Biology subjects

Smith, C. P.

Publications and source records attributed to Smith, C. P..

8 recordsLinked to original sources

bioRxiv2025

Global analysis of the cold-shock response in the model antibiotic producing actinomycete, Streptomyces coelicolor A3(2)

Cold-shock adaptation is essential for the survival of soil-dwelling sessile streptomycetes exposed to fluctuating environmental temperatures, yet the precise regulatory mechanisms underlying this response remain poorly understood. Here, we investigated the global transcriptional and translational responses of the model actinomycete, Streptomyces coelicolor A3(2), to cold-shock using integrated RNA-seq and polysome profiling. Cold-shock treatment in minimal liquid medium triggered significant transcriptional changes in 811 genes. Notably, three operons, encoding a CspA homologue, a DEAD-box helicase, and a cystathionine-{beta}-synthase (CBS) domain-containing protein and/or a protein of unknown function (SCO5921-SCO5918, SCO4684-SCO4686, and SCO3731-SCO3733) were identified as central players of the cold-shock response, exhibiting up to 2,000-fold transcriptional induction. Systems-level transcriptomic analysis further revealed the cold-shock induced activation of pathways associated with gluconeogenesis, coenzyme A metabolism, phenylacetate degradation, lipid raft remodelling, and extracellular functions, pointing to extensive metabolic reprogramming coordinated with membrane adaptation during cold acclimation. Polysome profiling unveiled strong translational potentiation of operonic genes downstream from the promoter proximal cspA homologue genes, a process potentially mediated by RNA secondary structures that overlap ribosome binding sites (RBSs). The pronounced induction of DEAD-box RNA helicases and CspA RNA chaperones is presumed to reflect their critical requirement for resolving excessive nucleic acid secondary structures inherent to the high G+C content genome of Streptomyces (>73% G+C), including the RBS-masking stem-loops within their own operons. Together, this study provides a comprehensive, system-level understanding of cold-shock adaptation in Streptomyces, highlighting a multi-layered regulatory architecture that couples metabolic reprogramming with RNA structure-dependent translational control to mitigate thermal stress. IMPORTANCEThis study characterizes the cold-shock response of the model actinomycete, S. coelicolor A3(2), for the first time at both the transcriptome and translatome levels. Combined with a machine-learning based iModulon framework, our findings provide critical insights on both metabolic adaptation and multi-layered regulatory mechanisms, including transcriptional networks and RNA structure-dependent translational control. Beyond advancing our fundamental understanding of cold acclimation in Streptomyces, we identified several putative cis-acting regulatory elements within the intergenic regions between the primary cold-shock genes (SCO4684 and SCO5921) and their downstream DEAD-box helicase-encoding genes. These regulatory elements, coupled with the exceptionally robust transcriptional and translational induction of the core cold-shock operons, significantly expands the synthetic biology toolkit for Streptomyces. Ultimately, these molecular components hold substantial potential for exploitation in optimizing and manipulating cryptic antibiotic biosynthetic gene clusters within this bacterial genus of considerable industrial importance.

genomics↗

bioRxiv2023

HIV-1 resistance to islatravir/tenofovir combination therapy in wild-type or NRTI resistant strains of diverse HIV-1 subtypes

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4-ethynyl-2-fluoro-2-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies.

microbiology↗

bioRxiv2022

The RNA binding proteins ZFP36L1 and ZFP36L2 modulate transcriptional and post-transcriptional genome-wide effects of glucocorticoids

