Biology subjects

Lagator, M.

Publications and source records attributed to Lagator, M..

10 recordsLinked to original sources

bioRxiv2026

Using a lanthanide tagged nanobody to visualise Glial fibrillary acidic protein by secondary ion mass spectrometry

Lanthanide-labelled antibodies have been widely used for multiplex imaging of proteins directly from tissue using two approaches, imaging mass cytometry (IMC) and variants of secondary ion mass spectrometry (SIMS). Here we show the same approach can be taken using SIMS, but utilising lanthanide-labelled nanobodies instead. Nanobodies offer several advantages over conventional antibodies for antigen tagging, owing to their small size (12-15 kDa), high stability, and solubility. Their reduced molecular size enables greater diffusion through tissue, facilitating access to target sites and allowing them to reach regions that may be less accessible to larger antibodies. This enhanced accessibility can promote more effective localisation and increase binding to target antigens. In the work presented here, we produced a lanthanide-tagged nanobody and assessed its efficacy and compatibility with mass spectrometry imaging (MSI). We showed successful detection of Glial fibrillary acidic protein (GFAP) in tissue by correlating MSI of a lanthanide-tagged anti-GFAP nanobody with immunofluorescence microscopy imaging (IF). The use of nanobodies is an excellent alternative to current antibody tagging methods for SIMS and IMC.

molecular biology↗

bioRxiv2025

Strong promoters are mutationally robust

Mutational robustness is the persistence of a phenotype upon mutation. It facilitates molecular evolution and has been characterized in a variety of biological systems, but studies of prokaryotic promoters are limited. Prokaryotic promoters are non-coding DNA sequences that regulate transcription. The main housekeeping promoters ({sigma}70) share two sequence motifs called the -35 and the -10 box that are spaced 17{+/-}1 base pairs (bps) apart. The sequence of these boxes, their distance, and the existence of multiple nearby boxes can determine promoter strength, the ability of a promoter to drive high levels of transcription. Here we used computational modelling to show that the mutational robustness of {sigma}70 promoters - the persistence of promoter strength upon mutation - correlates with promoter strength itself. Mutational robustness is also influenced by potential overlaps between -10 and -35 boxes, and by specific positions within the boxes. It is higher when the boxes are exactly 17 bps apart. These findings can be partially explained by the flexibility of -35 boxes, which enables adjacent bases to create overlapping and new -35 boxes. Our work can help to engineer synthetic promoters for strength and robustness, and to understand the dynamics of promoter evolution.

evolutionary biology↗

bioRxiv2025

Identifying rare spontaneous mutations through wildtype E. coli population sequencing

Understanding the rate and types of mutations occurring in different populations is fundamental to our ability to predict and potentially manipulate evolution. Common techniques for microbial mutation rate estimation fall into 2 camps; rapid, locus specific, generally liquid-culture based fluctuation assays; and slow, genome-wide, generally solid-culture based mutation accumulation experiments. This constrains the hypotheses which can be tested, notably the mutagenic effects of liquid environments cannot be rapidly quantified at a genome-wide scale. One example of such effect is the negative association previously observed between population density and mutation rates at many marker loci using the fluctuation assay. Because homogeneous population density is specifically relevant in liquid culture, this association has not been tested at a truly genome-wide scale. We fill this methodological gap by developing a novel pipeline that relies on population sequencing to capture how mutation rates are affected by population density in liquid cultures. We simulate expected mutation counts in a growing population along with random sampling during sequencing to estimate the necessary sequencing coverage as [≥]1,000-fold. We then carry out PCR-free sequencing of 95 wildtype E. coli populations at this coverage, calling rare variants with both reference-based and reference-free methods. These variant-calling methods are prone to different sources of error which can be minimised by considering only mutations called by both pipelines. This approach identifies 119 mutations across all cultures, with (non)coding/(non)synonymous and mutational spectrum profiles consistent with being samples of spontaneous mutation unbiased by selection. The distribution of these mutations also supports the motivating hypothesis, finding that mutation counts across the genome are strongly negatively associated with population density. This demonstrates the utility of population sequencing for the rapid testing of many previously inaccessible evolutionary biology hypotheses.

