Biology subjects

Taylor, T. B.

Publications and source records attributed to Taylor, T. B..

6 recordsLinked to original sources

bioRxiv2025

Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent

CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages CRISPR-Cas interference was only partially protective in high nutrient conditions, yet completely protective in low nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.

microbiology↗

bioRxiv2024

GnT motifs: short nucleotide tracts of greater than or equal to 8bp that can increase T-G:C mutation rates >1000-fold in bacteria

Nucleotides across a genome do not mutate at equal frequencies. Instead, specific nucleotide positions can exhibit much higher mutation rates than the genomic average due to their immediate nucleotide neighbours. These mutational hotspots can play a prominent role in adaptive evolution, yet we lack knowledge of which short nucleotide tracts drive hotspots. In this work, we employ a combinatorial approach of experimental evolution with Pseudomonas fluorescens and bioinformatic analysis of various Salmonella species to characterise a short nucleotide motif ([≥]8bp) that drives T:A[->]G:C mutation rates >1000-fold higher than the average T[->]G rate in bacteria. First, we experimentally show that homopolymeric tracts ([≥]3) of G with a 3 T frequently mutate so that the 3 T is replaced with a G, resulting in an extension of the guanine tract, i.e., GGGGT [->] GGGGG. We then demonstrate that the potency of this T:A[->]G:C hotspot is dependent on the nucleotides immediately flanking the GnT motif. We find that the dinucleotide pair immediately 5 to a G4 tract and the nucleotide immediately 3 to the T strongly affect the T:A[->]G:C mutation rate, which ranges from [~]5-fold higher than the typical rate to >1000-fold higher depending on the flanking elements. Therefore the T:A[->]G:C hotspot motif is a product of several modular nucleotide components (1-4bp in length) which each exert a significant effect on the mutation rate of the GnT motif. This work advances our ability to accurately identify the position and quantify the mutagenicity of hotspot motifs predicated on short tracts of nucleotides.

molecular biology↗

bioRxiv2023

Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in the bacteria Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient rich (LB) and nutrient limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations) they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments. DATA SUMMARYAll data is available at OSF: DOI 10.17605/OSF.IO/8BT2W, and in supplementary materials.

evolutionary biology↗

bioRxiv2023

Regulatory network structure and environmental signals constrain transcription factor innovation

Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Rewiring - that is gaining or losing connections to transcriptional target genes - is a key mechanism by which transcription factors evolve and innovate. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for rewiring, we utilise a Pseudomonas fluorescens SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (fleQ). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rewire repeatably and reliably in a strict hierarchy - with NtrC the only evolved rewiring route in a {Delta}fleQ background, and PFLU1132 the only evolved rewiring route in a {Delta}fleQ{Delta}ntrC background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot fully explain this pattern, and mutations in rewired strains suggested high levels of transcription factor expression and activation drive rewiring. We predict that mutations that increase expression of the rewired transcription factor are vital to unlock rewiring potential. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in P. fluorescens. We show that in 5 additional RpoN-EBPs (HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining rewiring potential. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network rewiring.

evolutionary biology↗

bioRxiv2022

Evolutionary innovation through transcription factor promiscuity in microbes is constrained by pre-existing gene regulatory network architecture

The survival of a population during environmental shifts depends on whether the rate of phenotypic adaptation keeps up with the rate of changing conditions. A common way to achieve this is via change to gene regulatory network (GRN) connections - known as rewiring - that facilitate novel interactions and innovation of transcription factors. To understand the success of rapidly adapting organisms, we therefore need to determine the rules that create and constrain opportunities for GRN rewiring. Here, using an experimental microbial model system we reveal a hierarchy among transcription factors that are rewired to rescue lost function, with alternative rewiring pathways only unmasked after the preferred pathway is eliminated. We identify three key properties - high activation, high expression, and pre-existing low-level affinity for novel target genes - that facilitate transcription factor innovation via gain of functional promiscuity. Ease of acquiring these properties is constrained by pre- existing GRN architecture, which was overcome in our experimental system by both targeted and global network alterations. This work reveals the key properties that determine transcription factor evolvability, and as such, the evolution of GRNs.

evolutionary biology↗

bioRxiv2021

Extreme parallel evolution of flagellar motility is facilitated by synonymous sequence

Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes.

evolutionary biology↗