Biology subjects

Whitaker, R. J.

Publications and source records attributed to Whitaker, R. J..

15 recordsLinked to original sources

bioRxiv2026

Within-host population structure, migration, and parallel adaptive evolution of Pseudomonas aeruginosa in cystic fibrosis lung disease

Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of [~]1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Further, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection. IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial dataset containing 450 isolates from different lobes of the right lung across three timepoints from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

microbiology↗

bioRxiv2026

Provirus induction diversifies adaptive variation in Pseudomonas aeruginosa lysogen populations

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that forms chronic infections in people with cystic fibrosis. Often P. aeruginosa strains are lysogens, infected with proviruses, that are induced by cellular stressors which include challenge with antibiotics such as ciprofloxacin. We asked how infection of P. aeruginosa with Mu-like provirus DMS3 would alter the evolution of antibiotic resistance through serial transfer of six replicates of four strain backgrounds, with and without sublethal doses of ciprofloxacin over 20 days. Through population metagenome sequencing we found that inducing lysogen populations had significantly higher diversity in adaptive mutations than non-lysogens after 20 days. Lysogen populations have more co-existing mutations within the population and a higher number of mutations in adaptive genes, including antibiotic efflux pump and gyrase alleles, and CRISPR-Cas loci. These data suggest that viral induction shifts the adaptive regime of a clonal population from one of stepwise periodic selective sweeps to recurrent competing mutations. Our results demonstrate the way that provirus infection can shape the evolutionary trajectory of their hosts in an environment-dependent manner. SignificanceMost long-term chronic infections of Pseudomonas aeruginosa in cystic fibrosis patients trace their origin to a single ancestor. During long-term chronic infection the evolution of each unique ancestor strain, defined by its starting genotype, can dramatically impact the success of future treatment efforts. Often the common ancestral Pseudomonas aeruginosa genotype is infected by a virus; this in turn shapes the evolution of bacterial cells within the human island of the lung. This work shows that when the nested virus infecting Pseudomonas aeruginosa is the very common transposable Mu-like virus, it can cause the bacterial population to diversify in response to antibiotic treatment in ways that may complicate future antibiotic treatment and phage therapy.

microbiology↗

bioRxiv2025

Integron-associated structures underlie the accumulation of antibiotic resistance genes on plasmids beyond cassette arrays

Rising multidrug-resistant (MDR) bacteria threatens global public health. While antibiotic selection preserves MDR plasmids, how mobile genetic elements (MGEs) drive the antibiotic resistance gene (ARG) accumulation to form complex plasmid architectures remain elusive. Here, we demonstrate that MDR plasmid evolution follows a hierarchical scheme, where progressive combinations of integrons, IS26, and ISCR elements drive stepwise increases in ARG load. The combination of all these three MGEs were significantly linked to the highest ARG load on plasmids. In the presence of integrons or ISCR elements, ARG numbers in plasmids are strongly and positively correlated with IS26 copy numbers. Such correlations were further validated by Escherichia coli plasmids collected from human and pig wastewater. We propose an integron-IS26-ISCR (3I) framework with four MDR plasmid evolutionary stages of increasing ARG accumulation potential. The "3I" framework refines predictions of MDR plasmid emergence and spread, providing universal genetic markers for cost-effective surveillance.

microbiology↗

bioRxiv2025

Evolutionary trends in Bombella apis CRISPR-Cas systems

Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found in Bombella apis, a microbe associated with honey bee queens, brood, and royal jelly. Unlike other honey bee microbiome members, B. apis does not colonize the worker bee midgut or hindgut and has therefore been understudied with regards to its importance in the honey bee colony. However, B. apis appears to play beneficial roles in the colony, by protecting developing brood from fungal pathogens and by bolstering their development under nutritional stress. Previously we identified CRISPR-Cas systems as being acquired by B. apis in its transition to bee association, as they are absent in a sister clade. Here we assess the variation and distribution of CRISPR-Cas types across B. apis strains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the same B. apis genomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for each B. apis strain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that the B. apis CRISPR-Cas systems are dynamic, that microbes in the same niche have unique spacers which supports the functionality of these CRISPR-Cas systems, and that acquisition of new spacers may be occurring in multiple locations in the genome, allowing for a flexible antiviral arsenal for the microbe. IMPORTANCEHoney bee worker gut microbes have been implicated in everything from protection from pathogens to breakdown of complex polysaccharides in the diet. However, there are multiple niches within a honey bee colony that host a different group of microbes, including the acetic acid bacterium Bombella apis. B. apis is found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen digestive tract. The roles that B. apis may serve in these environments are just beginning to be discovered and include production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bol-stering their nutrition. Niche specificity in B. apis may be affected by the pressures of bacteriophage and other mobile elements which may target different strains in each specific bee environment. Studying the interplay between B. apis and its mobile genetic elements (MGEs) may help us better understand microbial community dynamics within the colony and the potential ramifications for the honey bee host.

