Biology subjects

Cusack, S. A.

Publications and source records attributed to Cusack, S. A..

5 recordsLinked to original sources

bioRxiv2026

MxtR/ErdR is a central regulator of short-chain fatty acid metabolism in Pseudomonas alloputida

Short-chain fatty acids (SCFAs) such as acetate and propionate represent important carbon and energy sources for environmental pseudomonads, requiring coordinated regulation to ensure efficient assimilation while maintaining metabolic homeostasis. In Pseudomonas alloputida KT2440, the two-component system MxtR/ErdR (CrbS/CrbR) is known to activate acetate assimilation through regulation of acsA-I. However, the full extent of the MxtR/ErdR regulon and its physiological role beyond acetate metabolism have remained unclear. We demonstrate that MxtR/ErdR directly regulates the methylcitrate cycle and coordinates both acetate and propionate metabolism. To define the MxtR/ErdR regulatory network, we combined comparative transcriptomics, physiological analyses, promoter-reporter assays, electrophoretic mobility shift assays (EMSAs) and targeted mutagenesis. Comparative transcriptomic analyses of the mxtR-H806N and {Delta}erdR mutants revealed extensive changes in gene expression, including coordinated downregulation of genes involved in propionate metabolism alongside genes associated with central carbon metabolism, transport, chemotaxis, and signal transduction. Consistent with these transcriptional changes, deletion of either mxtR or erdR abolished growth on propionate. Promoter-reporter assays and EMSAs demonstrated direct binding of ErdR to a conserved imperfect inverted repeat upstream of the prp gene cluster and prpE. Mutational analyses confirmed the functional importance of this binding motif for promoter activation. In addition, MxtR/ErdR contributed to pyruvate utilization through regulation of transport-associated genes, whereas deletion of downstream target genes only caused modest phenotypes, indicating that the physiological effects of MxtR/ErdR arise from coordinated regulation of multiple pathways. Our findings substantially expand the MxtR/ErdR regulon and identify this signaling system as a central regulator of SCFA homeostasis in P. alloputida KT2440.

microbiology↗

bioRxiv2025

Epitranscriptomic control of stress adaptations in Escherichia coli

Bacterial stress responses have been studied at the phenotypic, transcriptional, and translational levels, demonstrating the presence of an "alarm" phase immediately after stress exposure. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic RNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the alarm phase of stress adaptation.

microbiology↗

bioRxiv2024

Decoding the Escherichia coli epitranscriptome

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, and viral infections. In prokaryotes, regulation at the level of the epitranscriptome is still poorly understood. Here, we used Nanopore direct RNA sequencing of Escherichia coli to study RNA modifications and their changes under heat stress. With a single sequencing reaction, we detected most known modification types in rRNA, tRNA, and mRNA. RNA sequencing was complemented by a multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide PCR, and in vitro methylation. Known m5C and m6A sites in the rRNA were confirmed, but these types of modifications could not be localized in the mRNA. In response to heat stress, levels of m5C, m6A, and m6,6A increased in the 16S rRNA. Sequencing and mass spectrometry data demonstrated a decrease in tRNA modification abundance in the anticodon loop at 45 {degrees}C. In general, mRNA modifications at 37 {degrees}C were enriched in the coding regions of genes associated with general metabolism and RNA processing, which shifted to genes involved in cell wall synthesis and membrane transport under heat stress. This study provides new insights into the complexity of post-transcriptional regulation in bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/602490v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@11b9bc4org.highwire.dtl.DTLVardef@18f77feorg.highwire.dtl.DTLVardef@efdeaorg.highwire.dtl.DTLVardef@402859_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection

Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope (OE) membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of OE protein (OEP) functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed OE-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated three previously known phenotypes of cold-sensitive mutants, but also identified four mutants of additional oemiR OE loci. Altogether our study revealed a role of the OE to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of OEPs.

plant biology↗

bioRxiv2020

Genome-wide predictions of genetic redundancy in Arabidopsis thaliana

Genetic redundancy refers to a situation where an individual with a loss-of-function mutation in one gene (single mutant) does not show an apparent phenotype until one or more paralogs are also knocked out (double/higher-order mutant). Previous studies have identified some characteristics common among redundant gene pairs, but a predictive model of genetic redundancy incorporating a wide variety of features has not yet been established. In addition, the relative importance of these characteristics for genetic redundancy remains unclear. Here, we establish machine learning models for predicting whether a gene pair is likely redundant or not in the model plant Arabidopsis thaliana. Benchmark gene pairs were classified based on six feature categories: functional annotations, evolutionary conservation including duplication patterns and mechanisms, epigenetic marks, protein properties including post-translational modifications, gene expression, and gene network properties. The definition of redundancy, data transformations, feature subsets, and machine learning algorithms used affected model performance significantly. Among the most important features in predicting gene pairs as redundant were having a paralog(s) from recent duplication events, annotation as a transcription factor, downregulation during stress conditions, and having similar expression patterns under stress conditions. Predictions were then tested using phenotype data withheld from model building and validated using well-characterized, redundant and nonredundant gene pairs. This genetic redundancy model sheds light on characteristics that may contribute to long-term maintenance of paralogs that are seemingly functionally redundant, and will ultimately allow for more targeted generation of functionally informative double mutants, advancing functional genomic studies.

evolutionary biology↗