evolutionary biology↗

bioRxiv2025

Evolutionary potential of the Escherichia coli antimutator ΔnudJ is reduced via altered mutational spectrum

The rate of spontaneous mutation is a key factor in determining the capacity of a population to adapt to a novel environment, for example a bacterial population exposed to antibiotics. Genetic and environmental factors controlling the mutation rate commonly also cause shifts in the relative rates of different mutational classes, i.e. the spectrum of spontaneous mutations. When the mutational spectrum is altered, the relatively enriched and depleted mutations may differ in their fitness effects. Here we aim to explore how a reduced mutation rate and altered mutational spectrum can contribute to adaptation in Escherichia coli. We measure mutation rates across a set of Nudix hydrolase deletants, finding multiple strains with an antimutator phenotype. We focus on the antimutator {Delta}nudJ which can cause a 6-fold mutation rate reduction relative to the wildtype with an altered mutational spectrum. Not only does nudJ deletion reduce the probability of antibiotic resistance arising but, in the case of rifampicin, the accessible resistance mutations under this altered mutation spectrum have greater fitness costs. While this effect is, so far, specific to this drug, it opens up the possibility of reducing the likelihood of resistance mutations establishing through simultaneous modification of mutation rate and spectrum. The identification and characterisation of antimutator alleles such as {Delta}nudJ provides potential targets for antievolution drugs which could be applied during antibiotic treatment to inhibit NudJ activity. Such an approach could suppress spontaneous resistance evolution both through fewer resistance mutations and by limiting access to the fittest mutations.

evolutionary biology↗

bioRxiv2024

Linking Molecular Mechanisms to their Evolutionary Consequences: a primer

A major obstacle to predictive understanding of evolution stems from the complexity of biological systems, which prevents detailed characterization of key evolutionary properties. Here, we highlight some of the major sources of complexity that arise when relating molecular mechanisms to their evolutionary consequences and ask whether accounting for every mechanistic detail is important to accurately predict evolutionary outcomes. To do this, we developed a mechanistic model of a bacterial promoter regulated by two proteins, allowing us to connect any promoter genotype to six phenotypes that capture the dynamics of gene expression following an environmental switch. Accounting for the mechanisms that govern how this system works enabled us to provide an in-depth picture of how regulated bacterial promoters might evolve. More importantly, we used the model to explore which factors that contribute to the complexity of this system are essential for understanding its evolution, and which can be simplified without information loss. We found that several key evolutionary properties - the distribution of phenotypic and fitness effects of mutations, the evolutionary trajectories during selection for regulation - can be accurately captured without accounting for all, or even most, parameters of the system. Our findings point to the need for a mechanistic approach to studying evolution, as it enables tackling biological complexity and in doing so improves the ability to predict evolutionary outcomes.

evolutionary biology↗

bioRxiv2024

Evolution of intrinsic transcriptional terminators in their genomic context

Transcriptional gene expression relies on two fundamental processes - transcription initiation and termination. Transcriptional termination is essential for the coordinated control of gene expression. Intrinsic termination, the major mechanism of transcriptional termination in bacteria, relies on the sequence-dependent folding of nascent mRNA into a hairpin structure that enables RNA polymerase to dissociate from DNA. Despite their central role in regulating expression in prokaryotic genomes, the conservation of intrinsic terminators and the factors that shape their evolution remain poorly understood. Here, we combine comparative genomics with experimental measurements of terminator function, to study the conservation of intrinsic terminators between Escherichia coli and Salmonella enterica subsp. pullorum. While the two species are closely related, the sequence and function of most terminators are not conserved, with less than 20% of all terminators having an identifiable ortholog - in stark contrast to 60% for coding sequences. Terminators with higher sequence conservation also had more conserved function, indicative of stabilizing selection. The local genomic context shapes the evolution of intrinsic terminators, as their sequence conservation is dependent on the conservation of the upstream gene, while their function is affected by the distance to the downstream gene. Ultimately, any theory of gene regulatory network evolution ought to account for how transcriptional terminators evolve.

