Genomic and metagenomic analyses of the domestic mite Tyrophagus putrescentiae identify it as a widespread environmental contaminant and a host of a basal, mite-specific Wolbachia lineage (supergroup Q).

Tyrophagus putrescentiae (mould mite) is a global, microscopic trophic generalist that commonly occurs in various human-created habitats, causing allergies and damaging stored food. Its ubiquity and extraordinary ability to penetrate research samples or cultures through air currents or by active walking through tights spaces (such as treads of screw caps) may lead to sample contamination and introduction of its DNA to research materials in the laboratory. This prompts a thorough investigation into potential sequence contamination in public genomic databases. The trophic success of T. putrescentiae is primarily attributed to the symbiotic bacteria housed in specialized internal mite structures, facilitating adaptation to varied nutritional niches. However, recent work suggests that horizontal transfer of bacterial/fungal genes related to nutritional functionality may also contribute to the mite's trophic versatility. This aspect requires independent confirmation. Additionally, T. putrescentiae harbors an uncharacterized and genetically divergent bacterium, Wolbachia, displaying blocking and microbiome-modifying effects. The phylogenomic position and supergroup assignment of this bacterium are unknown. Here, we sequenced and assembled the T. putrescentiae genome, analyzed its microbiome, and performed detailed phylogenomic analyses of the mite-specific Wolbachia. We show that T. putrescentiae DNA is a substantial source of contamination of research samples. Its DNA may inadvertently be co-extracted with the DNA of the target organism, eventually leading to sequence contamination in public databases. We identified a diversity of bacterial species associated with T. putrescentiae, including those capable of rapidly developing antibiotic resistance, such as Escherichia coli. Despite the presence of diverse bacterial communities in T. putrescentiae, we did not detect any recent horizontal gene transfers in this mite species and/or in astigmatid (domestic) mites in general. Our phylogenomic analysis of Wolbachia recovered a basal, mite-specific lineage (supergroup Q) represented by two Wolbachia spp. from the mould mite and a gall-inducing plant mite. Fluorescence in situ hybridization confirmed the presence of Wolbachia inside the mould mite. The discovery of an early derivative Wolbachia lineage (supergroup Q) in two phylogenetically unrelated and ecologically dissimilar mites suggests that this endosymbiotic bacterial lineage formed a long-term association with mites. This finding provides a unique insight into the early evolution and host associations of Wolbachia. Further discoveries of Wolbachia diversity in acariform mites are anticipated.

Co-colonization of different species harboring KPC or NDM carbapenemase in the same host gut: insight of resistance evolution by horizontal gene transfer.

Introduction: The dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However, limited research has focused on the in-host transferability of carbapenem resistance. In this study, ten isolates were collected from gut specimens of five individuals, each hosting two different species, including Escherichia coli, Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae, or Citrobacter koseri. Methods: Species identification and antimicrobial susceptibility were determined by MALDI-TOF MS and broth microdilution method. Carbapenemase genes were detected and localized using PCR, S1-PFGE and southern blot. The transferability of carbapenemase genes between species was investigated through filter mating experiments, and the genetic contexts of the plasmids were analyzed using whole genome sequencing. Results and discussion: Our results revealed that each of the ten isolates harbored a carbapenemase gene, including bla NDM-5, bla NDM-1, or bla KPC-2, on a plasmid. Five different plasmids were successfully transferred to recipient cells of E. coli, K. pneumoniae or A. baumannii by transconjugation. The genetic contexts of the carbapenemase gene were remarkably similar between the two CRE isolates from each individual. This study highlights the potential for interspecies plasmid transmission in human gut, emphasizing the colonization of CRE as a significant risk factor for the dissemination of carbapenemase genes within the host. These findings underscore the need for appropriate intestinal CRE screening and colonization prevention.

The extent and characteristics of DNA transfer between plasmids and chromosomes.

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids and chromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes.

Deciphering the impact of organic loading rate and digestate recirculation on the occurrence patterns of antibiotics and antibiotic resistance genes in dry anaerobic digestion of kitchen waste.

