatch repair system in E. coli, in order to enable efficient mismatch repair. Hemi-methylated GATCs are further targeted by SeqA, which sequesters the replication origin and is thought to act in chromosome organization and segregation. These chromosome maintenance systems have been studied individually for many years, but little is known about their interplay. As SeqA and MutH target the same DNA motif, the question arises how these factors functionally interact with one another.We designed several experimental approaches to analyze the functional interaction between MutH and SeqA in E. coli, by investigating the efficiency of mismatch repair in presence and absence of SeqA. In addition to deletion mutants we designed and constructed synthetic secondary chromosomes. Based on the arrangement of GATC sites on these replicons, the binding of only MutH or both MutH and SeqA or none of these proteins is expected. The respective mutation rate should allow to assess the role of SeqA in mismatch repair. First mutation accumulation assays and next generation sequencing experiments showed that the mutation rates in a seqA deletion mutant are slightly increased compared to the wildtype. To support these results, further mutation accumulation assays are performed analyzing the synthetic secondary chromosomes.

Protein-protein interactions at the LapB interface, a central player in cell envelope synthesis

The asymmetric outer membrane (OM) of gram-negative bacteria consists of phospholipids (PL) as well as lipopolysaccharides (LPS), with LPS exclusively located in the outer leaflet. LPS levels in Escherichia coli are strictly controlled by post-translational regulation of LpxC, the key enzyme of LPS synthesis. LpxC is degraded by the membrane-bound protease FtsH. This process is regulated by the adaptor protein LapB (YciM), which binds LpxC and directs it towards FtsH. Further, based on periplasmic LPS levels, the activity of LapB itself is controlled by the anti-adaptor protein YejM. Our previous studies demonstrated that LapB interacts with other proteins in the lipopolysaccharide synthesis pathway, namely LpxA and LpxD (Möller et al., 2023). In addition, we found that LapB binds FabZ, the first enzyme in the phospholipid synthesis (PL) pathway. Since the proper balance between LPS and PL is mandatory for bacterial survival, LapB is believed to be a central player in cell envelope synthesis by serving as an anchor for the involved cytosolic enzymes. We now investigate, how the interaction between those proteins is navigated.We study the formation of protein complexes with purified LapB and its interaction partners to understand if the protein-protein interactions of LapB can occur simultaneously. Therefore, we use different in vitro approaches, such as size exclusion chromatography, blue native PAGE and pull downs. In addition, we employ targeted mutagenesis to investigate the interaction sites where LapB binds to its interaction partners, since it harbors nine tetratricopeptide repeat motifs (TPR) in its cytosolic domain. The results will provide insights into how LapB coordinates the assembly of a dynamic protein complex to orchestrate cell envelope biogenesis.

Influence of an SPFH protein on developmental programs in Bacillus subtilis

In its natural environment Bacillus subtilis is often exposed to life-threatening and fluctuating conditions. To respond to these environmental changes B. subtilis use alternative σ-factors which activate gene clusters to help to adapt to the given conditions. Perceiving these environmental changes is often facilitated by membrane-associated proteins. One notable protein complex is the PspA-YdjGHI complex, which encodes the SPFH (Stomatin/Prohibitin/Flotillin/HflK-C) protein, YdjI (1). Members of the widely conserved SPFH family are associated with regulation of membrane fluidity (2,3) and are known for their impact on a variety of membrane bound processes, including developmental processes in B. subtilis (4,5). Here, we show that YdjI is required for efficient motility and development of social communities. Absence of ydjI in a biofilm leads to changes in metabolism associated genes clusters, indicating that cells within the biofilm are starving. This results in a decreased secretion of BslA, one of the major matrix genes, preventing the formation of the hydrophobic coat and rendering the biofilm surface hydrophilic. Interestingly, phenotypic complementation is only achieved in a background strain that renders YdjI cytosolic suggesting that YdjI has dual functions depending on its subcellular localisation.(1) Savietto Scholz, A., et al. (2021) Front. Microbiol. 12: 754924(2) Bach, J.N. et al. (2013) Mol. Micro. 88:1205-1217(3) Zielińska, A., et al. (2020) Elife 9:e57179(4) Yepes A, et al. (2012) Mol Microbiol. 86(2):457-471(5) Dempwolff F, et al. (2012) J Bacteriol. 194(17):4652-61

Prof.

The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression

Previous studies on the transcriptional antisilencer VirB from S. flexneri have shown that it activates virulence gene expression by counteracting the nucleoid-organizing protein H-NS in a virS-dependent manner. The loading and spreading on the virS site lead to a local decrease in negative supercoiling, thereby destabilizing the H-NS complexes to activate gene expression. However, the underlying process remains unclear. In contrast to conventional transcriptional activators, VirB does not directly interact with the RNA polymerase (RNAP). VirB belongs to a novel class of molecular switches, which act as DNA sliding clamps that use cytidine triphosphate (CTP) binding and hydrolysis to control their opening and closing state. The newly discovered CTPase activity of ParB that allows its binding and sliding along the DNA raises the question of whether VirB uses a similar mode of action. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. CTP-binding mutations inhibit VirB loading in vitro, and abolish the formation of VirB nucleoprotein complexes as well as virulence gene expression in vivo. Therefore, VirB functions as a CTP-dependent molecular switch that regulates transcription through a loading-and-sliding mechanism during bacterial pathogenesis.

Prof.  Dr.

