Biology subjects

Tree, J. J.

Publications and source records attributed to Tree, J. J..

10 recordsLinked to original sources

bioRxiv2026

Disruption of cAMP Signalling and RNA Regulation Confers Cefiderocol Resistance in Acinetobacter baumannii

Cefiderocol is a siderophore-conjugated cephalosporin used to treat severe multidrug-resistant Gram-negative infections. Its recent prioritisation for US national preparedness underscores its clinical importance, yet resistance can emerge despite apparent in vitro susceptibility. Here, we identify two regulatory routes to cefiderocol resistance in carbapenem-resistant Acinetobacter baumannii (CRAB): mutation of the cAMP-producing adenylate cyclase CavA and truncation of the glycine-rich RNA-binding domain of the post-transcriptional regulator Hfq. These variants occurred in contemporary clinical isolates, conferred resistance in a murine CRAB lung-infection model and imposed minimal fitness costs. In Escherichia coli, deletion of the CavA orthologue cyaA increased competitive fitness during sub-inhibitory cefiderocol exposure. Hfq-CLASH and integrated omics revealed Hfq-dependent regulation of Fur-associated iron homeostasis in A. baumannii. Under iron limitation, truncated-Hfq induced a defence-primed state that attenuated siderophore uptake and reduced cefiderocol activity. These findings identify bacterial regulatory state and iron availability as determinants of cefiderocol susceptibility, with implications for resistance surveillance and susceptibility testing.

microbiology↗

bioRxiv2026

A cis-encoded RNA interaction controls sub-operonic spoVG expression in Staphylococcus aureus

Bacterial operons enable co-transcription of multiple genes, yet post-transcriptional mechanisms permitting differential regulation of individual genes within a polycistronic transcript remains incompletely understood. We demonstrate that a cis-encoded RNA-RNA interaction within the bicistronic yabJ-spoVG transcript forms an RNase III substrate that represses the transcription factor SpoVG in Staphylococcus aureus. Disruption of the cis-encoded regulation elevated monocistronic spoVG expression, reduced biofilm formation and cell wall peptidoglycan thickness, and increased sensitivity to cell wall-targeting antimicrobials in clinical vancomycin-intermediate S. aureus, consistent with phenotypes observed in an independent SpoVG overexpression strain. These findings uncover a previously undescribed mode of cis-regulatory control within a polycistronic operon and establish regulatory mRNA-mRNA interactions as a mechanism for sub-operonic gene regulation.

microbiology↗

bioRxiv2026

Molecular Determinants of Functional Bacterial sRNA-mRNA Interactions Revealed by Integrating RNA Interactomes and Interpretable Machine Learning

Bacterial small RNAs (sRNAs) regulate gene expression by base pairing with target mRNAs, yet transcriptome-wide interactome mapping has shown that many sRNA-mRNA interactions detected in vivo have modest or no regulatory effect using orthogonal reporter assays. The features that determine functional outcome remain poorly defined. Here, we integrated Hfq-CLASH interactome mapping with matched transcriptomic and proteomic profiling in Escherichia coli and developed an interpretable machine-learning framework to identify the determinants that distinguish functional from non-functional interactions. Using sequence, structural, thermodynamic, duplex and protein-occupancy features, transcriptomic and proteomic responses were predicted with above-chance performance, achieving AUCs of 0.78 and 0.74, respectively. Feature attribution revealed that physical pairing alone is insufficient for regulation; instead, regulatory outcome is shaped by a coordinated interplay between RNA secondary structure, thermodynamic accessibility and local protein-binding context. Target-side Hfq occupancy emerged as a positive predictor of functional regulation, whereas AR2-domain occupancy on the sRNA was associated with non-responsive interactions, suggesting that distinct ribonucleoprotein states may separate productive regulation from non-productive binding. These findings indicate that the regulatory fate of an sRNA-mRNA interaction is an emergent property of its biophysical context and protein-binding environment, rather than a direct consequence of physical pairing alone.

molecular biology↗

bioRxiv2026

The intrinsically disordered AR2 domain of RNase E binds mRNA translation initiation regions

Intrinsically disordered regions are widespread in RNA-processing machines. In Escherichia coli, RNase E uses its intrinsically disordered C-terminal domain (CTD) to recruit RNAs to the N-terminal catalytic domain, including mRNAs targeted by regulatory small RNAs (sRNAs), but the basis of substrate recognition and specificity is unclear. We engineered a protease-cleavable RNase E and used split-CRAC to isolate RNAs crosslinked to the AR2 sub-domain of the intrinsically disordered CTD fragment. AR2 preferentially engaged mRNAs and was depleted of sRNAs and sRNA-containing hybrids, supporting recognition of the mRNA. AR2 contacts concentrated on accessible A-rich motifs surrounding ribosome-binding sites and start codons, and purified AR2 recognised this motif in vitro. AR2 also contacted an AUAA motif in the rne translation-initiation region, and AR2 deletion increased RNase E abundance implicating this interaction in autoregulation. These findings define a relatively short AR2-binding motif and are consistent with CTD interactions with the 30S subunit that may provide additional specificity for a subset of mRNA translation initiation regions.