Glucocorticoids (GCs) are one of the most used anti-inflammatory drugs worldwide. Despite their widespread use, our understanding of their post-transcriptional effects remains poorly understood. The tristetraprolin (TTP) RNA binding protein (RBP) family (ZFP36, ZFP36L1 and ZFP36L2) has been implicated in inflammation regulation via binding to AU-rich elements (ARE) in mRNAs, with TTP being implicated in GC modulation. We hypothesised that ZFP36L1 and ZFP36L2 are part of the GC pathway and tested this hypothesis in bronchial epithelium, which commonly encounters GC in vivo upon inhalation. Our data show that dexamethasone, a commonly used GC, modulated the levels, subcellular localisation and RNA binding of ZFP36L1/L2. Employing Frac-seq (subcellular fractionation and RNA-sequencing), we show that GC modulated distinct subsets of RNAs in a subcellular-dependent manner. In addition to their mostly known transcriptional effects (116 differentially expressed genes, DEGs), GCs modified the binding to monosomes of myriad mRNAs (83 differentially bound genes, DBGs). We also demonstrate that ZFP36L1/L2 modulated gene expression mainly at the total cytoplasmic and polyribosome binding levels. ZFP36L1/L2 down-regulation led to an increase in ARE-containing mRNAs and a pronounced modification of the effects of GC on gene expression. We observed a small overlap of genes modulated by GCs when comparing control and ZFP36L1/L2 knockdown cells, in a subcellular-dependent manner Our data also suggest a novel role for these RBPs and GCs in epithelial biology via regulation of mRNAs encoding proteins important for epithelial cell function including cellular structure. We believe that our data has further implications in how we investigate gene expression. We show the power of employing sub-cellular fractionation when analysing genome-wide effects for known transcriptional modulators such as GCs, as well as a tool to demonstrate the extent of the effect of RBPs on gene expression modulation beyond total RNA levels.

molecular biology↗

bioRxiv2022

Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2.

Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a form of isolation, even when raised in a social environment. SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience a form of isolation.

neuroscience↗

bioRxiv2019

Vitamins D3 and D2 have marked but different global effects on gene expression in a rat oligodendrocyte precursor cell line

Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but there is uncertainty about what dose and form of vitamin D could improve the clinical course of MS. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with D3 at 24 h and 72 h following onset of differentiation. Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1,272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for gene ontology categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway. These findings should help to identify mechanisms mediating D3 action in MS.

genomics↗

bioRxiv2018

Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 in Streptomyces coelicolor A3(2)

The AbsA1-AbsA2 two component signalling system of Streptomyces coelicolor has long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of a{Delta} absA2 deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complemented{Delta} absA2 mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting absA2 on gene expression and to identify the in vivo AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of redZ and actII-orfIV AbsA2 binds to several previously unidentified sites within the cda biosynthetic gene cluster within and/or upstream of SCO3215 - SCO3216, SCO3217, SCO3229 - SCO3230, and SCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual biphasic ChIP peaks were observed with AbsA2 binding across the regulatory genes actII-orfIV and redZ and the absA2 gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis in Streptomyces coelicolor and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

genomics↗

bioRxiv2018

PGP-UK: a research and citizen science hybrid project in support of personalized medicine

Molecular analyses such as whole-genome sequencing have become routine and are expected to be transformational for future healthcare and lifestyle decisions. Population-wide implementation of such analyses is, however, not without challenges, and multiple studies are ongoing to identify what these are and explore how they can be addressed. Defined as a research project, the Personal Genome Project UK (PGP-UK) is part of the global PGP network and focuses on open data sharing and citizen science to advance and accelerate personalized genomics and medicine. Here we report our findings on using an open consent recruitment protocol, active participant involvement, open access release of personal genome, methylome and transcriptome data and associated analyses, including 47 new variants predicted to affect gene function and innovative reports based on the analysis of genetic and epigenetic variants. For this pilot study, we recruited ten participants willing to actively engage as citizen scientists with the project. In addition, we introduce Genome Donation as a novel mechanism for openly sharing previously restricted data and discuss the first three donations received. Lastly, we present GenoME, a free, open-source educational app suitable for the lay public to allow exploration of personal genomes. Our findings demonstrate that citizen science-based approaches like PGP-UK have an important role to play in the public awareness, acceptance and implementation of genomics and personalized medicine.

genomics↗

bioRxiv2017

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and translatome; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as potentiation. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

genomics↗