Abstract Predicting which receptor a phage binds to from genome sequence alone has remained an intractable challenge, principally because the experimental phenotypic data required to train and validate predictive models have not been available at sufficient scale. Here we address this by conducting 1,050 genome-wide genetic screens across 255 taxonomically diverse Escherichia coli dsDNA phages, assigning host receptors to 193 phages across 19 receptor classes. Comparative genomics and AlphaFold3 structural modelling resolved the sequence determinants of specificity to defined receptor-binding protein domains and individual residues. Machine learning models trained on this dataset predicted host receptor identity from phage genome sequence alone without prior annotation of receptor-binding genes, achieving perfect precision and greater than 80% recall on 49 independently validated phages, and yielding predictions for 1,060 of 1,875 E. coli phage genomes in NCBI. Domain swaps redirected receptor specificity as predicted, and a single amino acid substitution proved both necessary and sufficient to switch recognition between two distinct porins. These results demonstrate that systematic phenotyping at scale makes sequence-based prediction of molecular interaction specificity tractable, with direct implications for phage-based medicine, microbiome engineering and the broader challenge of inferring host-pathogen interaction outcomes from sequence.

The mobilome, defined as the collection of mobile genetic elements within a bacterial genome, plays a role in the adaptation of bacteria to abiotic and biotic drivers. In particular, prophages have been reported to contribute to bacterial resistance to virulent bacteriophages, to competitive interactions among bacterial hosts within microbial communities and to pathogenicity and virulence. It is, therefore, critical to better understand the role of prophages in distributing genes and functions within and among bacterial species to predict how bacteria adapt to their biotic environment. Pseudomonas syringae offers an ideal study system to ask these questions, both because of its broad range of lifestyles (spanning from environmental growth to plant pathogens) and its high intraspecies diversity. To examine the role of prophages in this species complex, we compared 587 genomes available from public databases and annotated the defence mechanisms, effectors and prophages in the genomes. We found that this species complex has an elaborate phage pandefensome consisting of 139 defence mechanisms. Assessing taxonomical signatures of the observed prophages uncovered broad differences in the types and numbers of genes encoded by different phage families, emphasizing how the evolutionary advantages conferred to hosts can depend on the prophage composition and offering insight into how these genes might disperse within a community. Our study highlights the intimate association of specific phage families with their hosts and their potential role in shaping key ecological traits of these important species.

Host-associated bacteria are found across the tree of life. In this opinion article, we propose that population genetics theory can be used to probe the conditions that form the path toward such symbioses. We illustrate how mutation-selection models generate insights into the maintenance of a symbiont under transmission between generations and from the environment. We outline how basic features of host population size and life history shape the fixation of a heritable symbiont in a host population, suggesting elevated fixation probabilities in long-lived hosts. Finally, whenever the fitness effects of the symbiont vary over time, reduced efficiency of selection increases the fixation of a deleterious symbiont. Our predictions of the properties of hosts, symbionts, and their ecological contexts that impact symbiont establishment frame expectations across systems.

Predicting infection heterogeneity requires looking beyond the host. Phage–bacteria coevolution in tomato fields maintains diverse bacterial strategies and explains uneven disease occurrence across plant populations.

Advanced genome editing technologies have enabled rapid, flexible rewriting of the Escherichia coli genome, but most have not been tested in other bacterial species. Recombitrons—a genome editing tool created by pairing modified, donor-producing bacterial retrons with single-stranded binding and annealing proteins—have increased the efficiency of recombineering to install flexible, precise edits in the prokaryotic chromosome. Here, to extend their utility outside of E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is variable across the species tested but functional in all of them, reaching editing efficiencies >20

Adaptations that enable plant-associated bacteria to fill disparate niches comprise a critical component of microbial diversity. Genes that confer locally adaptive bacterial traits, ranging from heavy metal resistance to pathogen or symbiont infectivity, often reside within mobile genetic elements (MGEs) that can move between genomes. While MGEs may speed microbial adaptation, they also have selfish fitness interests and potentially separate evolutionary trajectories from their host genome. MGEs can also impose physiological burdens and be limited in the transmissibility of function across hosts, which likely constrains bacterial local adaptation. Given these constraints, the prevalence of adaptive loci on potentially exploitative MGEs poses a dilemma: how do fitness conflicts and alignments between MGEs and the main replicon shape bacterial local adaptation and impact plant hosts? We synthesize research on ways MGEs confer rapid, niche-specific fitness advantages to bacteria, identify factors that promote or constrain bacterial adaptation, and highlight MGE impacts on plants. We focus on large, self-transmissible MGEs (islands, plasmids, and prophages; though we expect relevance to other MGEs as well) to better understand how MGEs bolster yet constrain bacterial local adaptation. We specifically explore the role of MGEs in shaping bacteria that themselves play a role in expanding or contracting the plant niche.

