Biology subjects

Geiser, J.

Publications and source records attributed to Geiser, J..

7 recordsLinked to original sources

bioRxiv2026

RNase E resolves toxic condensates by counteracting phase separation of Type II RhlB helicases

In many Proteobacteria, the RNA helicase RhlB is a component of the RNA degradosome, a multi-protein complex involved in RNA processing and degradation. Within this complex, RhlB interacts with the scaffold endoribonuclease RNase E. In Escherichia coli, allosteric activation of RhlB by RNase E has defined the current paradigm for RhlB regulation. Here we identify a distinct clade of RhlB helicases, exemplified by Pseudomonas aeruginosa RhlB, which we designate Type II. Unlike Type I RhlB, Type II RhlB helicases contain an N-terminal intrinsically disordered region that drives RNA-dependent liquid-liquid phase separation and enhances RhlB activity. Biochemical, structural, and functional analyses show that P. aeruginosa RNase E binds RhlB through an interface distinct from that described in the E. coli model and, rather than stimulating activity, antagonizes RhlB phase separation. Excessive RhlB condensation impairs bacterial growth at low temperature, and RNase E-mediated control of RhlB condensation maintains growth under these conditions. Together, these findings reveal that conserved RNA degradosome components can engage in distinct regulatory interactions across species and identify condensate dissolution as a novel mechanism regulating RNA helicase activity.

microbiology↗

bioRxiv2026

Intrinsic features of the RNase E membrane targeting sequence specify RNA degradosome organisation and activity

In bacteria, transcription and RNA degradation are physically separated via segregation of the main ribonucleolytic machinery - the RNA degradosome - into phase-separated or membrane-anchored molecular assemblies driven by RNase E. Despite the widespread conservation of an amphipathic membrane anchor (MTS) in RNase E, the regulatory information embedded within this sequence and its biological importance remain poorly understood. Here, we have studied the importance of the Pseudomonas aeruginosa RNase E MTS for bacterial fitness or virulence and assessed its interchangeability. We show that amphipathicity is dispensable for foci scaffolding but necessary for proper foci morphology, dynamics, and localisation, although sequence modulates foci behaviour. Loss of the MTS additionally causes a drastic sensitivity to high salinity and a consistent virulence defect in Galleria mellonella larvae. Moreover, transcriptomics and analysis of mRNA spatial organisation reveal that the MTS mutant has specific stabilisation of localised membrane protein-encoding transcripts, together with abnormal operon processing. Altogether, our study highlights the elegant MTS-mediated control of spatial organisation and target selection, shaping the transcriptome and bacterial stress response.

microbiology↗

bioRxiv2024

Critical functions and key interactions mediated by the RNase E scaffolding domain in Pseudomonas aeruginosa

The RNA degradosome is a bacterial multi-protein complex mediating mRNA processing and degradation. In Pseudomonadota, this complex assembles on the C-terminal domain (CTD) of RNase E through short linear motifs (SLiMs) that determine its composition and functionality. In the human pathogen Pseudomonas aeruginosa, the RNase E CTD exhibits limited similarity to that of model organisms, impeding our understanding of RNA metabolic processes in this bacterium. Our study systematically maps the interactions mediated by the P. aeruginosa RNase E CTD and highlights its critical role in transcript regulation and cellular functions. We identified the SLiMs crucial for membrane attachment, RNA binding and complex clustering, as well as for direct binding to the core components PNPase and RhlB. Transcriptome analyses of RNase E CTD mutants revealed altered expression of genes involved in quorum sensing, type III secretion, and amino acid metabolism. Additionally, we show that the mutants are impaired in cold adaptation, pH response, and virulence in an infection model. Overall, this work establishes the essential role of the RNA degradosome in driving bacterial adaptability and pathogenicity. Author summaryBacteria must rapidly adapt to changing environments, whether facing temperature shifts, nutrient scarcity, or antibiotic exposure. A key mechanism enabling this adaptability is the regulation of mRNA levels--the molecular blueprints for protein production. This process is governed by the RNA degradosome, a multi-protein complex that processes and degrades RNA to control gene expression. Although the RNA degradosome core function is conserved across bacteria, its composition and organization differ significantly between species, reflecting diverse lifestyles and environmental challenges each bacterium encounter. In this study, we investigated the RNA degradosome in Pseudomonas aeruginosa, a bacterial pathogen causing difficult-to-treat infections in humans. We identified its components and mapped regions within the complex essential for RNA binding, membrane attachment, and spatial organization. Disrupting these regions compromised P. aeruginosa ability to survive in cold conditions, respond to stress, and establish an infection. Through this work, we uncovered unique features of the P. aeruginosa RNA degradosome that distinguish it from those of other species, emphasizing the RNA degradosome critical role in bacterial adaptability and highlight it as a promising target for therapies against P. aeruginosa infections.

microbiology↗

bioRxiv2024

Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria

RNA helicases--central enzymes in RNA metabolism-- often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

molecular biology↗

bioRxiv2022

KaiC-like proteins improve stress resistance in environmental Pseudomonas species

KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria that are not known to have or need a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC-like proteins found in environmental Pseudomonas species. Using a bioinformatic approach, we describe the diversity and distribution of members of this protein family in the Pseudomonas genus and sketch, through comparative genomics, a conserved minimal interaction network comprising a histidine kinase and a response regulator. We then characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in sulfur uptake and alternative carbon source utilization in P. protegens. As expected, it physically interacts with its cognate histidine kinase and response regulator. Moreover, KaiC homologs are phosphorylated at one (P. putida) or two (P. protegens) sites and KaiC phosphorylation patterns change over time; however, in Pseudomonas species, changes in KaiC phosphorylation are driven by the age of the culture rather than by circadian cues as is the case in Cyanobacteria. In this study, through thorough bioinformatic and experimental analyses, we shed light onto the functional diversification and evolution of a unique protein family, diversely involved in bacterial rhythmic interactions with their environment. By so doing, we present a striking example of a protein whose general purpose is conserved in evolution, but whose molecular mechanics and participation in bacterial physiology can change dramatically across species.

microbiology↗

bioRxiv2021

The DEAD-box RNA helicases RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis

The RhlE DEAD-box RNA helicase protein family is widespread among Proteobacteria, but it is the least understood due to the lack of a clear biological function. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. RhlE1 and RhlE2 diverged during P. aeruginosa evolution; our data indicate that this resulted in a non-redundant biological role, a distinct molecular action and an enzymatic activity differentially stimulated by RNA. Whereas RhlE1 is specifically necessary for bacteria growth in cold, we show that RhlE2 acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts and multiple functionalities indispensable not only for P. aeruginosa environmental adaptation, but also for its virulence. The global action of RhlE2 relies on a unique C-terminal extension, which establishes an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery.

microbiology↗

bioRxiv2019

Encoding information in synthetic metabolomes

Biomolecular information systems offer numerous potential advantages over conventional semiconductor technologies. Downstream from DNA, the metabolome is an information-rich molecular system with diverse chemical dimensions which could be harnessed for information storage and processing. As a proof of principle of postgenomic data storage, here we demonstrate a workflow for representing abstract data in synthetic metabolomes. Our approach leverages robotic liquid handling for writing digital information into chemical mixtures, and mass spectrometry for extracting the data. We present several kilobyte-scale image datasets stored in synthetic metabolomes, which are decoded with accuracy exceeding 98-99% using multi-mass logistic regression. Cumulatively, >100,000 bits of digital image data was written into metabolomes. These early demonstrations provide insight into the benefits and limitations of postgenomic chemical information systems.

synthetic biology↗