Biology subjects

Grant, N. A.

Publications and source records attributed to Grant, N. A..

6 recordsLinked to original sources

bioRxiv2023

Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background

Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. choleraes pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previously, a transposon mutant library for V. cholerae was created to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed high lysis-specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. choleraes open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, in no instance did the luxO mutation transfer into the wildtype background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent. Significance StatementOrdered transposon mutant libraries are essential tools for catalyzing research by providing access to null mutants of all non-essential genes. Such a library was previously generated for Vibrio cholerae, but whole genome sequencing revealed that this library was made using a parent strain that is unable to exhibit cell-cell communication known as quorum sensing. Here, we utilize natural competence combined with a novel, high-throughput genomic DNA extraction method to regenerate the signaling incompetent V. cholerae ordered transposon mutant library in quorum sensing competent strain. Our library provides researchers with a powerful tool to understand V. cholerae biology within a genetic context that influences how it transitions from an environmentally benign organism to a disease-causing pathogen.

microbiology↗

bioRxiv2021

Adaptive phenotypic plasticity stabilizes evolution in fluctuating environments

Fluctuating environmental conditions are ubiquitous in natural systems, and populations have evolved various strategies to cope with such fluctuations. The particular mechanisms that evolve profoundly influence subsequent evolutionary dynamics. One such mechanism is phenotypic plasticity, which is the ability of a single genotype to produce alternate phenotypes in an environmentally dependent context. Here, we use digital organisms (self-replicating computer programs) to investigate how adaptive phenotypic plasticity alters evolutionary dynamics and influences evolutionary outcomes in cyclically changing environments. Specifically, we examined the evolutionary histories of both plastic populations and non-plastic populations to ask: (1) Does adaptive plasticity promote or constrain evolutionary change? (2) Are plastic populations better able to evolve and then maintain novel traits? And (3), how does adaptive plasticity affect the potential for maladaptive alleles to accumulate in evolving genomes? We find that populations with adaptive phenotypic plasticity undergo less evolutionary change than non-plastic populations, which must rely on genetic variation from de novo mutations to continuously readapt to environmental fluctuations. Indeed, the non-plastic populations undergo more frequent selective sweeps and accumulate many more genetic changes. We find that the repeated selective sweeps in non-plastic populations drive the loss of beneficial traits and accumulation of maladaptive alleles via deleterious hitchhiking, whereas phenotypic plasticity can stabilize populations against environmental fluctuations. This stabilization allows plastic populations to more easily retain novel adaptive traits than their non-plastic counterparts. In general, the evolution of adaptive phenotypic plasticity shifted evolutionary dynamics to be more similar to that of populations evolving in a static environment than to non-plastic populations evolving in an identical fluctuating environment. All natural environments subject populations to some form of change; our findings suggest that the stabilizing effect of phenotypic plasticity plays an important role in subsequent adaptive evolution.

evolutionary biology↗

bioRxiv2020

Changes in Cell Size and Shape During 50,000 Generations of Experimental Evolution with Escherichia coli

Bacteria adopt a wide variety of sizes and shapes, with many species exhibiting stereotypical morphologies. How morphology changes, and over what timescales, is less clear. Previous work examining cell morphology in an experiment with Escherichia coli showed that populations evolved larger cells and, in some cases, cells that were less rod-like. That experiment has now run for over two more decades. Meanwhile, genome sequence data are available for these populations, and new computational methods enable high-throughput microscopic analyses. Here, we measured stationary-phase cell volumes for the ancestor and 12 populations at 2,000, 10,000, and 50,000 generations, including measurements during exponential growth at the last timepoint. We measured the distribution of cell volumes for each sample using a Coulter counter and microscopy, the latter of which also provided data on cell shape. Our data confirm the trend toward larger cells, while also revealing substantial variation in size and shape across replicate populations. Most populations first evolved wider cells, but later reverted to the ancestral length-to-width ratio. All but one population evolved mutations in rod-shape maintenance genes. We also observed many ghost-like cells in the only population that evolved the novel ability to grow on citrate, supporting the hypothesis that this lineage struggles with maintaining balanced growth. Lastly, we show that cell size and fitness remain correlated across 50,000 generations. Our results suggest larger cells are beneficial in the experimental environment, while the reversion toward ancestral length-to-width ratios suggests partial compensation for the less favorable surface area-to-volume ratios of the evolved cells. ImportanceBacteria exhibit great morphological diversity, yet we have only a limited understanding of how their cell sizes and shapes evolve, and of how these features affect organismal fitness. This knowledge gap reflects, in part, the paucity of the fossil record for bacteria. Here, we revive and analyze samples extending over 50,000 generations from 12 populations of experimentally evolving Escherichia coli to investigate the relation between cell size, shape, and fitness. Using this "frozen fossil record" we show that all 12 populations evolved larger cells concomitant with increased fitness, with substantial heterogeneity in cell size and shape across the replicate lines. Our work demonstrates that cell morphology can readily evolve and diversify, even among populations living in identical environments.