microbiology↗

bioRxiv2025

Phage-encoded sRNA counteracts xenogenic silencing in pathogenic E. coli

Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogenic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet to be expressed they must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in multiple copies (up to nine) in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes and is enriched in E. coli strains that carry the locus of enterocyte effacement (LEE). We show that HnrS directly base-pairs with the ribosome-binding site of the hha mRNA, repressing its translation and thereby reducing Hha-enhanced H-NS silencing. This counter-silencing de-represses the LEE type III secretion system and concomitantly represses motility. Transcriptomic profiling further revealed that HnrS indirectly activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways that contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that prophage-encoded, multicopy hnrS provides a counter-silencing mechanism that reduces Hha-H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the core H-NS regulon. HnrS illustrates how mobile genetic elements deploy sRNAs to counteract xenogenic silencing and promote virulence gene expression, enhancing colonisation of the host. Importance statementHorizontally acquired genes are often silenced to protect bacterial genomes, but this defence also limits the expression of new traits. We identify a prophage-encoded small RNA, HnrS, that counteracts this restriction by repressing the xenogenic silencer Hha, lifting Hha-H-NS-mediated repression of virulence and metabolic genes. HnrS activates the locus of enterocyte effacement, nitrate/nitrite respiration, and nitric oxide resistance--pathways that help E. coli survive and colonise the inflamed gut. Our findings reveal an RNA-based counter-silencing mechanism encoded by mobile genetic elements, showing how phages can reprogram bacterial regulatory networks to promote adaptation and pathogenicity.

microbiology↗

bioRxiv2025

A Zur-dependent regulatory RNA involved in maintaining zinc homeostasis in Staphylococcus aureus

Small regulatory RNAs (sRNAs) are key drivers of bacterial adaptation to environmental fluctuations, including iron and manganese restriction imposed by the host. This study explored the repertoire of sRNAs produced by the human pathogen Staphylococus aureus in response to metal limitation. Two sRNAs, S1077 and ZinS (RsaX20), regulated by zinc (Zn) availability, were identified. Further investigations revealed that, similar to the cnt operon from which it derives, S1077 synthesis is controlled by the transcription factors Zur and Fur. In contrast, zinS transcription is solely repressed by Zur. Amongst the ZinS targets are several Zn-dependent enzymes, such as the alcohol dehydrogenase Adh, whose synthesis is negatively regulated by ZinS. Loss of ZinS does not alter staphylococcal metal accumulation, suggesting a role in a Zn-sparing response. Remarkably, zinS also encodes a small peptide, ZinP. Genomic analysis suggests that the regulatory portion of ZinS emerged from the 3 untranslated region of zinP in S. aureus and closely related species after horizontal gene transfer from phylogenetically distant organisms. All our findings demonstrate that sRNAs also facilitate bacterial adaptation to Zn limitation, and that genetic exchange and subsequent neofunctionalization have enabled S. aureus to adapt to metal-restricted environments.

microbiology↗

bioRxiv2023

The functional small RNA interactome of vancomycin tolerant Staphylococcus aureus

RNA-RNA interactome profiling techniques have expanded our understanding of sRNA-mRNA interactions in bacteria. However, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To specifically identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used SOMS to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating this clustering analysis with sRNA-mRNA interactome data generated in vancomycin tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating this translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin treatment. While RsaOI is not essential for vancomycin tolerance, we demonstrate regulation of the phosphocarrier protein HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI may serve as a regulator of carbon metabolism and cell wall turnover during cell wall stress exerted by vancomycin.

microbiology↗

bioRxiv2023

The 3'UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions

Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3 UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3 UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3 UTR was found to repress dapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3 UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3 UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3 UTR. Our findings reveal an extended regulatory network for vigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.

microbiology↗

bioRxiv2021

Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor

To sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohaemorrhagic E. coli (EHEC) iron-limitation induces transcription of the outermembrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37{degrees}C by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive sRNA CyaR and transcriptional terminator Rho. These results indicate that chuAS expression is regulated at the transcription initiation, transcript elongation, and translational level. The natural dependence of these processes creates a hierarchy of regulatory AND and OR logic gates that integrate information about the local environment. We show that the logic of the chuA regulatory circuit is activated under conditions that satisfy low iron AND (low glucose OR high temperature). We speculate that additional sensing of a gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuAS transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a conditional hierarchy of signal input.

microbiology↗

bioRxiv2018

Hfq CLASH uncovers sRNA-target interaction networks enhancing adaptation to nutrient availability

By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to very efficiently adapt to constant changes in their environment. To better understand how Escherichia coli acclimatizes to changes in nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover sRNA-target interactions. Strikingly, we uncovered hundreds of novel Hfq-mediated sRNA-target interactions at specific growth stages, involving many novel 3UTR-derived sRNAs and a plethora of sRNA-sRNA interactions. We discovered sRNA-target interaction networks that play a role in adaptation to changes in nutrient availability. We characterized a novel 3UTR-derived sRNA (MdoR), which is part of a regulatory cascade that enhances maltose uptake by (a) inactivating repressive pathways that block the accumulation of maltose transporters and (b) by reducing the flux of general porins to the outer membrane. Our work provides striking examples of how bacteria utilize sRNAs to integrate multiple regulatory pathways to enhance nutrient stress adaptation.

systems biology↗