Plasmids in the human gut reveal neutral dispersal and recombination that is overpowered by inflammatory diseases.

Plasmids are pivotal in driving bacterial evolution through horizontal gene transfer. Here, we investigated 3467 human gut microbiome samples across continents and disease states, analyzing 11,086 plasmids. Our analyses reveal that plasmid dispersal is predominantly stochastic, indicating neutral processes as the primary driver of their wide distribution. We find that only 20-25% of plasmid DNA is being selected in various disease states, constraining its distribution across hosts. Selective pressures shape specific plasmid segments with distinct ecological functions, influenced by plasmid mobilization lifestyle, antibiotic usage, and inflammatory gut diseases. Notably, these elements are more commonly shared within groups of individuals with similar health conditions, such as Inflammatory Bowel Disease (IBD), regardless of geographic location across continents. These segments contain essential genes such as iron transport mechanisms- a distinctive gut signature of IBD that impacts the severity of inflammation. Our findings shed light on mechanisms driving plasmid dispersal and selection in the human gut, highlighting their role as carriers of vital gene pools impacting bacterial hosts and ecosystem dynamics.

Phylogenetic analyses reveal insights into interdomain horizontal gene transfer of microbial lipases.

Microbial lipases play a pivotal role in a wide range of biotechnological processes and in the human skin microbiome. However, their evolution remains poorly understood. Accessing the evolutionary process of lipases could contribute to future applications in health and biotechnology. We investigated genetic events associated with the evolutionary trajectory of the microbial family LIP lipases. Using phylogenetic analysis, we identified two distinct horizontal gene transfer (HGT) events from Bacteria to Fungi. Further analysis of human cutaneous mycobiome members such as the lipophilic Malassezia yeasts and CUG-Ser-1 clade (including Candida sp. and other microorganisms associated with cutaneous mycobiota) revealed recent evolutionary processes, with multiple gene duplication events. The Lid region of fungal lipases, crucial for substrate interaction, exhibits varying degrees of conservation among different groups. Our findings suggest the adaptability of the fungal LIP family in various genetic and metabolic contexts and its potential role in niche exploration.

Programming Dynamic Division of Labor Using Horizontal Gene Transfer.

The metabolic engineering of microbes has broad applications, including biomanufacturing, bioprocessing, and environmental remediation. The introduction of a complex, multistep pathway often imposes a substantial metabolic burden on the host cell, restraining the accumulation of productive biomass and limiting pathway efficiency. One strategy to alleviate metabolic burden is the division of labor (DOL) in which different subpopulations carry out different parts of the pathway and work together to convert a substrate into a final product. However, the maintenance of different engineered subpopulations is challenging due to competition and convoluted interstrain population dynamics. Through modeling, we show that dynamic division of labor (DDOL), which we define as the DOL between indiscrete populations capable of dynamic and reversible interchange, can overcome these limitations and enable the robust maintenance of burdensome, multistep pathways. We propose that DDOL can be mediated by horizontal gene transfer (HGT) and use plasmid genomics to uncover evidence that DDOL is a strategy utilized by natural microbial communities. Our work suggests that bioengineers can harness HGT to stabilize synthetic metabolic pathways in microbial communities, enabling the development of robust engineered systems for deployment in a variety of contexts.

On the impact of incomplete taxon sampling on the relative timing of gene transfer events.

A recent study questioned the use of branch length methods to assess the relative timing of horizontal gene transfers because of the effects of so-called "ghost" lineages. This Formal Comment discusses key considerations regarding the potential effect of missing lineages when assessing relative timing of evolutionary events.

Rapid dissemination of host metabolism-manipulating genes via integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Salivary proteins potentially derived from horizontal gene transfer are critical for salivary sheath formation and other feeding processes.

