Search Results (98)

For gene expression analysis in complex microbiomes, utilizing both metagenomic and metatranscriptomic reads from the same sample enables advanced functional analysis. Due to their diversity, metagenomic contigs are often used as reference sequences instead of complete genomes. However, studies optimizing mapping strategies for both read types remain limited. In addition, although transcripts per million (TPM) is commonly used for normalization, few studies have evaluated the influence of ribosomal RNA (rRNA) in metatranscriptomic reads. This study compared Burrows–Wheeler Aligner–Maximal Exact Match (BWA-MEM) and Bowtie2 as mapping tools for metagenomic contigs. Even after optimizing Bowtie2 parameters, BWA-MEM showed higher efficiency in mapping both metagenomic and metatranscriptomic reads. Further analysis revealed that rRNA sequences contaminate predicted protein-coding regions in metagenomic contigs. When comparing TPM values across samples, contamination by rRNA led to an overestimation of TPM changes. This effect was more pronounced when the difference in rRNA content between samples was larger. These findings suggest that metatranscriptomic reads mapped to rRNA should be excluded before TPM calculations. This study highlights key factors influencing read mapping and quantification in gene expression analysis of complex microbiomes. The findings provide insights for improving analytical accuracy and advancing functional studies using both metagenomic and metatranscriptomic data. Full article

Dichorhavirus is a recently accepted plant virus genus within the family Rhabdoviridae. Species assigned to the genus consist of bi-segmented, negative sense, single-stranded RNA viruses and are transmitted by Brevipalpus spp. Currently, there are five recognized species and two unclassified members in the genus Dichorhavirus. Four out of seven-orchid fleck virus (OFV), citrus leprosis virus N, citrus chlorotic spot virus, and citrus bright spot virus-can infect citrus and produce leprosis disease-like symptoms. The E-probe Diagnostic for Nucleic Acid Analysis (EDNA) was developed to reduce computational effort and then integrated within Microbe-Finder (MiFi^®) online platform to design and evaluate e-probes in raw High Throughput Sequencing (HTS) data. During this study, Dichorhavirus genomes were downloaded from public databases and e-probes were designed using the MiProbe incorporated into the MiFi^® platform. Three different sizes of e-probes, 40, 60, and 80 nucleotides, were developed and selected based on whole genome comparisons with near-neighbor genomes. For curation, each e-probe was searched in the NCBI nucleotide sequence database using BLASTn. All the e-probes that had hits with non-target species with ≥90% identities were removed. The sensitivity and specificity of Dichorhavirus genus, species, strain, and variant-specific e-probes were validated in vivo using HTS meta-transcriptomic libraries generated from Dichorhavirus-suspected citrus, orchid, and ornamentals. Through downstream analysis of HTS data, EDNA not only detected the known hosts of OFV but also discovered an unknown host leopard plant (Farfugium japonicum), and the possible existence of a new ornamental strain of OFV in nature. Full article

Background/Objectives: Dental caries remains a prevalent chronic disease globally, driven by complex interactions between the host, diet, and microbial communities. This study employs a metatranscriptomic RNA-Seq analysis to explore the functional dynamics of the dentin microbiome in both healthy and carious teeth. By examining the transcriptional activity of bacterial communities, we aimed to identify key microbial species and molecular functions associated with caries progression. Methods: Samples from six patients (three healthy and three decayed teeth) were analyzed using the Illumina NovaSeq 2000 platform, with data processed through the SAMSA2 pipeline for taxonomic and functional annotation. Results: The differential expression analysis revealed significant upregulation of Streptococcus and Lactobacillus species, including S. mutans, S. sobrinus, and L. salivarius, in carious samples, highlighting their roles in acid production and carbohydrate metabolism. Additionally, Mycobacterium species, known for their biofilm-forming capabilities and acid tolerance, were upregulated in decayed teeth. The Gene Ontology (GO) enrichment analysis identified unique molecular functions and biological processes in carious teeth, such as carbohydrate metabolism, oxidative stress response, and bacterial cell wall biogenesis, which are critical for microbial survival in acidic environments. In contrast, healthy teeth exhibited functions related to homeostasis and nutrient acquisition, reflecting a balanced microbial community. Conclusions: The study underscores the polymicrobial nature of dental caries, with multiple bacterial species contributing to disease progression through diverse metabolic and stress-response mechanisms. These findings provide deeper insights into the ecological shifts within the oral microbiome during caries development, emphasizing the importance of a functional metatranscriptomic analysis in understanding the pathogenesis of dental caries. Full article