Unraveling the role of a new input receiver in a complex cyanobacterial circadian clock

Circadian rhythms are biological processes that synchronize an organism"s physiology and behavior with the 24-hour day-night cycle. The primary model organism for procaryotic circadian research is Synechococcus elongatus PCC 7942 (Synechococcus). At the core of this unicellular freshwater cyanobacterium"s timekeeping mechanism is a simple posttranslational oscillator consisting of the proteins KaiA, KaiB and KaiC. The clock of Synechocystis sp. PCC 6803 (Synechocystis), another unicellular freshwater cyanobacterium, is more complex (Köbler et al. 2024). Due to its genetic tractability, phototrophic lifestyle, and ability to grow hetero-/mixotrophically, Synechocystis is a model organism for synthetic biology, biotechnology and photosynthetic research. However, its circadian clock is less characterized than that of Synechococcus. While Synechocystis exhibits the core clock proteins KaiA1, KaiB1 and KaiC1, our recent discovery of the chimeric KaiA3 revealed a second fully functional oscillator consisting of KaiA3, KaiB3 and KaiC3. In both oscillators, KaiA stimulates the phosphorylation of KaiC while KaiB sequesters KaiA and leads to the dephosphorylation of KaiC. Additionally, these two oscillators are interconnected as KaiC1 interacts with KaiB3 and KaiC3 with KaiB1 (Wiegard et al. 2020). Both oscillators are required for the maintenance of the circadian rhythm and for the switch between phototrophic and heterotrophic growth. However, little is known about the input and output factors that integrate environmental signals and relay the status of the clock to downstream processes such as gene expression. Based on pull-down assays and bioinformatic analysis, we found two candidate kinases that could interact with the response receiver domain of KaiA3 and act as putative input factors for the clock. We are further investigating the interaction of these kinases with KaiA3 using yeast two-hybrid assays. Additionally, we measure the circadian backscatter oscillations of knockout mutants of these kinases to observe their effect on the circadian rhythm of Synechocystis. Our aim is to shed light on the complex circadian clock of Synechocystis, including its input factors, that leads to the metabolic flexibility of this model organism.

Morphological characterisation of star-shaped bacteria from the genus Stella

The physiological and morphological diversity of bacteria is enormous, ranging from simple rods or cocci to extraordinary asymmetrical cell shapes. In the second half of the last century, Heinz Schlesner (Kiel University) isolated over 500 strains based on their unusual morphology from various habitats and deposited them in his personal bacterial strain collection. From this collection, we investigated 17 strains, which are potential members of the genus Stella within the Alphaproteobacteria. Members of this genus are described to form unusually shaped cells, which resemble flat, six-pointed stars (Vasilyeva, 1970). In order to understand their peculiar cell structure and morphology better, we examined the cells employing multiple light microscopy techniques. Furthermore, the phylogenetic relationships of Stella spp. were analysed to complement the microscopic examination. In addition to their exceptional morphology, strains belonging to the genus Stella have been reported earlier to change their shape in response to the nutrient compositions of the surrounding media and other environmental factors (Vasilyeva et al., 1974). Therefore, we investigated the influence of different factors on their cell shape and growth.Vasilyeva, L. V. 1970. 'Звездообразный почвенный микроорганизм', Известия Академии Наук СССР Серия биологическая: 308-10.Vasilyeva, L. V., Lafitskaya, T. N., Aleksandrushkina, N. I., and Krasilnikova, E. N. 1974. 'Физиолого-биохимиеские особенности простекобактерий Stella humosa и Prosthecomicrobium sp.', Известия Академии Наук СССР Серия биологическая: 699-714.

Investigation of minor pilin functions in the cyanobacterium Synechocystis sp. PCC 6803

Type IV pili (TFP) are flexible and versatile fibers on the surface of many gram-negative bacteria, primarily composed of multiple copies of protein subunits called major pilins. In addition to the cyanobacterial major pilin PilA1, Synechocystis sp. PCC 6803 encodes less abundant pilin-like proteins, known as minor pilins, which are crucial for pilus assembly and facilitate various functions, including natural competence, aggregation, adhesion, and motility.Recent studies have highlighted the importance of different transcriptional units of minor pilins in distinct pilus functions. TU2300, which encodes PilA5 and PilA6, is essential for natural transformation but is dispensable for motility and flocculation. Similarly, we demonstrated that TU2301, which encodes PilA7 and PilA8, is crucial for natural transformation, but not for motility and flocculation. TU763, encoding PilA9-PilA12, is essential for motility and flocculation but dispensable for natural transformation. Investigations of the minor pilins PilX1, PilX2, and PilX3 indicate their importance for flocculation.The absence of extracellular PilA1 in mutant strains lacking specific sets of minor pilins suggests that TFP assembly depends on the presence of either PilA5-8 or PilA9-12. We hypothesized that two different priming complexes are assembled, forming the tip of a pilus fiber with distinct functions. Here, we provide mass spectrometry (MS) data for sheared pili fractions and co-immunoprecipitation of specific minor pilins to elucidate the composition of these distinct pilus subtypes.Furthermore, we visualized spatiotemporal dynamics of pilus subtypes by confocal microscopy employing a PilA1(T77C) cysteine mutant that can be stained with Alexa Fluor 488. The combination of this PilA1-variant with the deletion of minor pilin genes allowed us to detect phenotypic differences in cell surface piliation. Preliminary data showed long, less dynamic pilus fibers in pilA9-12 mutants, whereas pilA5-6 mutants exhibited short, dynamic fibers. The machine learning algorithm ILASTIK was used to automatically detect pili and analyze the pilus subtypes of knock-out mutants. We present a rapid and standardized method for analyzing the microscopic images of cells decorated with TFP.

Prokaryotic cell biology 1

Termin

Programm