evolutionary biology↗

bioRxiv2020

Maintenance of metabolic plasticity despite relaxed selection in a long-term evolution experiment with Escherichia coli

AO_SCPLOWBSTRACTC_SCPLOWTraits that are unused in a given environment are subject to processes that tend to erode them, leading to reduced fitness in other environments. Although this general tendency is clear, we know much less about why some traits are lost while others are retained, and about the roles of mutation and selection in generating different responses. We addressed these issues by examining populations of a facultative anaerobe, Escherichia coli, that have evolved for >30 years in the presence of oxygen, with relaxed selection for anaerobic growth and the associated metabolic plasticity. We asked whether evolution led to the loss, improvement, or maintenance of anaerobic growth, and we analyzed gene expression and mutational datasets to understand the outcomes. We identified genomic signatures of both positive and purifying selection on aerobic-specific genes, while anaerobic-specific genes showed clear evidence of relaxed selection. We also found parallel evolution at two interacting loci that regulate anaerobic growth. We competed the ancestor and evolved clones from each population in an anoxic environment, and we found that anaerobic fitness had not decayed, despite relaxed selection. In summary, relaxed section does not necessarily reduce an organisms fitness in other environments. Instead, the genetic architecture of the traits under relaxed selection and their correlations with traits under positive and purifying selection may sometimes determine evolutionary outcomes.

evolutionary biology↗

bioRxiv2020

A Simple Test to Infer Mode of Selection in Metagenomics Time Series of Evolving Asexual Populations

A general method to infer both positive and purifying selection during the real-time evolution of hypermutator pathogens would be broadly useful. To this end, we introduce a simple test to infer mode of selection (STIMS) from metagenomic time series of evolving microbial populations. We test STIMS on metagenomic data generated by simulations of bacterial evolution, and on metagenomic data spanning 62,750 generations of Lenskis long-term evolution experiment with Escherichia coli (LTEE). In both cases, STIMS recovers signals of positive and purifying selection on gold standard sets of genes. Using STIMS, we find strong evidence of ongoing positive selection on key regulators of the E. coli gene regulatory network, even in some hypermutator populations. STIMS also detects positive selection on regulatory genes in hypermutator populations of Pseudomonas aeruginosa that adapted to subinhibitory concentrations of colistin - an antibiotic of last resort - for just twenty-six days of laboratory evolution. Our results show that the fine-tuning of gene regulatory networks is a general mechanism for rapid and ongoing adaptation. The simplicity of STIMS, together with its intuitive visual interpretation, make it a useful test for positive and purifying selection in metagenomic data sets that track the evolution of hypermutator populations in real-time.

evolutionary biology↗

bioRxiv2020

Genomic and phenotypic evolution of Escherichia coli in a novel citrate-only resource environment

Evolutionary innovations allow populations to colonize new, previously inaccessible ecological niches. We previously reported that aerobic growth on citrate (Cit+) evolved in a population of Escherichia coli during adaptation to a minimal glucose medium containing citrate (DM25). Cit+ can grow in citrate-only medium (DM0), which is a novel environment for E. coli. To study adaptation to this new niche, we evolved one set of Cit+ populations for 2,500 generations in DM0 and a control set in DM25. We identified numerous parallel mutations, many mediated by transposable elements. Several lineages evolved multi-copy amplifications containing the maeA gene, constituting up to [~]15% of the genome. We also found substantial cell death in ancestral and evolved clones. Our results demonstrate the importance of copy-number variation and transposable elements in the refinement of the Cit+ trait. However, the observed mortality suggests a persistent evolutionary mismatch between E. coli physiology and a citrate-only environment.

evolutionary biology↗