Evolution is defined as the change in allele frequency over time as a result of either neutral processes, such as genetic drift, or as an adaptive process in response to selection. In contrast, ecological dynamics describe changes in population densities, species distributions, species interactions, and/or relative abundances within communities, all of which can also be the result of either stochastic or deterministic processes. Although the distinction between these patterns has long held for plants and animals, microbial community dynamics can blur the line between ecological and evolutionary processes, especially as they can occur on very similar timescales. Despite the importance of differentiating changes occurring within a population or strain from those occurring among populations, many common methodologies used to study microbiomes are not able to differentiate among them. In this review, we summarize the forces known to generate genetic diversity in bacterial genomes and describe the approaches used to study bacterial evolution from simple to more complex systems. We then explore how current methodologies and conceptual understanding can be applied to both understand and differentiate between the ecological and evolutionary processes in microbial communities. By highlighting lessons from longitudinal microbiome studies and experimental evolution, we explore the unique opportunities afforded by newer sequencing approaches and better sequencing resolution. Throughout, we identify the unique and outstanding challenges in studying these processes in microbiome systems and emphasize the great benefits in doing so to move forward our ability to better predict and manipulate microbiomes.

BACKGROUND:Parasites are foundational to ecosystem health both as indicator species of community productivity and as drivers of diversity. In bacterial communities, bacteriophage viruses can have such roles as they track and modulate the dynamic composition of bacterial hosts within an ecosystem. We aimed to test whether viromes can be used as broad signatures of microbiome health using previously published results across systems. METHODS:In this systematic review and meta-analysis, we searched PubMed, Google Scholar, Scopus, and Web of Science from Jan 22, 2022, to Sept 17, 2024, for peer-reviewed, primary literature published in English, using search terms "phage diversity", "microbiome", "virome", "virus", "phageome", "disease", and "dysbiosis". Inclusion criteria were: a comparison between a dysbiosis state and a healthy state in a human or animal host; a defined host organism and microbiome site; examination of the virome; an obtained measure of virome diversity (α, β, or both); use of statistical analysis to assess whether α or β diversity are changed in dysbiosis; and sufficient methodology description on viral isolation and on virus sequence analysis pipeline. We conducted a qualitative data analysis to assess factors explaining changes to virome diversity in dysbiosis. We then calculated response ratios for each study to test for overall patterns of virome α diversity change under disturbance. Finally, we conducted a quantitative analysis on studies from which we were able to obtain paired virome and bacteriome α diversity data to examine the correlation between these data in defined health compared with defined disturbance conditions. This study was not registered. FINDINGS:We identified a total of 74 studies for inclusion that spanned human (n=61), mouse (n=8), pig (n=3), dog (n=1), and cow (n=1) hosts and a diverse spectrum of infections and diseases. By comparing observed phage and bacterial diversity in microbiomes characterised by dysbiosis with those considered control populations, we were able to identify some key commonalities. Of the 69 studies that investigated changes to α diversity of the virome in dysbiosis, 28 (41%) reported significant changes, but with variable directional change. Of 38 datasets (from 30 studies) for which virome α diversity values were available, 22 (58%) gave a response ratio of less than 1 (α diversity decreases in dysbiosis) and 16 (42%) of more than 1 (α diversity increases in dysbiosis); however, in 27 (71%) datasets, 95% CIs overlapped with 1 (ie, no change in α diversity). We found shifting virome composition to be a more consistent signature of dysbiosis, with 47 (69%) of 68 studies reporting a significant change in viral β diversity with dysbiosis. 62 (89%) of 70 studies reported significant enrichment of system-specific viral taxa under dysbiosis. Our quantitative correlation analysis suggested that bacterial α diversity is a greater predictor of virome α diversity in healthy groups (mean r2=0·380; 95% CI 0·597-0·163) than in dysbiosis (mean r2=0·118, 0·223-0·012; sign test for asymmetric non-parametric data p=4·9 × 10-10). INTERPRETATION:Overall, although specific viral signatures of dysbiosis are likely to be highly disease-specific and condition-specific, we show that existing ecological theory shows promise in predicting the relationship between bacterial and phage diversity and in providing broad signatures of dysbiosis across disease systems. Our observation that the relationship between bacterial and phage diversity breaks down under disturbance suggests that this feature could be a useful signature of dysbiosis and that future studies incorporating the virome could provide opportunity to diagnose, treat, and better understand the causes of microbiome disturbance. FUNDING:There was no funding source for this study.