evolutionary biology↗

bioRxiv2024

High-throughput method rapidly characterizes hundreds of novel antibiotic resistance mutations

A fundamental obstacle to tackling the antimicrobial resistance crsisis is identifying mutations that lead to resistance in a given genomic background and environment. We present a high-throughput technique - Quantitative Mutational Scan Sequencing (QMS-Seq) - that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compared four E. coli strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identified 975 resistance mutations, many in genes and regulatory regions not previously associated with resistance. QMS-Seq revealed that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-Seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.

microbiology↗

bioRxiv2022

Not digested: algal glycans move carbon dioxide into the deep-sea

Marine algae annually synthesize gigatons of glycans from carbon dioxide, exporting it within sinking particles into the deep-sea and underlying sea floor, unless those glycans are digested before by bacteria. Identifying algal glycans in the ocean remains challenging with the molecular resolution of conventional analytic techniques. Whether algal glycans are digested by heterotrophic bacteria during downward transport, before they can transfer carbon dioxide from the ocean surface into the deep-sea or the sea floor, remains unknown. In the Red Sea Shaban Deep, where at 1500 m water depth a brine basin acts as a natural sediment trap, we found its high salt and low oxygen concentration accumulated and preserved exported algal glycans for the past 2500 years. By using monoclonal antibodies specific for glycan structures, we detected fucose-containing sulfated polysaccharide, {beta}-glucan, {beta}-mannan and arabinogalactan glycans, synthesized by diatoms, coccolithophores, dinoflagellates and other algae living in the sunlit ocean. Their presence in deep-sea sediment demonstrates these algal glycans were not digested by bacteria. Instead they moved carbon dioxide from the surface ocean into the deep-sea, where it will be locked away from the atmosphere at least for the next 1000 years. Considering their global synthesis, quantity and stability against degradation during transport through the water column, algal glycans are agents for carbon sequestration. Significance statementAlgae and plants use the greenhouse gas carbon dioxide to synthesize polymeric carbohydrates, or glycans, for energy storage, structural support and as protection against invasion by microbes. Glycans provide protection, are carbon sinks and enable carbon sequestration for as long as they are not digested by bacteria or other organisms, which releases the carbon dioxide back in to the atmosphere. In this study, we show that non-digested algal glycans sink into the deep ocean and into marine sediment. Thus, glycans are more than food for animals and prebiotics for bacteria, they are also molecules that remove carbon dioxide from the atmosphere and transfer it to the deep-sea, where it can be stored for 1000 years and longer.

biochemistry↗

bioRxiv2020

Structure and Evolution of Constitutive Bacterial Promoters

Predicting gene expression levels from any DNA sequence is a major challenge in biology. Using libraries with >25,000 random mutants, we developed a biophysical model that accounts for major features of {sigma}70-binding bacterial promoters to accurately predict constitutive gene expression levels of any sequence. We experimentally and theoretically estimated that 10-20% of random sequences lead to expression and 82% of non-expressing sequences are one point mutation away from a functional promoter. Generating expression from random sequences is pervasive, such that selection acts against {sigma}70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. The pervasiveness of {sigma}70- binding sites, which arises from the structural features of promoters captured by our biophysical model, implies that their emergence is unlikely the limiting step in gene regulatory evolution.

evolutionary biology↗

bioRxiv2019

Experimental Determination of Evolutionary Barriers to Horizontal Gene Transfer

Horizontal gene transfer, the acquisition of genes across species boundaries, is a major source of novel phenotypes. Several barriers have been suggested to impede the likelihood of horizontal transmission; however experimental evidence is scarce. We measured the fitness effects of genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli, and found that most result in strong fitness costs. Previously identified evolutionary barriers -- gene function and the number of protein-protein interactions -- did not predict the fitness effects of transferred genes. In contrast, dosage sensitivity, gene length, and the intrinsic protein disorder significantly impact the likelihood of a successful horizontal transfer. While computational approaches have been successful in describing long-term barriers to horizontal gene transfer, our experimental results identified previously underappreciated barriers that determine the fitness effects of newly transferred genes, and hence their short-term eco-evolutionary dynamics.

microbiology↗