Search for: All records

ABSTRACT Multipartite bacterial genome organization can confer advantages, including coordinated gene regulation and faster genome replication, but is challenging to maintain.Agrobacterium tumefacienslineages often contain a circular chromosome (Ch1), a linear chromosome (Ch2), and multiple plasmids. We previously observed that in some stocks of the C58 lab model, Ch1 and Ch2 were fused into a linear dicentric chromosome. Here we analyzedAgrobacteriumnatural isolates from the French Collection for Plant-Associated Bacteria and identified two strains distinct from C58 with fused chromosomes. Chromosome conformation capture identified integration junctions that were different from the C58 fusion strain. Genome-wide DNA replication profiling showed that both replication origins remained active. Transposon sequencing revealed that partitioning systems of both chromosome centromeres were essential. Importantly, the site-specific recombinase XerCD is required for the survival of the strains containing the fusion chromosome. Our findings show that replicon fusion occurs in natural environments and that balanced replication arm sizes and proper resolution systems enable the survival of such strains. IMPORTANCEMost bacterial genomes are monopartite with a single, circular chromosome. However, some species, likeAgrobacterium tumefaciens, carry multiple chromosomes. Emergence of multipartite genomes is often related to adaptation to specific niches, including pathogenesis or symbiosis. Multipartite genomes confer certain advantages; however, maintaining this complex structure can present significant challenges. We previously reported a laboratory-propagated lineage ofA. tumefaciensstrain C58 in which the circular and linear chromosomes fused to form a single dicentric chromosome. Here we discovered two geographically separated environmental isolates ofA. tumefacienscontaining fused chromosomes with integration junctions different from the C58 fusion chromosome, revealing the constraints and diversification of this process. We found that balanced replication arm sizes and the repurposing of multimer resolution systems enable the survival and stable maintenance of dicentric chromosomes. These findings reveal how multipartite genomes function across different bacterial species and the role of genomic plasticity in bacterial genetic diversification. 

ABSTRACT The broadly conserved ParB protein performs crucial functions in bacterial chromosome segregation and replication regulation. The cellular function of ParB requires it to dimerize, recognizeparSDNA sequences, clamp on DNA, then slide to adjacent sequences through nonspecific DNA binding. How ParB coordinates nonspecific DNA binding and sliding remains elusive. Here, we combine multiplein vitrobiophysical and computational tools andin vivoapproaches to address this question. We found that the five conserved lysine residues in the C-terminal domain of ParB play distinct roles in proper positioning and sliding on DNA, and their integrity is crucial for ParB’sin vivofunctions. Many proteins with diverse cellular activities need to move on DNA while loosely bound. Our findings reveal the detailed molecular mechanism by which multiple flexible basic residues enable DNA binding proteins to efficiently slide along DNA. 

ABSTRACT Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found inBombella apis.B. apisis an important honey bee symbiont, found primarily in larvae, queens, and hive compartments.B. apisis found in the worker bee gut but is not considered a core member of the bee microbiome and has therefore been understudied with regard to its importance in the honey bee colony. However,B. apisappears 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 byB. apisin its transition to bee association, as they are absent in a sister clade. Here, we assess the variation and distribution of CRISPR-Cas types acrossB. apisstrains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the sameB. apisgenomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for eachB. apisstrain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that theB. apisCRISPR-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 different groups of microbes, including the acetic acid bacteriumBombella apis.B. apisis found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen’s digestive tract. The roles thatB. apismay serve in these environments are just beginning to be discovered and include the production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bolstering their nutrition. Niche specificity inB. apismay be affected by the pressures of bacteriophage and other mobile elements, which may target different strains in each specific bee environment. Studying the interplay betweenB. apisand 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. 

Abstract Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops, while replisomes duplicate entire chromosomes. These essential molecular machines must collide frequently in every cell cycle, yet how such collisions are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in theBacillus subtilisgenome, we engineered head-on and head-to-tail collisions between SMC complexes and the replisome. Replisome progression was monitored by genome-wide marker frequency analysis, and SMC translocation was monitored by time-resolved ChIP-seq and Hi-C. We found that SMC complexes do not impede replisome progression. By contrast, replisomes restrict SMC translocation regardless of collision orientations. Combining experimental data with simulations, we determined that SMC complexes are blocked by the replisome and then released from the chromosome. Occasionally, SMC complexes can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion in vivo, with implications for processes such as chromosome segregation, DNA repair, and gene regulation that require dynamic chromosome organization in all organisms. 

ABSTRACT Microorganisms often inhabit environments that are suboptimal for growth and reproduction. To survive when challenged by such conditions, individuals engage in dormancy, where they enter a metabolically inactive state. For this persistence strategy to confer an evolutionary advantage, microorganisms must be able to resuscitate and reproduce when conditions improve. Among bacteria in the phylum Actinomycetota, dormancy can be terminated by resuscitation-promoting factor (Rpf), an exoenzyme that hydrolyzes glycosidic bonds in the peptidoglycan of cell walls. We characterized Rpf fromMicrococcusKBS0714, a bacterium isolated from agricultural soil. The protein exhibited high substrate affinityin vitro, even though resuscitation was maximized in live-cell assays at micromolar concentrations. Site-directed mutations at conserved catalytic sites significantly reduced or eliminated resuscitation, as did the deletion of repeating motifs in a lectin-encoding linker region. We then tested the effects of recombinant Rpf fromMicrococcusKBS0714 on a diverse set of dormant soil bacteria. Patterns of resuscitation mapped onto strain phylogeny, which reflected core features of the cell envelope. Additionally, the direction and magnitude of the Rpf effect were associated with functional traits, in particular, aspects of the moisture niche and biofilm production, which are critical for understanding dormancy and the persistence of microbial populations in soils. These findings expand our understanding of how Rpf may affect seed bank dynamics with implications for the diversity and functioning of microorganisms in terrestrial ecosystems. IMPORTANCEDormancy is a process whereby individuals enter a reversible state of reduced metabolic activity. In fluctuating environments, dormancy protects individuals from unfavorable conditions, enhancing fitness and buffering populations against extinction. However, waking up from dormancy is a critical yet risky decision. Some bacteria resuscitate stochastically, while others rely on environmental cues or signals from neighboring cells to transition back to active growth. Resuscitation-promoting factor (Rpf) is an exoenzyme that cleaves bonds in the peptidoglycan of bacterial cell walls, facilitating dormancy termination and enabling regrowth. Although this family of proteins has been well characterized in model organisms and clinically relevant strains, our study characterizes Rpf from a soil bacterium and examines its effects on resuscitation across a diverse collection of bacteria, linking it to functional traits that may influence dormancy dynamics in both natural and managed ecosystems. 

