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

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RNA-mediated adaptation to a new chromosome

RNA biology

Approximately 10% of bacterial species possess more than one main chromosome, an architecture named multipartite genome. Such arrangement enables quicker adaptation to new environmental niches. Vibrio cholerae, for example, has two chromosomes: Chr1 and Chr2. While genes encoding for essential cell functions and pathogenicity are located on Chr1, Chr2 encodes genes involved in specialized metabolism and pathogenicity. A novel giant replicon (named hereafter Chr3) has been found in a recent isolate of Vibrio cholerae. Despite being present in various Vibrionaceae, the function of Chr3 remains elusive.To shed light on the impact of Chr3 on its host, we performed transcriptional and post-transcriptional analysis. Comparative transcriptomics showed that Chr3 is conditionally expressed, influencing Chr1 and Chr2 gene expression mostly in the exponential growth phase. Global analysis of RNA duplex formation using RIL-Seq (RNA-interaction-by-ligation-and-sequencing) revealed a vast network of Hfq-assisted interactions between Chr3 and the other two chromosomes. In combination with RIP-Seq (RNA immunoprecipitation and sequencing) analysis, we found 74 sRNAs (small regulatory RNA) encoded on Chr3. Whilst the majority of the newly annotated sRNAs target genes encoded on Chr3, Vcr313 targets genes on primary chromosomes involved in tRNA metabolism. We further show that Chr3 impacts quorum sensing, possesses a functional typeI-F CRISPR-Cas, and contains a more specialized Hfq-like RNA-binding protein. Overall, our data shows ample interactions between all three chromosomes suggesting they are equally involved in post transcriptional gene control. Moreover, the new set of advantageous genes encoded on Chr3 makes it an important entity to expand the adaptation capacities of Vibrio cholerae.

Abstract author

Short lecture

Understanding the link between potassium homeostasis and c-di-AMP signalling in Streptomyces

Signal transduction & gene regulation

QuestionSoil bacteria such as Streptomyces are often exposed to osmotic stress during rainfall and drought and thus have evolved diverse strategies for adaptation. In many Firmicutes, the nucleotide second messenger c-di-AMP controls ion and osmolyte transport to maintain osmotic homeostasis. While the synthesis and degradation of c-di-AMP by the deadenylate cyclase DisA and the phosphodiesterase AtaC, respectively, are well studied, our knowledge about c-di-AMP effectors and functions in these Actinobacteria is very limited. Our goal is to understand the link between c-di-AMP and ion homeostasis, specifically focussing on the Kdp system involving the sensor histidine kinase KdpD, the response regulator KdpE, and the high affinity potassium transporter KdpFABC.Methods Mutagenesis was used to generate a set of mutants carrying deletions in genes encoding the different components of the Kdp system, c-di-AMP metabolism, or a combination of both. The resulting mutants were used to investigate development and resistance towards potassium stress by phenotypical analysis. Complementation experiments with different KdpD variants were performed to further understand the KdpD dependent phenotypes. Using a bacterial-two-hybrid library, screens for novel KdpD interaction partners were initiated.ResultsOur phenotypical analysis revealed that disruption of the Kdp system in S. venezuelae wildtype has no effect on the development or resistance towards potassium stress. However, strikingly, double mutants carrying mutations in kdpD and either in disA or ataC showed a strong delay in development and a growth defect. Interestingly, the observed phenotypes were independent of KdpE and KdpFABC, which suggests that KdpD can act via a non-classical pathway. Complementation studies with different KdpD variants showed that kinase- and phosphatase-inactive versions of KdpD were not able to fully complement the developmental defects, supporting the hypothesis that in Streptomyces KdpD affects the phosphorylation status of a yet unknown interaction partner.ConclusionOur data demonstrate that KdpD is important for Streptomyces development and balanced growth, particularly when c-di-AMP homeostasis is disturbed. Since the observed phenotypes where independent of KdpE and KdpFABC, we hypothesize that KdpD controls the activity of response regulators other than KdpE and aim at identification of novel intraction partners of the histidine kinase.

The role of polyphosphate in Pseudomonas aeruginosa mediated killing of Staphylococcus aureus

Pseudomonas aeruginosa and Staphylococcus aureus have frequently been co-isolated from patients with cystic fibrosis or burn/chronic wounds, fueling the interest in studying the mechanistic details of this interaction. It was reported that both pathogens generate molecules that modulate their interaction with significant impact on disease outcomes. P. aeruginosa outcompetes S. aureus in vitro and in vivo, which is mediated by several P. aeruginosa virulence factors. We recently discovered that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role for the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. PolyP is composed of long chains of phosphate (Pi) molecules linked via phosphoanhydrid bonds, is found in all three domains of life but only in bacteria have the enzymes of polyP metabolism been well studied. Microbial polyP synthesis is catalyzed by polyP kinase (Ppk) while polyP is degraded by exopolyphosphatase (Ppx) into Pi molecules. Bacteria lacking Ppk (i.e. ∆polyP) are defective in virulence, biofilm formation, and oxidative stress response. We found that P. aeruginosa ∆polyP cells are less detrimental to S. aureus growth and survival while the Gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high levels of polyP. Moreover, P. aeruginosa polyP production is stimulated by S. aureus through a heat-sensitive secretion product. The polyP-dependent phenotype of P. aeruginosa-mediated killing of S. aureus could at least in part be direct, as we detected polyP in the spent media, which causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect. We show that P. aeruginosa pyocyanin production is regulated by polyP on the transcriptional level, which harms S. aureus through membrane damage and potentially the generation of reactive oxygen species, resulting in the increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the Gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources. A more in-depth understanding of how polyP influences interspecies interactions is critical, as targeting polyP synthesis in bacteria such as P. aeruginosa may have a significant impact on other microorganisms and potentially result in dynamic changes in the microbial composition.

