Biology subjects

Gallie, J.

Publications and source records attributed to Gallie, J..

13 recordsLinked to original sources

bioRxiv2026

Codon degeneracy contributes to divergent fitness effects of rare tRNAs with A-starting anticodons

Transfer RNA (tRNA) repertoires vary greatly across genomes, shaped by genetic drift and selection. A peculiar pattern across prokaryotes is the near-complete absence of tRNAs with unmodified adenine at the 34th (wobble) position (i.e., tRNAANN). Each of these tRNAs are just a single mutation away from several other tRNAs. Hence, their persistent absence suggests fundamental but hitherto unclear constraints. We engineered 36 Escherichia coli strains expressing tRNAs carrying each theoretically possible ANN anticodon to determine their functionality and fitness effects. Notably, there was no evidence of broad toxicity due to these tRNAs. All five tRNAANN tested underwent post-transcriptional maturation and all seven tested compensated for the deletion of their respective native tRNABNN (carrying G, C or U at the 34th position), demonstrating that tRNAANN are translationally active. Furthermore, tRNAANN from four-fold degenerate (4D) codon boxes were unmodified and were generally neutral or beneficial, whereas tRNAANN from two-fold degenerate (2D) boxes underwent A34-to-I34 modification and were more likely to impair fitness. We suggest superwobbling by tRNAANN -- decoding an entire four-codon set -- as one mechanism underlying these differential fitness effects. Maximal degeneracy in 4D boxes buffers or exploits tRNAANN superwobbling via synonymous decoding, whereas constrained degeneracy in 2D boxes renders it deleterious, likely through amino acid misincorporation. Thus, these differential fitness effects, sharpens the paradox of neutral or beneficial yet absent 4D tRNAANN, while beginning to empirically unravel underlying causes for the absence of 2D tRNAANN.

evolutionary biology↗

bioRxiv2026

Compensatory evolution facilitates loss of prfB autoregulation in Pseudomonas fluorescens SBW25

Understanding why some traits are maintained whereas others are repeatedly lost is a central question in evolutionary biology. Here, we address this problem through an analysis of the evolutionary dynamics of autoregulation of the prfB gene, which encodes peptide-chain release factor 2 (RF2), a key factor in bacterial translation termination. RF2 recognizes UGA and UAA stop codons and catalyzes the release of the completed polypeptide. In many species, prfB contains an internal UGA stop codon, triggering premature translation termination by RF2 itself. Complete RF2 translation depends on a +1 programmed ribosomal frameshifting (PRF) event on the internal stop codon, which occurs more frequently when RF2 levels are low, resulting in autoregulation of prfB expression. While widespread, this autoregulatory mechanism has been lost in multiple bacterial lineages. We combined phylogenetics, experimental evolution and molecular genetics to investigate the evolutionary forces behind this loss. We found no significant correlation between PRF loss and stop codon usage using phylogenetically informed analyses, and PRF disruption in Pseudomonas fluorescens SBW25 had no detectable fitness effect. However, engineered mutations that reduced frameshifting caused fitness defects, which were compensated by two classes of mutation: (i) mutations that impair specific ribosomal proteins, and (ii) single-nucleotide deletions in prfB that adjust the reading frame and bypass the internal stop codon. These results suggest that compensatory mutations facilitate the loss of prfB autoregulation under RF2-limiting conditions. We discuss three potential scenarios that could account for this process.

evolutionary biology↗

bioRxiv2024

Empirical evidence of a role for insertion sequences in the repair of DNA breaks in bacterial genomes

Insertion Sequences (ISs) are mobile pieces of DNA that are widespread in bacterial genomes. IS movements typically involve (i) excision of the IS element, (ii) cutting of the target site DNA, and (iii) IS element insertion. This process generates a new copy of the IS element, as well as a short duplication at the target site. It has been noted that, when observing extant IS element copies in a genome, occasionally no Target Site Duplication (TSD) is readily identifiable. This has been attributed to degeneration of the TSD at some point after the insertion event. Here, we provide evidence that some IS movement events - namely, those that occur in association with large-scale genome rearrangements - occur without generating TSDs. In support of this hypothesis, we provide two direct, empirical observations of such IS transposition events: an IS481 movement occurring with a large duplication in Pseudomonas fluorescens SBW25, and an IS5/IS1182 movement plus a large deletion in Escherichia coli C. Additionally, we use sequencing data from the Lenski long-term evolution experiment to provide a further 14 examples of IS150 movements in E. coli B that are associated with large deletions and do not carry TSDs. Overall, our results indicate that some IS elements can insert into, and thus repair, existing DNA breaks in bacterial genomes.

evolutionary biology↗

bioRxiv2022

Large-scale duplication events underpin population-level flexibility in bacterial tRNA gene copy number