As parasites of bacteria, phages can regulate microbiome diversity and composition and may therefore affect susceptibility to pathogens and disease. Many infectious diseases are associated with altered bacteriophage communities, but observational studies alone do not allow us to determine when altered phage community composition is a contributor to disease risk, a response to infection, or simply an indicator of dysbiosis. To address this question directly, we used size-selective filtration to deplete plant-associated microbial communities of phages, then challenged plants with the bacterial pathogen Pseudomonas syringae. Plants with phage-depleted microbiomes were more susceptible to infection, an effect that could not be explained by direct effects of the phage communities on either P. syringae or the plant host. Moreover, the presence of phages was most impactful when the phage communities were isolated from neighboring field locations rather than from the same host plant as the bacteria, possibly suggesting that moderate rates of lysis maintain a community structure that is most resistant to pathogen invasion. Overall, our results support the idea that phage communities contribute to plant defenses by modulating the microbiome.

There is overwhelming evidence that the microbiome can be important to host physiology and fitness. As such, there is interest in and some theoretical work on understanding when hosts and microbiomes (co)evolve so that microbes benefit hosts and hosts favour beneficial microbes. However, the outcome of evolution likely depends on how microbes benefit hosts. Here, we use adaptive dynamics to investigate how host and symbiont evolution depend on whether symbionts increase host lifespan or host reproduction in a simple model of host and symbiont dynamics. In addition, we investigate 2 ways hosts release (and transmit) symbionts: by releasing symbionts steadily during their lifetime or by releasing them at reproduction, potentially increasing symbionts' chances of infecting the host's offspring. The former is strict horizontal transmission, whereas the latter is also a form of indirect or "pseudovertical" transmission. Our first key result is that the evolution of symbionts that benefit host fecundity requires pseudovertical transmission, while the evolution of symbionts that benefit host lifespan does not. Furthermore, our second key result is that when investing in host benefits is costly to the free-living symbiont stage, intermediate levels of pseudovertical transmission are needed for selection to favour beneficial symbionts. This is true regardless of fitness effects because release at reproduction increases the free-living symbiont population, which increases competition for hosts. Consequently, hosts could evolve away from traits that favour beneficial symbionts. Generally, our work emphasizes the importance of different forms of vertical transmission and fitness benefits in host, microbiome, and holobiont evolution as highlighted by our prediction that the evolution of fecundity-increasing symbionts requires parent-to-offspring transmission.

Advanced genome editing technologies have enabled rapid and flexible rewriting of the Escherichia coli genome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing in E. coli have not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering in E. coli by enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyond E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain.

Given the multitude of challenges Earth is facing, sustainability science is of key importance to our continued existence. Evolution is the fundamental biological process underlying the origin of all biodiversity. This phylogenetic diversity fosters the resilience of ecosystems to environmental change, and provides numerous resources to society, and options for the future. Genetic diversity within species is also key to the ability of populations to evolve and adapt to environmental change. Yet, the value of evolutionary processes and the consequences of their impairment have not generally been considered in sustainability research. We argue that biological evolution is important for sustainability and that the concepts, theory, data, and methodological approaches used in evolutionary biology can, in crucial ways, contribute to achieving the UN Sustainable Development Goals (SDGs). We discuss how evolutionary principles are relevant to understanding, maintaining, and improving Nature Contributions to People (NCP) and how they contribute to the SDGs. We highlight specific applications of evolution, evolutionary theory, and evolutionary biology's diverse toolbox, grouped into four major routes through which evolution and evolutionary insights can impact sustainability. We argue that information on both within-species evolutionary potential and among-species phylogenetic diversity is necessary to predict population, community, and ecosystem responses to global change and to make informed decisions on sustainable production, health, and well-being. We provide examples of how evolutionary insights and the tools developed by evolutionary biology can not only inspire and enhance progress on the trajectory to sustainability, but also highlight some obstacles that hitherto seem to have impeded an efficient uptake of evolutionary insights in sustainability research and actions to sustain SDGs. We call for enhanced collaboration between sustainability science and evolutionary biology to understand how integrating these disciplines can help achieve the sustainable future envisioned by the UN SDGs.