PII signaling superfamily: a new paradigm for controlling cellular metabolism

Because of their photosynthesis-dependent lifestyle, cyanobacteria evolved sophisticated regulatory mechanisms to adapt to oscillating day-night metabolic changes, which is very crucial for their multicellularity lifestyle. How they coordinate the metabolic switch between autotrophic and heterotrophic metabolism in light/dark cycles is poorly understood. Members of the PII signaling superfamily adeptly monitor and synchronize the cell's carbon, nitrogen, energy, redox, and diurnal states, primarily by binding interdependently to adenyl-nucleotides, including charged nucleotides (ATP, ADP, and AMP) and second messengers such as cAMP, and c-di-AMP. These proteins also undergo a variety of posttranslational modifications, such as phosphorylation, adenylation, uridylation, carboxylation, and disulfide bond formation, which further provide cues on the metabolic state of the cell. Serving as precise metabolic sensors, PII superfamily proteins transmit this information to diverse cellular targets, establishing dynamic regulatory assemblies that fine-tune cellular homeostasis. This highlights PII signaling superfamily as a central switch-point in cyanobacterial cell physiology.

New insights in the transcriptional regulation of the Type IVa pilus machine of Myxococcus xanthus

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for a variety of processes including surface motility. T4aP-dependent motility depends on cycles of T4aP extension, surface adhesion, and retraction. Because the T4aP adheres strongly to the surfaces, cells are pulled forward during retractions.T4aP-dependent motility is one of two distinct motility systems, which are used by Myxococcus xanthus to move across surfaces forming spreading, predatory colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. T4aP of M. xanthus are composed of thousands of copies of the major pilin PilA and tipped by a complex composed of four minor pilins and the PilY1 adhesins. Interestingly, M. xanthus encodes three sets of minor pilins and PilY1 adhesins, and based on high confidence AlphaFold-Multimer model of these tip complexes, the different N-terminal domains of the PilY1 proteins, make the ultimate tip. Thus, M. xanthus has the potential to assemble T4aP with three different tip complexes involved in adhesion.Based on RNAseq analyses, cluster_1 and cluster_3 genes are expressed in presence of nutrients, while cluster_2 genes are not expressed. By contrast, during starvation on a surface, cluster_2 genes are expressed, expression of cluster_3 genes increases, while expression of cluster_1 genes decreases. These transcriptomic data indicate that expression of the three clusters is regulated in response to nutrients and/or surface attachment. This is in agreement with whole cell proteomics data, which show that (i) cells grown on an agar surface accumulate predominantly cluster_3 proteins, (ii) T4aP prepared from cells grown on an agar surface are predominantly composed of PilA and cluster_3 proteins, while (iii) cells grown in suspension accumulate similar amounts of cluster_1 and cluster_3 proteins.We asked how the expression of the corresponding genes is regulated. To this end, we focused on the NtrC-like transcriptional regulators PilR and PilR2, which are essential for T4aP-formation and T4aP-dependent motility. Our whole-cell proteomics data suggest that PilR is important for expression of pilA while PilR2 is important for expression of cluster_1 & _3 genes. ChIP-qPCR experiments confirmed a specific enrichment of PilR-FLAG at the pilA-promoter and of PilR2-FLAG at the promoter of cluster_1 and cluster_3. Using ChIP-Seq and RNA-Seq we now aim to explore their regulons in more detail.

Conformational dynamics of the cytoplasmic acid-stress sensor AdiY of Escherichia coli

The arginine-dependent (Adi) system is a critical component of Escherichia coli's enzyme-based acid resistance mechanisms, enabling the bacterium to survive in highly acidic environments such as the human digestive tract¹. This system consists of the arginine decarboxylase AdiA and the arginine/agmatine antiporter AdiC, which work in tandem to combat intra- and extracellular acidification². Central to the precise regulation of this system is AdiY, a transcriptional activator belonging to the AraC/XylS family. AdiY controls the expression of the adiA and adiC genes in response to acidic conditions, particularly during anaerobic growth³. In this study, we used an in-silico approach to identify a set of amino acids within the C-terminal DNA-binding domain of AdiY that appear to play a key role in regulating its activity. We have tested the activation profile of a series of AdiY variants with amino acid replacements at these positions. Various biochemical techniques were used, including the measurement of in vitro DNA-binding affinity by Surface Plasmon Resonance (SPR) spectroscopy and the assessment of promoter activation in vivo. Our observations revealed that wild-type AdiY exhibits increased DNA binding affinity to its target promoters at acidic pH (5.8 to 6.0), which is attributed to pH-dependent conformational changes. Conversely, AdiY is unable to bind its target promoters at physiological pH. These findings provide new insights into the functional dynamics of AdiY as a soluble cytoplasmic pH sensor and highlight its importance in the cellular response to intracellular pH fluctuations. Schwarz, J., Schumacher, K., Brameyer, S. & Jung, K. Bacterial battle against acidity. FEMS Microbiol. Rev. 46, 1–27 (2022).Schumacher, K., Brameyer, S. & Jung, K. Bacterial acid stress response: from cellular changes to antibiotic tolerance and phenotypic heterogeneity. Curr. Opin. Microbiol. 75, 102367 (2023).Stim-Herndon, K. P., Flores, T. M. & Bennett, G. N. Molecular characterization of adiY, a regulatory gene which affects expression of the biodegradative acid-induced arginine decarboxylase gene (adiA) of Escherichia coli. Microbiology 142, 1311–1320 (1996).

RNA biology & gene regulation

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