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

PD Dr.

Chair

Prof. Dr.

EF-P and its paralog EfpL (YeiP) differentially control translation of proline-containing sequences

Prokaryotic physiology and metabolism

Polyproline sequences are deleterious to cells because they stall ribosomes. In bacteria, EF-P plays an important role in overcoming such polyproline sequence-induced ribosome stalling. Additionally, numerous bacteria possess an EF-P paralog called EfpL (also known as YeiP) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its role in the translational stress response. Through ribosome profiling, we analyze the EfpL arrest motif spectrum and find additional sequences beyond the canonical polyproline motifs that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that EfpL can sense the metabolic state of the cell via lysine acylation. Overall, our work characterizes the role of EfpL in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.

Short lecture

Fundamental quantitative insights into bacterial spore electrophysiology

Cells harness out-of-equilibrium electrochemical gradients to power bioenergetics, homeostasis and signaling. In contrast, electrophysiological features and functions in dormant bacterial spores remain obscure.Here, we introduce and apply a non-invasive combined experimental-theoretical approach that overcomes limitations of classical techniques, thereby enabling fundamental biophysical insights into the electrophysiology of Bacillus subtilis spores. To this end, we monitor individual spore pH dynamics upon microfluidics-controlled environmental fluctuations using genetically encoded fluorescent reporters. Our observations indicate that external pH or salinity changes trigger transitions of internal spore pH to new equilibria within hours to days. Fitting a thermodynamic model of passive proton flux to time series data allowed pH sensor calibration and quantitation of spore pH and voltage changes. Our estimates suggest that ionic environmental changes can alter spore pH and electric potential slowly but substantially.Thus, dormant spores are open passive systems responding and equilibrating to changing ionic environments on faster-than-lifetime timescales. Thereby, a spore's electrophysiological state is dynamic and relies on environmental factors.

Environmental impacts on antibiotic resistance in E. coli strains Nissle, MG1655, and W

Question: Antibiotic resistance is a critical public health issue, with extensive resources devoted to discovering new methods to combat it. Recent studies indicate that antibiotic resistance observed in laboratory conditions may differ significantly from clinical scenarios. Additionally, cross-protection against stressors such as acidity may enhance antibiotic resistance and induce cross-sensitivity. Leveraging this phenomenon could optimize antibiotic treatment strategies to reduce resistance; however, differences between experimental and clinical conditions suggest that the clinical implications of cross-sensitivity warrant further investigation. This study aims to compare the antibiotic resistance profiles of E. coli strains Nissle, MG1655, and W under varied environmental conditions that mimic the human gastrointestinal tract, including acidic environments, oxygen and nutrient limitations. Alongside minimal inhibitory concentration (MIC) determination for different antibiotic classes, we investigate the promoter activity of key stress-response pathways in response to antibiotics. This integrated approach seeks to elucidate the relationship between stress pathway activation, at the single-cell level, and antibiotic efficacy across diverse strains.Methods: MICs for antibiotics - such as ampicillin, chloramphenicol, nalidixic acid, trimethoprim, oxytetracycline, polymyxin B, and norfloxacin - were determined for each strain under various abiotic stress conditions. Additionally, we assessed promoter activity of selected stress-response pathways through fluorescence microscopy to investigate their heterogeneous activation and correlation between stress response and antibiotic resistance.Results: Significant variability in MIC values was observed among strains and conditions. For instance, MG1655 and Nissle exhibited increased resistance to nalidixic acid under low-nutrient conditions, while pre-exposure to acidic conditions elevated resistance to ampicillin in MG1655 and E. coli strain W.Conclusion: This study underscores the distinct antibiotic resistance and stress-response profiles of E. coli strains Nissle, MG1655, and W under diverse environmental conditions. The findings provide insights that may refine the clinical and experimental use of E. coli strains, support targeted antimicrobial strategies, and enhance understanding of bacterial stress responses.

Prof.

Spatial and temporal dynamics of pneumococcal cell wall under host immune response revealed by super-resolution microscopy

Prokaryotic cell biology

Introduction: The cell wall of Streptococcus pneumoniae is a complex, multi-layered structure that surrounds the bacterial cell, providing rigidity, and protection. It also plays a crucial role in host-pathogen interactions by anchoring key proteins involved in immune evasion. The cell wall is composed of a thick peptidoglycan (PG) layer attached to teichoic acids, and lipoteichoic acids. The PG itself is formed of long chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units, which are cross-linked by short peptide chains.Objectives: Understanding the organization and synthesis of the PG layer and its interactions with cell wall anchored proteins is essential for comprehending how the organism is protected against external cues.Methods: To explore the relationship between cell wall protein anchoring and the rate of cell wall synthesis, we examined the spatial and temporal distribution of the surface-exposed pneumococcal protein PspA on the reference strain D39 (WT). The subcellular localization was accessed by super-resolution structured illumination microscopy (SIM). The super-resolution 3D image stacks were processed using HuygensPro and Imaris, whereas the 2D images were analysed using custom-developed workflows written in the graphical image processing language JIPipe. We then compared the PG layer synthesis rates between the WT and a PspA mutant strain (ΔpspA). To spatially distinguish between new and old PG and thusly address the temporal resolution, we sequentially labelled new PG with three various fluorescent D-amino acid (FDAA) probes. Throughout the experiments, we grew bacteria in a regular media and under an immunological challenging condition (i.e., presence of serum).Results: PspA showed a homogenous distribution along the cell surface with the exception of the septal area. Interestingly, when bacteria were exposed to human serum, the PspA relocated to a predominantly mid-cell, septal region. We observed that PG was asymmetrically synthesized. Marked differences occurred in the labelling pattern of ΔpspA when growing in the serum-containing media. There was minimal overlap between old and new PG in the WT strain but this was different in the ΔpspA strain.Conclusions: We observed for the first time a dynamic spatial positioning of pneumococcal proteins during an infection scenario, thus opening new ways to reassess how pathogens exploit their protein machineries to evade the immune system.