The complement of tRNA genes within a genome is typically considered to be a (relatively) stable characteristic of an organism. Here we demonstrate that bacterial tRNA gene set composition can be more flexible than previously appreciated, particularly regarding tRNA gene copy number. We report the high-rate occurrence of spontaneous, large-scale, tandem duplication events in laboratory populations of the bacterium Pseudomonas fluorescens SBW25. The identified duplications are up to [~]1 Mb in size ([~]15 % of the wildtype genome) and are predicted to change the copy number of up to 917 genes, including several tRNA genes. The observed duplications are inherently unstable: they occur, and are subsequently lost, at extremely high rates. We propose that this unusually plastic type of mutation provides a mechanism by which tRNA gene set diversity can be rapidly generated, while simultaneously preserving the underlying tRNA gene set in the absence of continued selection. That is, if a tRNA set variant provides no fitness advantage, then high-rate segregation of the duplication ensures the maintenance of the original tRNA gene set. However, if a tRNA gene set variant is beneficial, the underlying duplication fragment(s) may persist for longer and provide raw material for further, more stable, evolutionary change.

evolutionary biology↗

bioRxiv2022

E. coli-{varphi}X174 genotype to phenotype map reveals flexibility and diversity in LPS structures

Phage therapy is a promising method for the treatment of multi-drug-resistant bacterial infections. However, its long-term efficacy depends on understanding the evolutionary effects of the treatment. Current knowledge of such evolutionary effects is lacking, even in well-studied systems. We used the bacterium Escherichia coli C and its bacteriophage {Phi}X174, which infects cells using host lipopolysaccharide (LPS) molecules. We first generated 31 bacterial mutants resistant to {Phi}X174 infection. Based on the genes disrupted by these mutations, we predicted that these E. coli C mutants collectively produce eight unique LPS structures. We then developed a series of evolution experiments to select for {Phi}X174 mutants capable of infecting the resistant strains. During phage adaptation, we distinguished two types of phage resistance: one that was easily overcome by {Phi}X174 with few mutational steps ("easy" resistance), and one that was more difficult to overcome ("hard" resistance). We found that increasing the diversity of the host and phage populations could accelerate the adaptation of phage {Phi}X174 to overcome the hard resistance phenotype. From these experiments, we isolated 16 {Phi}X174 mutants that, together, can infect all 31 initially resistant E. coli C mutants. Upon determining the infectivity profiles of these 16 evolved phages, we uncovered 14 distinct profiles. Given that only eight profiles are anticipated if the LPS predictions are correct, our findings highlight that the current understanding of LPS biology is insufficient to accurately forecast the evolutionary outcomes of bacterial populations infected by phage.

evolutionary biology↗

bioRxiv2022

Distinguishing between canonical and non-canonical tRNA genes reveals that Thermococcaceae adhere to the standard archaeal tRNA gene set

Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

genomics↗

bioRxiv2022

The layered costs and benefits of translational redundancy

The rate and accuracy of translation hinges upon multiple components - including transfer RNA (tRNA) pools, tRNA modifying enzymes, and rRNA molecules - many of which are redundant in terms of gene copy number or function. It has been hypothesized that the redundancy evolves under selection, driven by its impacts on growth rate. However, we lack empirical measurements of the fitness costs and benefits of redundancy, and we have poor understanding of how this redundancy is organized across components. We manipulated redundancy in multiple translation components of Escherichia coli by deleting 28 tRNA genes, 3 tRNA modifying systems, and 4 rRNA operons in various combinations. We find that redundancy in tRNA pools is beneficial when nutrients are plentiful, and costly under nutrient limitation. This nutrient-dependent cost of redundant tRNA genes stems from upper limits to translation capacity and growth rate, and therefore varies as a function of the maximum growth rate attainable in a given nutrient niche. The loss of redundancy in rRNA genes and tRNA modifying enzymes had similar nutrient-dependent fitness consequences. Importantly, these effects are also contingent upon interactions across translation components, indicating a layered hierarchy from copy number of tRNA and rRNA genes to their expression and posttranscriptional modification. Overall, our results indicate both positive and negative selection on redundancy in translation components, depending on a species evolutionary history with feasts and famines.

evolutionary biology↗

bioRxiv2021

Evolutionary flexibility in routes to mat formation by Pseudomonas

Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharide(s), the production of which is often controlled by the secondary messenger c-di-GMP. Mechanisms of mat formation have been particularly well characterized in Pseudomonas fluorescens SBW25; mutations that lead to an increase in c-di-GMP production by diguanylate cyclases (WspR, AwsR, or MwsR) result in the secretion of cellulose, and mat formation. Here, we characterize and compare mat formation in two close relatives of SBW25: Pseudomonas simiae PICF7 and Pseudomonas fluorescens A506. We find that PICF7 - the strain more closely related to SBW25 - can form mats through mutations affecting the activity of the same three diguanylate cyclases as SBW25. However, instead of cellulose, these mutations activate the production of the Pel exopolysaccharide. We also provide evidence for at least two further - as yet uncharacterized - routes to PICF7 mat formation. P. fluorescens A506, while retaining the same mutational routes to mat formation as SBW25 and PICF7, forms mats by a semi-heritable mechanism that likely culminates in Pga and/or Psl production. Overall, our results demonstrate a high level of evolutionary flexibility in the molecular and structural routes to mat formation, even among close relatives.

