Engineering simplified microbial communities that retain function and ecological cohesiveness remains a major challenge because the taxa and interactions required for community establishment are rarely known a priori. Here, we experimentally evaluated drift-driven microbiome simplification as an alternative strategy for generating reduced and reconstructable microbial consortia. Using the tomato rhizosphere as a model system, three source microbial communities were subjected to different dilution bottlenecks and serial propagation. Increasing dilution was the main determinant of community composition and simplification, while repeated passage produced additional reductions in ASV richness, phylogenetic diversity, and evenness. Importantly, diversity loss was not accompanied by a uniform deterioration in bacterial colonization or plant performance, indicating that substantial simplification can occur without a parallel collapse in these system-level properties. Candidate Minimal Microbiome prototypes were selected from endpoint communities, reconstructed as synthetic communities, and their ecological cohesiveness was evaluated through invasion experiments. The reconstructed communities strongly restricted the establishment of the original complex microbial fractions, although invasion success depended markedly on invader identity. The drift-derived communities were at least as resistant to invasion as an independently designed bottom-up synthetic community. Together, these results provide experimental support for drift-driven simplification as a strategy to generate reduced microbial communities whose dominant members can be isolated, reconstructed, and experimentally evaluated. By allowing ecological assembly to generate candidate community configurations before cultivation and reconstruction, this approach provides a complementary route to rational bottom-up design and function-directed top-down microbiome engineering.

ABSTRACT The rhizosphere microbiome, one of the most diverse and metabolically active microbial ecosystems known, plays fundamental roles in plant health and productivity. However, the ecological dynamics occurring during the transition between germination and the establishment of the first true leaves, a developmental window associated with the onset of active photosynthesis and rapid root expansion, remain poorly understood. Here, we investigated rhizosphere microbiome assembly during the first four weeks of tomato development by sampling communities arising from seven distinct natural soil inocula twice weekly to obtain fine-scale temporal resolution. Bacterial load, richness, evenness and phylogenetic diversity all increased significantly during plant development, indicating progressive increases in rhizosphere ecosystem complexity. In addition, diverse initial microbial communities differentially influenced both host plant development and the bacterial carrying capacity of the resulting rhizosphere ecosystem. Although temporal effects on rhizosphere microbiome composition were significant, assembly trajectories remained strongly constrained by the initial inoculum. Temporal analysis nevertheless revealed significant taxonomic turnover despite limited global compositional restructuring. In particular, Proteobacteria and Pseudomonadaceae decreased over time, whereas Actinobacteria, Acidobacteria and Streptomycetaceae increased. However, communities did not become progressively more similar or divergent over time. Altogether, our results indicate that early rhizosphere microbiome assembly involves rapid ecological succession within inoculum-constrained compositional trajectories, with early copiotrophic Proteobacteria progressively giving rise to more diverse and phylogenetically structured communities. These findings suggest that the first weeks of plant development may represent a critical ecological window for microbiome-based manipulation strategies in agriculture.

ABSTRACT The rhizosphere microbiome plays fundamental roles in plant health and productivity, yet the ecological rules governing microbiome assembly remain poorly understood. Here, we investigated early rhizosphere community assembly in tomato using a replicated combinatorial community coalescence framework, in which seven distinct natural bacterial communities were inoculated individually and in all possible pairwise and triplet combinations. Single-inoculum communities clustered according to inoculum identity, indicating a strong effect of source community composition on assembly trajectories. However, when all communities were analyzed jointly, samples formed a continuous compositional landscape with no clear evidence of discrete community states. Despite major differences in source community composition, rhizosphere communities consistently converged toward a highly similar uneven rank–abundance structure, with two ASVs accounting for 50% and a median of nineteen ASVs for 90% of total abundance. While assembly was dominated by a very small number of Pseudomonas ASVs, limited evidence of alternative dominant states was observed. Increasing inoculum complexity did not increase stochasticity but instead promoted stronger convergence toward a global rhizosphere compositional centroid. Moreover, dominance hierarchies emerging from community coalescence closely mirrored the distance of source communities to this centroid. The two most dominant communities originated from orchard soils, suggesting that historical contingency and prior adaptation to horticultural crop rhizospheres may influence competitive success. Together, these results are consistent with the existence of a canonical rhizosphere attractor in composition and abundance architecture, with patterns consistent with assembly occurring under a limited number of dominant ecological niches imposed by the tomato rhizosphere.