Functional characterization of the peptidoglycan recycling pathway in Caulobacter crescentus

Most bacteria possess a peptidoglycan (PG) cell wall that is required to withstand the turgor pressure, maintain cell shape and establish a physical barrier against the environment. The PG layer needs to be constantly remodeled to enable the cells to elongate, grow and divide.During this process, small PG fragments are released into the periplasm. As these fragments can be used as a source for new PG building blocks, many bacteria take great effort in recycling these degradation products. Enterobacteria, such as Escherichia coli, for instance, reuse up to 60 % of released PG fragments. To this end, the turnover products are transported into the cytoplasm and further degraded by a set of PG recycling-specific enzymes, which separate the two sugar moieties and degrade the peptide stem.While E. coli and other Gammaproteobacteria have rather simple shapes, the class of Alphaproteobacteria shows a variety of different cell shapes. In these organisms, correct PG remodeling and therefore also the availability of PG precursors seems to be even more important to maintain the complex cell shapes. On organism belonging to the Alphaproteobacteria is C. crescentus, a crescent-shaped bacterium that is characterized by a biphasic life cycle involving to morphological distinct cell types and asymmetric cell division.Here, we identified that C. crescentus also has a functional PG recycling pathway, which is, although not necessary for general growth, essential to maintain proper cell shape. Additionally, we observed that PG recycling is needed to maintain the natural ß-lactam resistance of C. crescentus. By characterizing the so far unknown PG recycling mechanism in Alphaproteobacteria, identifying its relevance for the cells and understanding the connection between ß-lactam resistance and PG recycling, we aim to gain novel insights which can help uncovering potential treatments against pathogenic Alphaproteobacteria such as Bartonella or Brucella species.

YhaM, a novel player of SOS response?

A reliable response to DNA damage is essential for living organisms to survive 1. The consequences of DNA damage range from reduced fitness to various diseases such as cancer 2,3.Bacillus subtilis (B. subtilis) reacts to DNA damage by invoking the SOS response, which is controlled and directed by the protein RecA and the transcriptional repressor LexA4,5.A gen-operon that has been understudied concerning SOS response contains yhaO-sbcE-yhaM. YhaM (yhaM) has been characterized as a member of a family of 3'-exoribonucleases bearing hydrolytic ribonuclease activity. It is characterized by its unique composition and arrangement of domains. The protein consists of an N-terminal oligonucleotide domain (OB) and a C-terminal metal-dependent phosphohydrolase domain (HD). The involvement of YhaM could be demonstrated for different pathways of RNA degradation 6,7. The specific function of YhaM is not clear 8.Here, we introduce YhaM as a new member of the bacterial SOS response machinery.Structural characterization revealed the formation of a hexamer ring where the individual monomers are assembled in a head-to-tail conformation. The hexamer interface is formed by the phosphohydrolase domain (HD) and the oligonucleotide binding domains decorate the opening of the resulting pore. Our biochemical data, in combination with in vivo experiments, led to a hypothesis where the OB domain acts as a scanner for hydrolysis identifying chromosomal locations activity. We observed that the loss of yhaM leads to a Mitomycin C (MMC) sensitive phenotype. Microscopy analyses showed that YhaM accumulates together with RecA during DNA damage, especially when double-strand breaks are formed by MMC, and has a role in the repair mechanism. This suggests that YhaM has its site of action on DNA, which is further corroborated by the finding that the overproduction of YhaM results in a replication block and highly condensed DNA.Therefore, we introduce YhaM as a new member of bacterial SOS response machinery, based on its structural and catalytic properties.Friedberg, et al. American Society for Microbiology Press (2005)Funchain, et al. Genetics (2000)Tubbs, et al. Cell (2017)Goranov, et al. Journal of bacteriology (2006)Au, et al. Journal of bacteriology (2005)Redko, et al. Journal of bacteriology (2010)Oussenko, et al. Journal of bacteriology (2002)Bechhofer, et al. Critical reviews in biochemistry and molecular biology (2019)

Prokaryotic cell biology & Prokaryotic physiology and metabolism
Cellular responses and stress mechanisms

Termin

Chair

Themen

Programm

Prokaryotic cell biology & Prokaryotic physiology and metabolismCellular responses and stress mechanisms

Termin

Chair

Themen

Programm

Prokaryotic cell biology & Prokaryotic physiology and metabolism
Cellular responses and stress mechanisms