genomics↗

bioRxiv2024

Plasmid transmission dynamics and evolution of partner quality in a natural population of Rhizobium leguminosarum

Many bacterial traits important to host-microbe symbiosis are determined by genes carried on extrachromosomal replicons such as plasmids, chromids, and integrative and conjugative elements. Multiple such replicons often coexist within a single cell and, due to horizontal mobility, have patterns of variation and evolutionary histories that are distinct from each other and from the bacterial chromosome. In nitrogen-fixing Rhizobium, genes carried on multiple plasmids make up almost 50% of the genome, are necessary for the formation of symbiosis, and underlie bacterial traits including host plant benefits. Thus the genomics and transmission of plasmids in Rhizobium underlie the ecology and evolution of this important model symbiont. Here we leverage a natural population of clover-associated Rhizobium in which partner quality has declined in response to long-term nitrogen fertilization. We use 62 novel, reference-quality genomes to characterize 257 replicons in the plasmidome and study their genomics and transmission patterns. We find that, of the four most frequent plasmid types, two (types II & III) have more stable size, larger core genomes, and track the chromosomal phylogeny (display more vertical transmission), while others (types I & IV - the symbiosis plasmid, or pSym) vary substantially in size, shared gene content, and have phylogenies consistent with frequent horizontal transmission. We also find differentiation in pSym subtypes driven by long-term nitrogen fertilization. Our results highlight the variation in plasmid transmission dynamics within a single symbiont and implicate plasmid horizontal transmission in the evolution of partner quality. IMPORTANCEUnderstanding how bacterial genes move through natural populations is critical for understanding how bacterial traits evolve. The nitrogen-fixing bacterium Rhizobium leguminosarum lives in symbiosis with plants and is a model for studying how gene transmission from one cell to another on mobile genetic elements called plasmids impacts the evolution of bacteria and plants. Here we characterize the genomes of a natural bacterial population, then use novel approaches to show that mechanisms of plasmid gene transmission varies across multiple plasmid types possessed by R. leguminosarum. We find that changes in plasmid genes are associated with the decline of symbiotic partner quality in strains isolated from environments undergoing long-term fertilization. Together, these results underscore the importance of plasmid evolution in shaping ecosystem processes like nitrogen cycling. Our study provides a framework for probing the plasmid dynamics within natural bacterial populations and how plasmid transmission affects genetic diversity and ecological interactions in bacteria.

genomics↗

bioRxiv2023

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 populations as they resolve CRISPR autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage, and show that lysogen evolution is profoundly impacted by CRISPR autoimmunity and phage transposition around the chromosome. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in exponential phase corresponded to a difference in the type of CRISPR self-targeting resolution, mediated either by host mutation or phage transposition. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that have lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts. ImportanceThe chronic opportunistic multi-drug resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of CRISPR-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

microbiology↗

bioRxiv2022

CRISPR comparison toolkit (CCTK): Rapid identification, visualization, and analysis of CRISPR array diversity

CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, the historical record of spacer acquisitions and deletions represented by arrays has not previously been used to reconstruct strain histories. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.

bioinformatics↗

bioRxiv2021

Prediction of prophages and their host ranges in pathogenic and commensal Neisseria species

The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one other, primarily via transformation and homologous recombination, and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from other prophages and MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCEDrug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus, and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) have been identified and characterized in Neisseria species. In this study, we identified a large number of candidate phages integrated within the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.

microbiology↗

bioRxiv2020

Discovering the Molecular Determinants of Phaeobacter inhibens susceptibility to Phaeobacter phage MD18

Bacteriophage technologies have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. Here, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and dissected its mechanism of infection. Phaeobacter phage MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. Consistent with this finding, the sequence of the MD18 revealed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, GO term enrichment, and protein associations reveals that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens. ImportanceBacteriophages are useful non-antibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identifies Phaeobacter phage MD18, the first documented phage of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggests that the P haeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identifies genes that modulate host susceptibility to phage MD18 and implicates the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

microbiology↗

bioRxiv2019

The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification

As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested protection networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the protection network plays a key role in the coevolutionary dynamics.