Plant-microbe interactions occur mainly in the rhizosphere, a hot spot of microbial activity and diversity, and the outcome of these interactions can significantly affect plant productivity. A better understanding of the rhizosphere microbiome is, therefore, required to develop successful knowledge-based microbiome modification strategies that help improve plant productivity. Here, we investigated the community assembly process of the tomato rhizosphere microbiome and its potential composition-function relationships through the lens of a conceptual framework based on the phylogenetically constrained assembly of microbial communities. In this study, we grew tomato plantlets on different soil types with high replication and assessed community composition using high-throughput sequencing of the 16S rRNA bacterial marker gene and community function based on the predicted minimal metagenome of the microbial ecosystem. We also conducted additional computer simulations to examine the relationship between phylogenetic clustering and community function. We observed a systematic enrichment in the rhizosphere in terms of phylogeny and predicted functional content and were able to delimit the phylogenetic signal in the ecosystem owing to the presence of 12 functionally coherent phylogenetic core groups in all samples. These groups, which accounted for a large fraction of the total community, belonged to the Alpha, Beta, and Gammaproteobacteria, Actinobacteria, and Bacilli classes and presented exclusive functions related to important ecosystem services. Furthermore, our analyses indicated that these groups accounted for a large proportion of the ecosystem's predicted minimal metagenome. Overall, our study suggests that community assembly followed coupled phylo-functional selection, a phenomenon that likely occurs in other rhizosphere microbiomes. This knowledge not only advances our understanding of how community composition-phylogeny-function relationships drive the assembly process of the rhizosphere microbiome but also will help guide the design of synthetic rhizosphere microbiomes for both research and commercial purposes.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ABSTRACT Plant-microbe interactions occur mainly in the rhizosphere, a hot spot of microbial activity and diversity. Given that the outcome of such interactions can significantly impact plant productivity, we require a better understanding of the rhizosphere microbiome if knowledge-based microbiome modification strategies are to be successfully deployed in the future. Here, we aimed to gain a better understanding of the assembly process of the tomato rhizosphere microbiome and its potential composition-function relationships. Among other things, we studied community assembly through the lens of a conceptual framework for the phylogenetically constrained assembly of microbial communities, while assessing community function based on the predicted minimal metagenome of the microbial ecosystem. We observed a systematic enrichment in terms of phylogeny and predicted functional content in the rhizosphere and were able to delimit phylogenetic signal in the ecosystem with 12 functionally coherent phylogenetic groups present in all samples which together accounted for a large fraction of the total community. Our analyses indicated that these groups included a significantly larger content of the ecosystem’s minimal metagenome than expected by chance. Thus, our study suggests that community assembly followed coupled phylo-functional selection independent of host genetics, and we expect the same phenomenon to occur in other rhizosphere microbiomes. This knowledge provides a thrust in our understanding of how community composition-phylogeny-function relationships drive the assembly process of the rhizosphere microbiome and should help guide the design of synthetic rhizosphere microbiomes for both research and commercial purposes.

Metagenomic analysis can generate hypotheses about microbiome interactions and function, yet mechanistic understanding is only possible through precise experimentation manipulating its microbiota composition. The high-throughput isolation of microbiome members thus represents a core resource in this field of research.