Abstract Studying host-associated microbiome assembly is key to understanding microbial and host evolution and health. While honey bee microbiomes have been central models for such investigations among pollinators, they overlook the diversity of eusocial dynamics and multi- kingdom interactions. Stingless bees, highly eusocial managed bees that rely on yeast for larval development, offer a valuable complementary system to study microbiome assembly, and within an eco-evolutionary framework. Using amplicon sequencing, metagenomics, and microbial experiments, we investigate the drivers of stingless bee microbiome assembly. We reveal a spatially structured, site-adapted microbiome, where high microbial influx hive components are segregated from the brood, which harbors a stable, multi-kingdom community. We show that the brood microbiome is not only physically protected but also actively maintained through highly selective bacterial-fungal interactions. Our findings uncover multi-layered mechanisms shaping an eusocial insect microbiome, from host biology to cross-kingdom interactions, while providing critical insights into microbiome maintenance of important pollinators. 

Abstract Microorganisms associated with plants can affect nutrient and water acquisition, plant defenses, and ecological interactions, with effects on plant growth that range from beneficial to antagonistic. In Glycine max (soybean), many studies have examined the soil microbiome and the legume–rhizobium relationship, but little is known about foliar endophytes, their effects on plant biomass and fitness, and how plants respond to their presence. To address these questions, we inoculated Glycine max with field-collected isolates of previously isolated, dominant strains of Methylobacterium and Colletotrichum in either sterile or non-sterile soil. We then used RNAseq to compare the transcriptomic responses of plants to single- and co-inoculation of endophytes. We found that all endophyte treatments increased soybean growth compared to control, but only in sterile soil. These results suggest context-dependency, with endophytes serving as facultative mutualists under stress or nutrient deprivation. Similarly, transcriptomic analyses revealed that soybean defense and stress responses depended on the interaction of endophytes; Methylobacterium elicited the strongest response but was modulated by the presence of Colletotrichum. Our findings highlight the environmentally dependent effects of co-existing endophytes within soybean leaves. 

ABSTRACT Microbial host populations evolve traits conferring specific resistance to viral predators via various defence mechanisms, while viruses reciprocally evolve traits to evade these defences. Such coevolutionary dynamics often involve diversification promoted by negative frequency‐dependent selection. However, microbial traits conferring competitive asymmetries can induce directional selection, opposing diversification. Despite extensive research on microbe–virus coevolution, the combined effect of both host trait types and associated selection remains unclear. Using a CRISPR‐mediated coevolutionary system, we examine how the co‐occurrence of both trait types impacts viral evolution and persistence, previously shown to be transient and nonstationary in computational models. A stochastic model incorporating host competitive asymmetries via variation of intrinsic growth rates reveals that competitively advantaged host clades generate the majority of immune diversity. Greater asymmetries extend viral extinction times, accelerate viral adaptation locally in time and augment long‐term local adaptation. These findings align with previous experiments and provide further insights into long‐term coevolutionary dynamics. 

ABSTRACT Much of life on Earth is at the mercy of currents and flow. Residence time (τ) estimates how long organisms and resources remain in a system based on the ratio of volume (V) to flow rate (Q). Shortτshould promote immigration but limit species establishment, while longτshould favour species that survive on limited resources. Theory suggests these opposing forces shape the abundance, diversity and function of flowing systems. We experimentally tested how residence time affects a lake microbial community by exposing chemostats to aτgradient spanning seven orders of magnitude. Microbial abundance, richness and evenness increased non‐linearly withτ, while functions like productivity and resource consumption declined. Taxa formed distinct clusters of short‐ and long‐τspecialists consistent with niche partitioning. Our findings demonstrate that residence time drives biodiversity and community function in flowing habitats that are commonly found in environmental, engineered and host‐associated ecosystems. 

Abstract Despite the queen’s crucial reproductive role in honey bee colonies, queen diet and feeding behavior remain remarkably enigmatic, with most studies assuming they are solely fed nutritious glandular secretions (i.e., royal jelly) by workers. This colors our understanding of basic honey bee biology and how governmental agencies assess pesticide risk. We hypothesized that adult queens also consume honey and pollen. Through experiments with queenright laboratory microcolonies fed with marked diets, we demonstrate that queens are fed pollen and nectar by workers and can also feed directly. We then measured pollen content in mature, unmanipulated queens sacrificed from 43 conventional field colonies from two distinct geographical regions. Similar to workers, we found pollen in almost all queens guts, though at expectedly lower quantities than in young workers. These findings suggest queens have a more complex, dynamic diet than previously thought, raising new questions about how dietary habits and feeding behaviors influence pesticide risk and other aspects of queen biology.