evolutionary biology↗

bioRxiv2021

Host-parasite coevolution promotes innovation through deformations in fitness landscapes

During the struggle for survival, populations occasionally evolve new functions that give them access to untapped ecological opportunities. Theory suggests that coevolution between species can promote the evolution of such innovations by deforming fitness landscapes in ways that open new adaptive pathways. We directly tested this idea by using high throughput gene editing-phenotyping technology (MAGE-Seq) to measure the fitness landscape of a virus, bacteriophage {lambda}, as it coevolved with its host, the bacterium Escherichia coli. Through computer simulations of {lambda}s evolution on the empirical fitness landscape, we showed that {lambda} was more likely to evolve to use a new receptor if it experienced a shift in its fitness landscape caused by coevolution. This result was further validated by additional laboratory experiments. This study provides direct evidence for the role of coevolution in driving evolutionary novelty and provides a quantitative framework for predicting evolution in coevolving ecological communities.

evolutionary biology↗

bioRxiv2021

Sustained coevolution of phage Lambda and Escherichia coli involves inner as well as outer membrane defenses and counter-defenses

Bacteria often evolve resistance to phage through the loss or modification of cell-surface receptors. In Escherichia coli and phage {lambda}, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyze another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair {lambda}s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that {lambda} evolved an additional counter-defense that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral {lambda}, but some evolved phage grew well on the deletion mutants, indicating they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfills the model of an inverse-gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated. IMPACT STATEMENTLaboratory studies of coevolution help us understand how host defenses and pathogen counter-defenses change over time, which is often essential for predicting the future dynamics of host-pathogen interactions. One particular model, termed "inverse-gene-for-gene" coevolution, predicts that coevolution proceeds through alternating steps, whereby hosts lose the features exploited by pathogens, and pathogens evolve to exploit alternative features. Using a classic model system in molecular biology, we describe the nature and timing of a previously overlooked step in the coevolution of E. coli and bacteriophage lambda. Our work demonstrates that this mode of coevolution can profoundly re-shape the interactions between bacteria and phage.

microbiology↗

bioRxiv2020

The birth of a bacterial tRNA gene

Organisms differ in the types and numbers of tRNA genes that they carry. While the evolutionary mechanisms behind tRNA gene set evolution have been investigated theoretically and computationally, direct observations of tRNA gene set evolution remain rare. Here, we report the evolution of a tRNA gene set in laboratory populations of the bacterium Pseudomonas fluorescens SBW25. The growth defect caused by deleting the single-copy tRNA gene, serCGA, is rapidly compensated by large-scale (45-290 kb) duplications in the chromosome. Each duplication encompasses a second, compensatory tRNA gene (serTGA) and is associated with a rise in tRNA-Ser(UGA) in the mature tRNA pool. We postulate that tRNA-Ser(CGA) elimination increases the translational demand for tRNA-Ser(UGA), a pressure relieved by increasing serTGA copy number. This work demonstrates that tRNA gene sets can evolve through duplication of existing tRNA genes, a phenomenon that may contribute to the presence of multiple, identical tRNA gene copies within genomes.

evolutionary biology↗

bioRxiv2018

Repeated phenotypic evolution by different genetic routes: the evolution of colony switching in Pseudomonas fluorescens SBW25

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25 (Beaumont et al., 2009). The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere (Beaumont et al., 2009; Gallie et al., 2015). Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these - a non-synonymous point mutation in the housekeeping sigma factor rpoD - is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB (Beaumont et al., 2009). A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression (Remigi et al., 2018). We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosome expression, and ultimately an increase in CAP expression.

evolutionary biology↗

bioRxiv2017

Identification And Characterization Of Domesticated Bacterial Transposases

Selfish genetic elements (SGEs), such as insertion sequences (ISs) and transposons are found in most genomes. Transposons are usually identifiable by their high copy number within genomes. In contrast REP associated tyrosine transposases (RAYTs), a recently described class of bacterial transposase, are typically present at just one copy per genome. This suggests that RAYTs no longer copy themselves and thus they no longer function as a typical transposase. Motivated by this possibility we interrogated thousands of fully sequenced bacterial genomes in order to determine patterns of RAYT diversity, their distribution across chromosomes and accessory elements, and rate of duplication. RAYTs encompass exceptional diversity and are divisible into at least five distinct groups. They possess features more similar to housekeeping genes than insertion sequences, are predominantly vertically transmitted and have persisted through evolutionary time to the point where they are now found in 24% of all species for which at least one fully sequenced genome is available. Overall, the genomic distribution of RAYTs suggests that they have been co-opted by host genomes to perform a function that benefits the host cell.

evolutionary biology↗