Herbivorous insects employ an array of salivary proteins to aid feeding. However, the mechanisms behind the recruitment and evolution of these genes to mediate plant-insect interactions remain poorly understood. Here, we report a potential horizontal gene transfer (HGT) event from bacteria to an ancestral bug of Eutrichophora. The acquired genes subsequently underwent duplications and evolved through co-option. We annotated them as horizontal-transferred, Eutrichophora-specific salivary protein (HESPs) according to their origin and function. In Riptortus pedestris (Coreoidea), all nine HESPs are secreted into plants during feeding. The RpHESP4 to RpHESP8 are recently duplicated and found to be indispensable for salivary sheath formation. Silencing of RpHESP4-8 increases the difficulty of R. pedestris in probing the soybean, and the treated insects display a decreased survivability. Although silencing the other RpHESPs does not affect the salivary sheath formation, negative effects are also observed. In Pyrrhocoris apterus (Pyrrhocoroidea), five out of six PaHESPs are secretory salivary proteins, with PaHESP3 being critical for insect survival. The PaHESP5, while important for insects, no longer functions as a salivary protein. Our results provide insight into the potential origin of insect saliva and shed light on the evolution of salivary proteins.

Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

Fusobacterium nucleatum subsp. polymorphum recovered from malignant and potentially malignant oral disease exhibit heterogeneity in adhesion phenotypes and adhesin gene copy number, shaped by inter-subspecies horizontal gene transfer and recombination-derived mosaicism.

Fusobacterium nucleatum is an anaerobic commensal of the oral cavity associated with periodontitis and extra-oral diseases, including colorectal cancer. Previous studies have shown an increased relative abundance of this bacterium associated with oral dysplasia or within oral tumours. Using direct culture, we found that 75 % of Fusobacterium species isolated from malignant or potentially malignant oral mucosa were F. nucleatum subsp. polymorphum. Whole genome sequencing and pangenome analysis with Panaroo was carried out on 76 F. nucleatum subsp. polymorphum genomes. F. nucleatum subsp. polymorphum was shown to possesses a relatively small core genome of 1604 genes in a pangenome of 7363 genes. Phylogenetic analysis based on the core genome shows the isolates can be separated into three main clades with no obvious genotypic associations with disease. Isolates recovered from healthy and diseased sites in the same patient are generally highly related. A large repertoire of adhesins belonging to the type V secretion system (TVSS) could be identified with major variation in repertoire and copy number between strains. Analysis of intergenic recombination using fastGEAR showed that adhesin complement is shaped by horizontal gene transfer and recombination. Recombination events at TVSS adhesin genes were not only common between lineages of subspecies polymorphum, but also between different subspecies of F. nucleatum. Strains of subspecies polymorphum with low copy numbers of TVSS adhesin encoding genes tended to have the weakest adhesion to oral keratinocytes. This study highlights the genetic heterogeneity of F. nucleatum subsp. polymorphum and provides a new framework for defining virulence in this organism.

First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

Chitosan oligosaccharide accelerates the dissemination of antibiotic resistance genes through promoting conjugative plasmid transfer.

The dissemination of antibiotic resistance genes (ARGs), especially via plasmid-mediated horizontal gene transfer, poses a pervasive threat to global health. Chitosan-oligosaccharide (COS) is extensively utilized in medicine, plant and animal husbandry. However, their impact on microflora implies the potential to exert selective pressure on plasmid transfer. To explore the role of COS in facilitating the dissemination of ARGs via plasmid conjugation, we established in vitro mating models. The addition of COS to conjugation mixtures significantly enhanced the transfer of RP4 plasmid and mcr-1 positive IncX4 plasmid in both intra- and inter-specific. Phenotypic and transcriptome analysis revealed that COS enhanced intercellular contact by neutralizing cell surface charge and increasing cell surface hydrophobicity. Additionally, COS increased membrane permeability by inhibiting the Tol-Pal system, thereby facilitating plasmid conjugative transfer. Furthermore, COS served as the carbon source and was metabolized by E. coli, providing energy for plasmid conjugation through regulating the expression of ATPase and global repressor factor-related genes in RP4 plasmid. Overall, these findings improve our awareness of the potential risks associated with the presence of COS and the spread of bacterial antibiotic resistance, emphasizing the need to establish guidelines for the prudent use of COS and its discharge into the environment.