Organic loading rate (OLR) is crucial for determining the stability of dry anaerobic digestion (AD). Digestate recirculation contributes to reactor stability and enhances methane production. Nevertheless, the understanding of how OLR and digestate recirculation affect the abundance and diversity of antibiotics and antibiotic resistance genes (ARGs), as well as the mechanisms involved in the dissemination of ARGs, remains limited. This study thoroughly investigated this critical issue through a long-term pilot-scale experiment. The metabolome analyses revealed the enrichment of various antibiotics, such as aminoglycoside, tetracycline, and macrolide, under low OLR conditions (OLR ≤ 4.0 g·VS/L·d) and the reactor instability. Antibiotics abundance decreased by approximately 19.66-31.69 % during high OLR operation (OLR ≥ 6.0 g·VS/L·d) with digestate recirculation. The metagenome analyses demonstrated that although low OLR promoted reactor stability, it facilitated the proliferation of antibiotic-resistant bacteria, such as Pseudomonas, and triggered functional profiles related to ATP generation, oxidative stress response, EPS secretion, and cell membrane permeability, thereby facilitating horizontal gene transfer (HGT) of ARGs. However, under stable operation at an OLR of 6.0 g·VS/L·d, there was a decrease in ARGs abundance but a notable increase in human pathogenic bacteria (HPB) and mobile genetic elements (MGEs). Subsequently, during reactor instability, the abundance of ARGs and HPB increased. Notably, during digestate recirculation at OLR levels of 6.0 and 7.0 g·VS/L·d, the process attenuated the risk of ARGs spread by reducing the diversity of ARGs hosts, minimizing interactions among ARGs hosts, ARGs, and MGEs, and weakening functional profiles associated with HGT of ARGs. Overall, digestate recirculation aids in reducing the abundance of antibiotics and ARGs under high OLR conditions. These findings provide advanced insights into how OLR and digestate recirculation affect the occurrence patterns of antibiotics and ARGs in dry AD.

Microbial diversity and ecological complexity emerging from environmental variation and horizontal gene transfer in a simple mathematical model.

BACKGROUND: Microbiomes are generally characterized by high diversity of coexisting microbial species and strains, and microbiome composition typically remains stable across a broad range of conditions. However, under fixed conditions, microbial ecology conforms with the exclusion principle under which two populations competing for the same resource within the same niche cannot coexist because the less fit population inevitably goes extinct. Therefore, the long-term persistence of microbiome diversity calls for an explanation. RESULTS: To explore the conditions for stabilization of microbial diversity, we developed a simple mathematical model consisting of two competing populations that could exchange a single gene allele via horizontal gene transfer (HGT). We found that, although in a fixed environment, with unbiased HGT, the system obeyed the exclusion principle, in an oscillating environment, within large regions of the phase space bounded by the rates of reproduction and HGT, the two populations coexist. Moreover, depending on the parameter combination, all three major types of symbiosis were obtained, namely, pure competition, host-parasite relationship, and mutualism. In each of these regimes, certain parameter combinations provided for synergy, that is, a greater total abundance of both populations compared to the abundance of the winning population in the fixed environment. CONCLUSIONS: The results of this modeling study show that basic phenomena that are universal in microbial communities, namely, environmental variation and HGT, provide for stabilization and persistence of microbial diversity, and emergence of ecological complexity.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

CRISPR-Cas3 and type I restriction-modification team up against blaKPC-IncF plasmid transfer in Klebsiella pneumoniae.

OBJECTIVE: We explored whether the Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification (R-M) systems are compatible and act together to resist plasmid attacks. METHODS: 932 global whole-genome sequences from GenBank, and 459 K. pneumoniae isolates from six provinces of China, were collected to investigate the co-distribution of CRISPR-Cas, R-M systems, and blaKPC plasmid. Conjugation and transformation assays were applied to explore the anti-plasmid function of CRISPR and R-M systems. RESULTS: We found a significant inverse correlation between the presence of CRISPR and R-M systems and blaKPC plasmids in K. pneumoniae, especially when both systems cohabited in one host. The multiple matched recognition sequences of both systems in blaKPC-IncF plasmids (97%) revealed that they were good targets for both systems. Furthermore, the results of conjugation assay demonstrated that CRISPR-Cas and R-M systems in K. pneumoniae could effectively hinder blaKPC plasmid invasion. Notably, CRISPR-Cas and R-M worked together to confer a 4-log reduction in the acquisition of blaKPC plasmid in conjugative events, exhibiting robust synergistic anti-plasmid immunity. CONCLUSIONS: Our results indicate the synergistic role of CRISPR and R-M in regulating horizontal gene transfer in K. pneumoniae and rationalize the development of antimicrobial strategies that capitalize on the immunocompromised status of KPC-KP.

Horizontal gene transfer plays a crucial role in the development of antibiotic resistance in an antibiotic-free shrimp farming system.