Parasites are foundational to ecosystem health both as indicator species of community productivity but also as drivers of diversity. In bacterial communities, bacteriophage viruses can play such a role as they track the dynamic composition of bacterial hosts, and in the case of lytic phages, confer a growth advantage to lower abundance bacteria while adapting to more common ones. We set out to test whether viromes can be used as signatures of microbiome health using previously published results across systems. By comparing observed phage and bacterial diversity between microbiomes characterized by disturbance (so-called dysbiosis) and those considered control populations, we were able to identify some key commonalities. While just under half of studies report significant changes in viral species richness in dysbiosis, just under two thirds of studies find the viral composition to shift in dysbiosis, with specific viral taxa enrichment acting as a common signature of dysbiosis. Our analyses also suggest that the positive relationship between bacteriome and virome alpha diversity observed in health breaks down under microbiome disturbance. Overall, while specific viral signatures of dysbiosis are likely to be highly disease- and condition-specific, existing ecological theory shows clear promise in predicting and explaining microbiome health. Future data on bacteria-phage diversity relationships may provide us with much needed opportunity to diagnose, treat, and better understand the causes of dysbiosis. Evidence before this study Being able to identify signatures of microbiome health (or lack thereof) has the potential to improve the way we diagnose and treat disease. To do this, the bacterial microbiome is traditionally characterised at the 16S taxonomic level, and changes in composition are linked to changes in disease status. More recently, the field of viromics has gained attention, and studies have begun to probe the relationship between the virome and health or disturbance (‘dysbiosis’). This work has focused to date on finding single phages that indicate presence of known pathogens, or in a few cases the relationship between viral diversity and disease. To our knowledge, no work has yet sought to identify a common signature of dysbiosis or find commonalities across systems that suggest a role for phages in dysbiosis. Decades of ecological theory has shown how parasites can shape the ecology and evolution of their hosts, and here we argue that bacteriophage viruses have the potential to shape these same processes within microbial communities. The motivation for the current work was thus to ask whether existing ecological theory could help us identify viral signatures of dysbiosis in the microbiome. Added value of this study This study employed a systematic review and meta-analysis to test whether and when phage communities can be used as signatures of microbiome health. To do this, we synthesized previously published results that measure composition of the virome between bacterial microbiomes characterised by health or dysbiosis. We found a total of 39 studies across human, mouse, pig and cow hosts that spanned a diverse spectrum of dysbioses, including bacterial infections, viral infections, and varied diseases such as cancer, cirrhosis, and inflammatory bowel disease, and identified a number of commonalities. Just under half of these studies reported a significant change in viral species richness in dysbiosis, and just under two thirds reported the viral composition to shift in dysbiosis. While the vast majority of studies report an enrichment of specific viral taxa associated with dysbiosis, there is little overlap among studies regarding the identity of these enriched taxa. Finally, our analysis provides evidence that the positive relationship between bacteriome and virome alpha diversity breaks down in dysbiosis. Implications of all available evidence Synthesis of the available evidence suggests that while looking for specific viral taxa as signatures may be limited to associations that are highly disease or condition specific, there is promise for the use of existing ecological theory in predicting and explaining microbiome health when considering compositional changes in the virome. Prospective studies should look to expand the data we have on bacteria-phage relationships at the level of species richness and community compositions, and we argue that more routinely investigating the virome or phageome, in addition to collecting 16S taxonomic descriptions of the microbial community, would help improve our ability to identify signatures of microbiome health. These viral signatures may offer early warning signs of microbiome disturbance and disease. This has clear relevance to our ability to diagnose, treat, and understand the underlying causes of disease. ### Competing Interest Statement The authors have declared no competing interest.

Microorganisms, including bacteria, archaea, viruses, fungi, and protists, are essential to life on Earth and the functioning of the biosphere. Here, we discuss the key roles of microorganisms in achieving the United Nations Sustainable Development Goals (SDGs), highlighting recent and emerging advances in microbial research and technology that can facilitate our transition toward a sustainable future. Given the central role of microorganisms in the biochemical processing of elements, synthesizing new materials, supporting human health, and facilitating life in managed and natural landscapes, microbial research and technologies are directly or indirectly relevant for achieving each of the SDGs. More importantly, the ubiquitous and global role of microbes means that they present new opportunities for synergistically accelerating progress toward multiple sustainability goals. By effectively managing microbial health, we can achieve solutions that address multiple sustainability targets ranging from climate and human health to food and energy production. Emerging international policy frameworks should reflect the vital importance of microorganisms in achieving a sustainable future.