Phage-plasmids promote recombination and emergence of phages and plasmids.

Phages and plasmids are regarded as distinct types of mobile genetic elements that drive bacterial evolution by horizontal gene transfer. However, the distinction between both types is blurred by the existence of elements known as prophage-plasmids or phage-plasmids, which transfer horizontally between cells as viruses and vertically within cellular lineages as plasmids. Here, we study gene flow between the three types of elements. We show that the gene repertoire of phage-plasmids overlaps with those of phages and plasmids. By tracking recent recombination events, we find that phage-plasmids exchange genes more frequently with plasmids than with phages, and that direct gene exchange between plasmids and phages is less frequent in comparison. The results suggest that phage-plasmids can mediate gene flow between plasmids and phages, including exchange of mobile element core functions, defense systems, and antibiotic resistance. Moreover, a combination of gene transfer and gene inactivation may result in the conversion of elements. For example, gene loss turns P1-like phage-plasmids into integrative prophages or into plasmids (that are no longer phages). Remarkably, some of the latter have acquired conjugation-related functions to became mobilisable by conjugation. Thus, our work indicates that phage-plasmids can play a key role in the transfer of genes across mobile elements within their hosts, and can act as intermediates in the conversion of one type of element into another.

Horizontal gene transfer after faecal microbiota transplantation in adolescents with obesity.

BACKGROUND: Horizontal gene transfer (HGT) describes the transmission of DNA outside of direct ancestral lineages. The process is best characterised within the bacterial kingdom and can enable the acquisition of genetic traits that support bacterial adaptation to novel niches. The adaptation of bacteria to novel niches has particular relevance for faecal microbiota transplantation (FMT), a therapeutic procedure which aims to resolve gut-related health conditions of individuals, through transplanted gut microbiota from healthy donors. RESULTS: Three hundred eighty-one stool metagenomic samples from a placebo-controlled FMT trial for obese adolescents (the Gut Bugs Trial) were analysed for HGT, using two complementary methodologies. First, all putative HGT events, including historical HGT signatures, were quantified using the bioinformatics application WAAFLE. Second, metagenomic assembly and gene clustering were used to assess and quantify donor-specific genes transferred to recipients following the intervention. Both methodologies found no difference between the level of putative HGT events in the gut microbiomes of FMT and placebo recipients, post-intervention. HGT events facilitated by engrafted donor species in the FMT recipient gut at 6 weeks post-intervention were identified and characterised. Bacterial strains contributing to this subset of HGT events predominantly belonged to the phylum Bacteroidetes. Engraftment-dependent horizontally transferred genes were retained within recipient microbiomes at 12 and 26 weeks post-intervention. CONCLUSION: Our study suggests that novel microorganisms introduced into the recipient gut following FMT have no impact on the basal rate of HGT within the human gut microbiome. Analyses of further FMT studies are required to assess the generalisability of this conclusion across different FMT study designs and for the treatment of different gut-related conditions. Video Abstract.

Regulation of quorum sensing for the manipulation of conjugative transfer of antibiotic resistance genes in wastewater treatment system.

The emergence and transmission of antibiotic resistance genes (ARGs) through plasmid-mediated conjugation has become a significant worldwide public health threat. Biofilms are widely recognized as the primary reservoirs for ARGs, providing favorable conditions for horizontal gene transfer. Quorum sensing (QS) plays a critical role in bacterial biofilm formation, which further influences the spread of bacterial resistance. In this study, we examined the effects of vanillin, a QS inhibitor (QSI), at subinhibitory concentrations (sub-MICs) ranging from 0 - 0.1 g/L, on the transfer of ARGs between Escherichia coli and Pseudomonas aeruginosa. Our findings indicated that vanillin at sub-MICs inhibited the conjugative transfer frequency of the RP4 plasmid. This inhibition was supported by the downregulation of plasmid transfer genes. The suppression of conjugation can mainly be attributed to the inhibition of biofilm formation, the synthesis of extracellular polymeric substances (EPS), and the secretion of virulence factors, all of which are regulated by the bacterial QS system. On the other hand, the levels of ROS and cell membrane permeability were not primary explanations for this phenomenon. Furthermore, vanillin also reduced the conjugative transfer frequency of ARGs in wastewater effluent, providing a potential approach to alleviate bacterial resistance in water environments. These findings underscore the regulatory role of QSI in controlling ARGs transfer and have significant implications for manipulating the dissemination of bacterial resistance in the environment.