Synanthropic birds might play an important role as reservoirs of many zoonotic endoparasites; however, little information is available on many parasites and their prevalence. Here, we use an approach based on targeted metagenomic detection through the use of DNA metabarcoding of faecal samples to screen for circulating parasites in alien parakeets (Myiopsitta monachus and Psittacula krameri) and urban landfill-feeding storks (Ciconia ciconia) and gulls (Larus fuscus). We focus especially on potentially zoonotic parasites, with the aim of better understanding the zoonotic risk that these birds’ faeces may pose. We detected a total of 23 genera of eukaryotic parasites: six fungi, three protists, five nematodes, two cestodes and seven trematodes. Among them, six stood out for their relevance to human health: Cryptococcus spp., Aspergillus spp. and Candida spp. (fungi); Cryptosporidium spp. (a protist); and Ascaris spp. and Halicephalobus spp. (nematodes). In parakeets, we detected Cryptococcus spp. and Ascaris spp., the latter being detected in 10–20% of the samples. In the White Stork and the Lesser Black-backed Gull, we found a high prevalence of Aspergillus spp. (in 15% and 50% of the samples, respectively) and Candida spp. (in 63% and 82% of the samples, respectively), and the presence of Cryptosporidium spp. in 10% of the samples. We detected Halicephalobus spp. in one gull sample (2%). Our results show that synanthropic birds may act as vectors and reservoirs of zoonotic parasites and their faeces could pose a risk to human health associated with the zoonotic parasites present in them. This should be taken into account when developing management plans for urban populations of these bird species.

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.

In the present work, we present the draft genome sequence of a new putative Arthrobacter species associated with the tomato rhizosphere.

The isolation and characterization of additional phages is crucial for adding reliable viral sequences with relevant biological information to viral databases. In this study, we present the complete genomes of two Arthrobacter phages obtained from different soil samples.

The dispersion of microorganisms through the atmosphere is a continual and essential process that underpins biogeography and ecosystem development and function. Despite the ubiquity of atmospheric microorganisms globally, specific knowledge of the determinants of atmospheric microbial diversity at any given location remains unresolved. Here we describe bacterial diversity in the atmospheric boundary layer and underlying soil at twelve globally distributed locations encompassing all major biomes, and characterise the contribution of local and distant soils to the observed atmospheric community. Across biomes the diversity of bacteria in the atmosphere was negatively correlated with mean annual precipitation but positively correlated to mean annual temperature. We identified distinct non-randomly assembled atmosphere and soil communities from each location, and some broad trends persisted across biomes including the enrichment of desiccation and UV tolerant taxa in the atmospheric community. Source tracking revealed that local soils were more influential than distant soil sources in determining observed diversity in the atmosphere, with more emissive semi-arid and arid biomes contributing most to signatures from distant soil. Our findings highlight complexities in the atmospheric microbiota that are relevant to understanding regional and global ecosystem connectivity.

ABSTRACTThe application of CRISPR/Cas9 to improve genome engineering efficiency of large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desire locus has not been reported yet. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiency depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex viruses (HSV) type 1 and 2, to optimize a CRISPR/Cas9 method that can be versatilely used for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep-sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and color-based selection of recombinants. This application of CRISPR/Cas9 reduces time and labor of screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desire locus in less than two weeks.DATA SUMMARYRaw sequence reads are available at the European Bioinformatics Institute (EMBL-EBI) European Nucleotide Archive (ENA) as Bioproject ID PRJEB32151 and PRJEB32152.Six supplementary figures, eleven supplementary tables and supplementary methods are available with the online version of this article. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Atmospheric transport is critical to dispersal of microorganisms between habitats, and this underpins resilience in terrestrial and marine ecosystems globally. A key unresolved question is whether microorganisms assemble to form a taxonomically distinct, geographically variable, and functionally adapted atmospheric microbiota. This question is made more complex by the unique challenges of separating potential contaminants from atmospheric signal, particularly given the ultra-low biomass of air and the long durations of sampling where contamination may occur. Here we adopted a comprehensive data filtering approach to mitigate contamination and characterise inter-continental patterns of microbial taxonomic and functional diversity in air within and above the atmospheric boundary layer and in underlying soils for 596 globally sourced samples. Bacterial and fungal assemblages in air were taxonomically structured and deviated significantly from purely stochastic assembly. Patterns differed with location and reflected climate, underlying surface cover and environmental filtering. Source-tracking indicated a complex recruitment process involving local soils plus globally distributed inputs from drylands and the phyllosphere. Assemblages displayed stress-response and metabolic traits relevant to survival in air, and taxonomic and functional diversity were correlated with macroclimate and atmospheric variables. Our findings highlight complexity in the atmospheric microbiota that is key to understanding regional and global ecosystem connectivity.