Many infectious diseases are associated with altered communities of bacteriophage viruses (phages). As parasites of bacteria, phages can regulate microbiome diversity and composition and may therefore affect disease susceptibility. Yet observational studies alone do not allow us to determine whether altered phage profiles are a contributor to disease risk, a response to infection, or simply an indicator of dysbiosis. To address this question, we used size-selective filtration to separate plant-associated microbial communities from their respective phages, then transplanted them together or separately onto tomato plants that we subsequently challenged with the bacterial pathogen Pseudomonas syringae . Microbial and phage communities together were more disease-protective than either component was alone, an effect that could not be explained by direct effects of phages on either P. syringae or the plant host. Moreover, the protective effect of phages was strongest when microbial and phage communities were isolated from neighboring field locations (allopatric phages), rather than from the same host plant (sympatric phages). This suggests a Goldilocks effect in which moderate rates of phage lysis maintain a microbiome community structure that is most resistant to pathogen invasion. Overall, our results support the idea that phage communities contribute to plant defenses by modulating the microbiome.### Competing Interest StatementThe authors have declared no competing interest.

The design and use of synthetic communities, or SynComs, is one of the most promising strategies for disentangling the complex interactions within microbial communities, and between these communities and their hosts. Compared to natural communities, these simplified consortia provide the opportunity to study ecological interactions at tractable scales, as well as facilitating reproducibility and fostering interdisciplinary science. However, the effective implementation of the SynCom approach requires several important considerations regarding the development and application of these model systems. There are also emerging ethical considerations when both designing and deploying SynComs in clinical, agricultural or environmental settings. Here we outline current best practices in developing, implementing and evaluating SynComs across different systems, including a focus on important ethical considerations for SynCom research.

Following on from the discovery that innate immune pathways are shared widely across the tree of life comes another surprise: Hobbs et al. show that viruses targeting animals and bacteria also use highly conserved tools to fight back. Why such mechanisms remain seemingly unchanged despite the rapid coevolution among hosts and pathogens is now a key open question for the field.

The mobilome, defined as the collection of mobile genetic elements within a bacterial genome, plays a critical role in the adaptation of bacteria to abiotic and biotic drivers. In particular, prophages have been reported to contribute to bacterial resistance to virulent bacteriophages, the competitive interaction of bacterial hosts within microbial communities, and in pathogenicity and virulence. It is therefore critical to better understand the role of prophages in distributing genes and functions within and among bacterial species to predict how bacteria adapt to their biotic environment. Pseudomonas syringae offers an ideal study system to ask these questions both because of its broad range of lifestyles (spanning from environmental growth to plant pathogens) and its high intraspecies diversity. To examine the role of the mobilome in this species complex, we compared 590 genomes available from public databases and annotated the defense mechanisms, effectors, and prophages in the genomes. We found that this species complex has an elaborate phage pandefensome consisting of 139 defense mechanisms. Host-associated P. syringae isolates were found to have both elaborate phage defensomes and effectoromes. Assessing taxonomical signatures of the observed prophages uncovered broad differences in the types and numbers of genes encoded by different phage families, emphasizing how the evolutionary advantages conferred to hosts will depend on the prophage composition and offering insight to how these genes might disperse within a community. Our study highlights the intimate association of phage families with their hosts and uncovers their key role in shaping ecology for this widespread species complex. Significance statement The bacterial accessory genome, including the mobilome and prophages, plays a critical role in shaping bacterial adaptation to abiotic and biotic drivers. These prophages are widespread across bacterial taxa and likely maintained because of their evolutionary advantage. Our ability to predict how a bacterial population will evolve over time requires a better understanding of where key functional traits arrive. To address this question, we assessed prophage-encoded phage defenses and effector across Pseudomonas syringae . We show that prophages carrying these genes belong to specific phage taxa with differences in the types of genes encoded. This emphasizes the evolutionary advantage of these prophages, offering a framework to uncover how these genes disperse within microbial communities and their role in pathogen evolution. ### Competing Interest Statement The authors have declared no competing interest.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease. IMPORTANCE Global change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.