Co-opting bacterial viruses for DNA exchange: structure and regulation of gene transfer agents.

Horizontal gene transfer occurs via a range of mechanisms, including transformation, conjugation and bacteriophage transduction. Gene transfer agents (GTAs) are an alternative, less-studied route for interbacterial DNA exchange. Encoded within bacterial or archaeal genomes, GTAs assemble into phage-like particles that selflessly package and transmit host DNA to recipient bacteria. Several unique features distinguish GTAs from canonical phages such as an inability to self-replicate, thus producing non-infectious particles. GTAs are also deeply integrated into the physiology of the host cell and are maintained under tight host-regulatory control. Recent advances in understanding the structure and regulation of GTAs have provided further insights into a DNA transfer mechanism that is proving increasingly widespread across the bacterial tree of life.

Genome analysis of a novel avian atadenovirus reveals a possible horizontal gene transfer.

We report the discovery and characterization of a novel adenovirus, Zoothera dauma adenovirus (ZdAdV), from a wild bird species, Zoothera dauma (Scaly thrush). This new atadenovirus was discovered by metagenomic sequencing without virus cultivation. Analyses of the full genome sequence revealed that this new virus is a distinct member of the genus Atadenovirus and represents a novel species. ZdAdV has a genome of 34,760 bp with 28 predicted genes and 39% GC content. ZdAdV is the first atadenovirus to contain ORF19, a gene previously found only in aviadenoviruses. Phylogenetic analysis of ORF19 suggests that it was acquired by ZdAdV through horizontal gene transfer from an aviadenovirus. By analyzing all orthologous genes of aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus, we also found potential horizontal gene transfer for the E4 gene in Pigeon aviadenovirus B. Our study widens our knowledge concerning the genetic diversity and evolutionary history of atadenoviruses and their potential for cross-species transmission.

An ocean of diffusible information.

In the ocean, free-living bacteria exist in a dilute world where direct physical interactions between cells are relatively rare. How then do they exchange genetic information via horizontal gene transfer (HGT)? Lücking et al. have explored the world of marine 'protected extracellular DNA' (peDNA), and find that extracellular vesicles (EVs) are likely to play an important role.

Multiple horizontal gene transfer events have shaped plant glycosyl hydrolase diversity and function.

Plant glycosyl hydrolases (GHs) play a crucial role in selectively breaking down carbohydrates and glycoconjugates during various cellular processes, such as reserve mobilization, pathogen defense, and modification/disassembly of the cell wall. In this study, we examined the distribution of GH genes in the Archaeplastida supergroup, which encompasses red algae, glaucophytes, and green plants. We identified that the GH repertoire expanded from a few tens of genes in early archaeplastidians to over 400 genes in modern angiosperms, spanning 40 GH families in land plants. Our findings reveal that major evolutionary transitions were accompanied by significant changes in the GH repertoire. Specifically, we identified at least 23 GH families acquired by green plants through multiple horizontal gene transfer events, primarily from bacteria and fungi. We found a significant shift in the subcellular localization of GH activity during green plant evolution, with a marked increase in extracellular-targeted GH proteins associated with the diversification of plant cell wall polysaccharides and defense mechanisms against pathogens. In conclusion, our study sheds light on the macroevolutionary processes that have shaped the GH repertoire in plants, highlighting the acquisition of GH families through horizontal transfer and the role of GHs in plant adaptation and defense mechanisms.

Detecting horizontal gene transfer with metagenomics co-barcoding sequencing.