evolutionary biology↗

bioRxiv2019

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1-3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

biochemistry↗

bioRxiv2019

Modeling the control of bacterial infections via antibiotic-induced proviruses

Most bacteria and archaea are infected by latent viruses that change their physiology and responses to environmental stress. We use a population model of the bacteria-phage relationship to examine the role that latent phage play on the bacterial population over time in response to antibiotic treatment. We demonstrate that the stress induced by antibiotic administration, even if bacteria are resistant to killing by antibiotics, is sufficient to control the infection under certain conditions. This work expands the breadth of understanding of phage-antibiotic synergy to include both temperate and chronic viruses persisting in their latent form in bacterial populations.\n\nImportanceAntibiotic-resistance is a growing concern for management of common bacterial infections. Here we show that antibiotics can be effective at sub-inhibitory levels when bacteria carry latent phage. Our findings suggest that specific treatment strategies based on the identification of latent viruses in individual bacterial strains may be an effective personalized medicine approach to antibiotic stewardship.

ecology↗

bioRxiv2018

Revealing S-layer Functions in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus

The crystalline surface layer (S-layer), consisting of two glycoproteins SlaA and SlaB, is considered to be the exclusive component of the cell envelope outside of the cytoplasmic membrane in Sulfolobus species. Although biochemically and structurally characterized, the S-layer in vivo functions remain largely elusive in Archaea. Here, we investigate how the S-layer genes contribute to the S-layer architecture and affect cellular physiology in a crenarchaeal model, Sulfolobus islandicus M.16.4. Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact, partially, or completely detached SlaA are observed for the {triangleup}slaB mutant. Importantly, we identify a novel S-layer-associated protein M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Additionally, we find that mutants deficient in SlaA form large cell aggregates and the individual cell size varies significantly. The slaB gene deletion also causes noticeable cellular aggregation, but the size of those aggregates is smaller when compared to {triangleup}slaA and {triangleup}slaAB mutants. We further show the {triangleup}slaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. Finally, we demonstrate that the {triangleup}slaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells where the cell cycle is tightly regulated. Together these data suggest that the lack of slaA results in either cell fusion or irregularities in cell division. Our studies provide novel insights into the physiological and cellular functions of the S-layer in Archaea.\n\nSignificanceRediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to understanding the archaeal cell is the S-layer which is ubiquitous in Archaea but whose in vivo function is unknown. In this study, we genetically dissect how the two well-known S-layer genes as well as a newly identified S-layer-associated-protein-encoding gene contribute to the S-layer architecture in a hyperthermophilic crenarchaeal model S. islandicus. We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or cell fusion.

microbiology↗

bioRxiv2018

The essential genome of the crenarchaeal model Sulfolobus islandicus

Sulfolobus islandicus is a model experimental system in the TACK superphylum of the Archaea, a key lineage in the evolutionary history of cell biology. Here we report a genome-wide identification of the repertoire of genes essential to S. islandicus growth in culture. We confirm previous targeted gene knockouts, uncover the non-essentiality of functions assumed to be essential to the Sulfolobus cell, including the proteinaceous S-layer, and highlight key essential genes whose functions are yet to be determined. Phyletic distributions illustrate the potential transitions that have occurred during the evolution of this contemporary archaeal cell and highlight the sets of genes that may have been associated with each transition. We use this comparative context as a lens to focus future research on archaea-specific uncharacterized essential genes for which future functional data would provide valuable insights into the evolutionary history of the contemporary cell.

microbiology↗

bioRxiv2018

Viral Fitness Across a Continuum from Lysis to Latency

The prevailing paradigm in ecological studies of viruses and their microbial hosts is that the reproductive success of viruses depends on the proliferation of the \"predator\", i.e., the virus particle. Yet, viruses are obligate intracellular parasites, and the virus genome - the actual unit of selection - can persist and proliferate from one cell generation to the next without lysis or the production of new virus particles. Here, we propose a theoretical framework to quantify the invasion fitness of viruses using an epidemiological cell-centric metric that focuses on the proliferation of viral genomes inside cells instead of virus particles outside cells. This cell-centric metric enables direct comparison of viral strategies characterized by obligate killing of hosts (e.g., via lysis), persistence of viral genomes inside hosts (e.g., via lysogeny), and strategies along a continuum between these extremes (e.g., via chronic infections). As a result, we can identify environmental drivers, life history traits, and key feedbacks that govern variation in viral propagation in nonlinear population models. For example, we identify threshold conditions given relatively low densities of susceptible cells and relatively high growth rates of infected cells in which lysogenic and other chronic strategies have higher potential viral reproduction than lytic strategies. Altogether, the theoretical framework helps unify the ongoing study of eco-evolutionary drivers of viral strategies in natural environments.

ecology↗