Antibiotic selective pressure in aquaculture systems often results in the antibiotic resistance genes (ARGs) proliferation. Nonetheless, a paucity of data exists concerning the mechanisms of ARGs development in aquaculture systems without the influences of antibiotics. This study utilized metagenomic approaches to elucidate the dynamics and transfer mechanisms of ARGs throughout the aquaculture of Pacific white shrimp. A marked change in the resistome was observed throughout the aquaculture without antibiotics. The total ARGs relative abundance increased from 0.05 to 0.33 by day 90 of cultivation, with even higher in mixed wastewater (0.44). Both bacterial communities and mobile genetic elements play pivotal roles in the development of ARGs. Metagenome-assembled genomes showed enrichment of environmentally intrinsic ARGs on chromosomes including macB and mdtK. The plasmid-mediated horizontal transfer was recognized as a principal factor contributing to the rise of ARGs, particularly for tetG and floR, and this led to an escalation of resistance risk, peaking at a risks core of 35.43 on day 90. This study demonstrates that horizontal gene transfer plays a crucial role in ARGs development without antibiotic pressure, which can provide a theoretical foundation for controlling ARGs proliferation in aquaculture systems.

Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Unveiling the microevolution of antimicrobial resistance in selected Pseudomonas aeruginosa isolates from Egyptian healthcare settings: A genomic approach.

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.

Multi-omics insights into the function and evolution of sodium benzoate biodegradation pathway in Benzoatithermus flavus gen. nov., sp. nov. from hot spring.

Biodegradation stands as an eco-friendly and effective approach for organic contaminant remediation. However, research on microorganisms degrading sodium benzoate contaminants in extreme environments remains limited. In this study, we report to display the isolation of a novel hot spring enriched cultures with sodium benzoate (400 mg/L) as the sole carbon source. The results revealed that the phylum Pseudomonadota was the potential sodium benzoate degrader and a novel genus within the family Geminicoccaceae of this phylum. The isolated strain was named Benzoatithermus flavus SYSU G07066T and was isolated from HNT-2 hot spring samples. Genomic analysis revealed that SYSU G07066T carried benABC genes and physiological experiments indicated the ability to utilize sodium benzoate as a sole carbon source for growth, which was further confirmed by transcriptomic data with expression of benABC. Phylogenetic analysis suggested that Horizontal Gene Transfer (HGT) plays a significant role in acquiring sodium benzoate degradation capability among prokaryotes, and SYSU G07066T might have acquired benABC genes through HGT from the family Acetobacteraceae. The discovery of the first microorganism with sodium benzoate degradation function from a hot spring enhances our understanding of the diverse functions within the family Geminicoccaceae. This study unearths the first novel genus capable of efficiently degrading sodium benzoate and its evolution history at high temperatures, holding promising industrial applications, and provides a new perspective for further exploring the application potential of hot spring "microbial dark matter".

Two high-quality Prototheca zopfii genomes provide new insights into their evolution as obligate algal heterotrophs and their pathogenicity.

The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Prototheca zopfii Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although P. zopfii maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two P. zopfii genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of Prototheca and non-photosynthetic organisms. The two high-quality P. zopfii genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity. IMPORTANCE: The genus Prototheca, characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii. The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations.

Bacteria contain a wide variety of innate and adaptive immune systems which provide protection to the host against invading genetic material, including bacteriophages (phages). It is becoming increasingly clear that bacterial immune systems are frequently lost and gained through horizontal gene transfer (HGT). However, how and when new immune systems can become established in a bacterial population has remained largely unstudied. We developed a joint epidemiological and evolutionary model that predicts the conditions necessary for the spread of a CRISPR-Cas immune system into a bacterial population lacking this system. We find that whether bacteria carrying CRISPR-Cas will spread (increase in frequency) into a bacterial population depends on the abundance of phages and the difference in the frequency of phage resistance mechanisms between bacteria carrying a CRISPR-Cas immune system, and those not (denoted as ${f}_{\Delta }$). Specifically, the abundance of cells carrying CRISPR-Cas will increase if there is a higher proportion of phage resistance (either via CRISPR-Cas immunity or surface modification) in the CRISPR-Cas possessing population than in the cells lacking CRISPR-Cas. We experimentally validated these predictions using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model. Specifically, by varying the initial ratios of different strains of bacteria that carry alternative forms of phage resistance we confirmed that the spread of cells carrying CRISPR-Cas through a population can be predicted based on phage density and the relative frequency of resistance phenotypes. Understanding which conditions promote the spread of CRISPR-Cas systems helps to predict when and where these defences can establish in bacterial populations after a horizontal gene transfer event, both in ecological and clinical contexts.

Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic.

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

Understanding the rapid spread of antimicrobial resistance genes mediated by IS26.

Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends. We retrieved 2656  translocatable  IS 26 -bounded  units with  ARGs (tIS26-bUs-ARGs) in complete bacterial genomes from the NCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that  IS 26 -bounded  units (IS26-bUs) mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26-bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of "passenger" genes. These observations extend our knowledge of the IS26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Integration of large and diverse angiosperm DNA fragments into Asian Gnetum mitogenomes.