Horizontal gene transfer (HGT) is the process through which genetic information is transferred between different genomes and that played a crucial role in bacterial evolution. HGT can enable bacteria to rapidly acquire antibiotic resistance and bacteria that have acquired resistance is spreading within the microbiome. Conventional methods of characterizing HGT patterns include short-read metagenomic sequencing (short-reads mNGS), long-read sequencing, and single-cell sequencing. These approaches present several limitations, such as short-read fragments, high amounts of input DNA, and sequencing costs, respectively. Here, we attempt to circumvent present limitations to detect HGT by developing a metagenomics co-barcode sequencing workflow (MECOS) and applying it to the human and mouse gut microbiomes. In addition to that, we have over 10-fold increased contig length compared to short-reads mNGS; we also obtained exceeding 30 million paired reads with co-barcode information. Applying the novel bioinformatic pipeline, we integrated this co-barcoding information and the context information from long reads, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Specifically, we detected approximately 3,000 HGT blocks in individual samples, encompassing ~6,000 genes and ~100 taxonomic groups, including loci conferring tetracycline resistance through ribosomal protection. MECOS provides a valuable tool for investigating HGT and advance our understanding on the evolution of natural microbial communities within hosts.IMPORTANCEIn this study, to better identify horizontal gene transfer (HGT) in individual samples, we introduce a new co-barcoding sequencing system called metagenomics co-barcoding sequencing (MECOS), which has three significant improvements: (i) long DNA fragment extraction, (ii) a special transposome insertion, (iii) hybridization of DNA to barcode beads, and (4) an integrated bioinformatic pipeline. Using our approach, we have over 10-fold increased contig length compared to short-reads mNGS, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Our results indicate the presence of approximately 3,000 HGT blocks, involving roughly 6,000 genes and 100 taxonomic groups in individual samples. Notably, these HGT events are predominantly enriched in genes that confer tetracycline resistance via ribosomal protection. MECOS is a useful tool for investigating HGT and the evolution of natural microbial communities within hosts, thereby advancing our understanding of microbial ecology and evolution.

Duplicated antibiotic resistance genes reveal ongoing selection and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) and gene duplication are often considered as separate mechanisms driving the evolution of new functions. However, the mobile genetic elements (MGEs) implicated in HGT can copy themselves, so positive selection on MGEs could drive gene duplications. Here, we use a combination of modeling and experimental evolution to examine this hypothesis and use long-read genome sequences of tens of thousands of bacterial isolates to examine its generality in nature. Modeling and experiments show that antibiotic selection can drive the evolution of duplicated antibiotic resistance genes (ARGs) through MGE transposition. A key implication is that duplicated ARGs should be enriched in environments associated with antibiotic use. To test this, we examined the distribution of duplicated ARGs in 18,938 complete bacterial genomes with ecological metadata. Duplicated ARGs are highly enriched in bacteria isolated from humans and livestock. Duplicated ARGs are further enriched in an independent set of 321 antibiotic-resistant clinical isolates. Our findings indicate that duplicated genes often encode functions undergoing positive selection and horizontal gene transfer in microbial communities.

Genes for cooperation are not more likely to be carried by plasmids.

Cooperation is prevalent across bacteria, but risks being exploited by non-cooperative cheats. Horizontal gene transfer, particularly via plasmids, has been suggested as a mechanism to stabilize cooperation. A key prediction of this hypothesis is that genes which are more likely to be transferred, such as those on plasmids, should be more likely to code for cooperative traits. Testing this prediction requires identifying all genes for cooperation in bacterial genomes. However, previous studies used a method which likely misses some of these genes for cooperation. To solve this, we used a new genomics tool, SOCfinder, which uses three distinct modules to identify all kinds of genes for cooperation. We compared where these genes were located across 4648 genomes from 146 bacterial species. In contrast to the prediction of the hypothesis, we found no evidence that plasmid genes are more likely to code for cooperative traits. Instead, we found the opposite-that genes for cooperation were more likely to be carried on chromosomes. Overall, the vast majority of genes for cooperation are not located on plasmids, suggesting that the more general mechanism of kin selection is sufficient to explain the prevalence of cooperation across bacteria.