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

All trypanosomatid genomes are colonized by non-LTR retrotransposons which exhibit a highly conserved 77-nt sequence at their 5' ends, known as the Pr77-hallmark (Pr77). The wide distribution of Pr77 is expected to be related to the gene regulation processes in these organisms as it has promoter and HDV-like ribozyme activities at the DNA and RNA levels, respectively. The identification of Pr77 hallmark-bearing retrotransposons and the study of the associations of mobile elements with relevant genes have been analyzed in the genomes of six strains of Trypanosoma cruzi belonging to different discrete typing units (DTUs) and with different geographical origins and host/vectors. The genomes have been sequenced, assembled and annotated. BUSCO analyses indicated a good quality for the assemblies that were used in comparative analyses. The results show differences among the six genomes in the copy number of genes related to virulence processes, the abundance of retrotransposons bearing the Pr77 sequence and the presence of the Pr77 hallmarks not associated with retroelements. The analyses also show frequent associations of Pr77-bearing retrotransposons and single Pr77 hallmarks with genes coding for trans-sialidases, RHS, MASP or hypothetical proteins, showing variable proportion depending on the type of retroelement, gene class and parasite strain. These differences in the genomic distribution of active retroelements and other Pr77-containing elements have shaped the genome architecture of these six strains and might be contributing to the phenotypic variability existing among them.

Herpes simplex virus serotype 2 (HSV-2) is a ubiquitous human pathogen that causes recurrent genital infections and ulcerations. Many HSV-2 strains with different biological properties have been identified, but only the genomes of HSV-2 strains HG52, SD90e and 333 have been reported as complete and fully characterized sequences. We de novo assembled, annotated and manually curated the complete genome sequence of HSV-2 strain MS, a highly neurovirulent strain, originally isolated from a multiple sclerosis patient. We resolved both DNA ends, as well as the complex inverted repeats regions present in HSV genomes, usually undisclosed in previous published partial herpesvirus genomes, using long reads from Pacific Biosciences (PacBio) technology. Additionally, we identified isomeric genomes by determining the alternative relative orientation of unique fragments in the genome of the sequenced viral population. Illumina short-read sequencing was crucial to examine genetic variability, such as nucleotide polymorphisms, insertion/deletions and sequence determinants of strain-specific virulence factors. We used Illumina data to fix two disrupted open reading frames found in coding homopolymers after PacBio assembly. These results support the combination of long- and short-read sequencing technologies as a precise and effective approach for the accurate de novo assembly and curation of complex microbial genomes.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.

Abstract Atmospheric transport is critical to dispersal of microorganisms between habitats and this underpins resilience in terrestrial and marine ecosystems globally. A key unresolved question is whether microorganisms assemble to form a taxonomically distinct, geographically variable, and functionally adapted atmospheric microbiome. Here we characterised globalscale patterns of microbial taxonomic and functional diversity in air within and above the atmospheric boundary layer and in underlying soils. Bacterial and fungal assemblages in air were taxonomically structured and deviated significantly from purely stochastic assembly. Patterns differed with location and reflected underlying surface cover and environmental filtering. Source-tracking indicated a complex recruitment process involving local soils plus globally distributed inputs from drylands and the phyllosphere. Assemblages displayed stressresponse and metabolic traits relevant to survival in air, and taxonomic and functional diversity were correlated with macroclimate and atmospheric variables. Our findings highlight complexity in the atmospheric microbiome that is key to understanding regional and global ecosystem connectivity.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.