BACKGROUND: Horizontal gene transfer (HGT) events have rarely been reported in gymnosperms. Gnetum is a gymnosperm genus comprising 25‒35 species sympatric with angiosperms in West African, South American, and Southeast Asian rainforests. Only a single acquisition of an angiosperm mitochondrial intron has been documented to date in Asian Gnetum mitogenomes. We wanted to develop a more comprehensive understanding of frequency and fragment length distribution of such events as well as their evolutionary history in this genus. RESULTS: We sequenced and assembled mitogenomes from five Asian Gnetum species. These genomes vary remarkably in size and foreign DNA content. We identified 15 mitochondrion-derived and five plastid-derived (MTPT) foreign genes. Our phylogenetic analyses strongly indicate that these foreign genes were transferred from diverse eudicots-mostly from the Rubiaceae genus Coptosapelta and ten genera of Malpighiales. This indicates that Asian Gnetum has experienced multiple independent HGT events. Patterns of sequence evolution strongly suggest DNA-mediated transfer between mitochondria as the primary mechanism giving rise to these HGT events. Most Asian Gnetum species are lianas and often entwined with sympatric angiosperms. We therefore propose that close apposition of Gnetum and angiosperm stems presents opportunities for interspecific cell-to-cell contact through friction and wounding, leading to HGT. CONCLUSIONS: Our study reveals that multiple HGT events have resulted in massive amounts of angiosperm mitochondrial DNA integrated into Asian Gnetum mitogenomes. Gnetum and its neighboring angiosperms are often entwined with each other, possibly accounting for frequent HGT between these two phylogenetically remote lineages.

Anthropogenic pollution may enhance natural transformation in water, favouring the spread of antibiotic resistance genes.

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Polyvinyl chloride microplastics in the aquatic environment enrich potential pathogenic bacteria and spread antibiotic resistance genes in the fish gut.

Microplastics and antibiotics coexist in aquatic environments, especially in freshwater aquaculture areas. However, as the second largest production of polyvinyl chloride (PVC) in the world, the effects of co-exposure to microplastics particles and antibiotics on changes in antibiotic resistance gene (ARG) profiles and the microbial community structure of aquatic organism gut microorganisms are poorly understood. Therefore, in this study, carp (Cyprinus carpio) were exposed to single or combined PVC microplastic contamination and oxytetracycline (OTC) or sulfamethazine (SMZ) for 8 weeks. PVC microplastics can enrich potential pathogenic bacteria, such as Enterobacter and Acinetobacter, among intestinal microorganisms. The presence of PVC microplastics enhanced the selective enrichment and dissemination risk of ARGs. PVC microplastics combined with OTC (OPVC) treatment significantly increased the abundance of tetracycline resistance genes (1.40-fold) compared with that in the OTC exposure treatment, revealing an obvious co-selection effect. However, compared with those in the control group, the total abundance of ARGs and MGEs in the OPVC treatment groups were significantly lower, which was correlated with the reduced abundances of the potential host Enterobacter. Overall, our results emphasized the diffusion and spread of ARGs are more influenced by PVC microplastics than by antibiotics, which may lead to antibiotic resistance in aquaculture.

Horizontally transferred glycoside hydrolase 26 may aid hemipteran insects in plant tissue digestion.

Glycoside hydrolases are enzymes that break down complex carbohydrates into simple sugars by catalyzing the hydrolysis of glycosidic bonds. There have been multiple instances of adaptive horizontal gene transfer of genes belonging to various glycoside hydrolase families from microbes to insects, as glycoside hydrolases can metabolize constituents of the carbohydrate-rich plant cell wall. In this study, we characterize the horizontal transfer of a gene from the glycoside hydrolase family 26 (GH26) from bacteria to insects of the order Hemiptera. Our phylogenies trace the horizontal gene transfer to the common ancestor of the superfamilies Pentatomoidea and Lygaeoidea, which include stink bugs and seed bugs. After horizontal transfer, the gene was assimilated into the insect genome as indicated by the gain of an intron, and a eukaryotic signal peptide. Subsequently, the gene has undergone independent losses and expansions in copy number in multiple lineages, suggesting an adaptive role of GH26s in some insects. Finally, we measured tissue-level gene expression of multiple stink bugs and the large milkweed bug using publicly available RNA-seq datasets. We found that the GH26 genes are highly expressed in tissues associated with plant digestion, especially in the principal salivary glands of the stink bugs. Our results are consistent with the hypothesis that this horizontally transferred GH26 was co-opted by the insect to aid in plant tissue digestion and that this HGT event was likely adaptive.