Walnut canker is a common disease in the Xinjiang Uygur autonomous region of China, which is caused by Cytospora chrysosperma. To date, there is no effective control measure for this disease. Infection with mycoviruses has been widely proven to reduce the virulence of plant pathogenic fungi, with some mycoviruses even serving as potential biological control agents for plant diseases. In this study, mycoviruses associated with 31 strains of C. chrysosperma from Xinjiang Uygur autonomous region were identified by metatranscriptomic sequencing. Seven new mycoviruses were identified by BLAST and RT-PCR analysis, which were Botrytis cinerea partitivirus 5 (BcPV5), Gammapartitivirus sp-XJ1 (GVsp-XJ1), Botoulivirus sp-XJ2 (BVsp-XJ2), Luoyang Fusar tick virus 2 (LfTV2), Leptosphaeria biglobosa narnavirus 17 (LbNV17), Sclerotinia sclerotiorum narnavirus 6 (SsNV6), and Cytospora ribis mitovirus (CrMV3). Among these, BcPV5, GVsp-XJ1, BVsp-XJ2, CrMV3, and LfTV2 were found to co-infect C. chrysosperma strain WS-11 and significantly reduce both the colony growth rate and virulence of the host. After co-culturing the virus-free WS-FV strain with WS-11, the colony growth rate and virulence of the derivative strain were also decreased. These results provide potential biocontrol resources for the control of walnut canker. Full article

The shallow-sea hydrothermal vent at Guishan Islet, located off the coast of Taiwan, serves as a remarkable natural site for studying microbial ecology in extreme environments. In April 2019, we investigated the composition of prokaryotic picoplankton communities, their gene expression profiles, and the dissolved inorganic carbon uptake efficiency. Our results revealed that the chemolithotrophs Thiomicrorhabdus spp. contributed to the majority of primary production in the waters affected by the hydrothermal vent plume. The metatranscriptomic analysis aligned with the primary productivity measurements, indicating the significant gene upregulations associated with carboxysome-mediated carbon fixation in Thiomicrorhabdus. Synechococcus and Prochlorococcus served as the prokaryotic photoautotrophs for primary productivity in the waters with lower influence from hydrothermal vent emissions. Thiomicrorhabdus and picocyanobacteria jointly provided organic carbon for sustaining the shallow-sea hydrothermal vent ecosystem. In addition to the carbon fixation, the upregulation of genes involved in the SOX (sulfur-oxidizing) pathway, and the dissimilatory sulfate reduction indicated that energy generation and detoxification co-occurred in Thiomicrorhabdus. This study improved our understanding of the impacts of shallow-sea hydrothermal vents on the operation of marine ecosystems and biogeochemical cycles. Full article

Salt stress poses a significant constraint on rice production, so further exploration is imperative to elucidate the intricate molecular mechanisms governing salt tolerance in rice. By manipulating the rhizosphere microbial communities or targeting specific microbial functions, it is possible to enhance salt tolerance in crops, improving crop yields and food security in saline environments. In this study, we conducted rice rhizospheric microbial amplicon sequencing and metatranscriptome analysis, revealing substantial microbiomic differences between the salt-tolerant rice cultivar TLJIAN and the salt-sensitive HUAJING. Fungal taxa including Hormiactis, Emericellopsis, Ceriosporopsis, Dirkmeia, and Moesziomyces predominated in the rhizosphere of salt-tolerant rice, while bacterial genera such as Desulfoprunum and Hydrogenophaga exhibited notable differences. Metatranscriptomic analysis identified 7192 differentially expressed genes (DEGs) in the two rice varieties, with 3934 genes being upregulated and 3258 genes being downregulated. Enrichment analyses in KEGG and GO pathways highlighted the majority of DEGs were associated with the “two-component system”, “sulfur metabolism”, and “microbial metabolism in diverse environments”. The interaction network of DEGs and microbial taxa revealed upregulation of transporters, transcriptional factors, and chaperones, such as ABC transporters and chaperonin GroEL, in the rhizosphere microbiomes of salt-tolerant varieties. Our multi-omics network analysis unveiled that fungi like Ceriosporopsis and Dirkmeria, along with bacteria such as Desulfoprunum, Rippkaea, and Bellilinea, showed a positive correlation with flavonoid synthesis in salt-tolerant rice. This study provides an in-depth exploration of the distinctive microbial communities associated with the rhizosphere of salt-tolerant rice varieties, shedding light on the complex interactions between these microbial consortia and their host plants under stress conditions. Full article

Antimicrobial resistance is a major global concern and economic threat, necessitating a reliable monitoring approach to understand its frequency and spread via the environment. Hospital wastewater serves as a critical reservoir for antimicrobial-resistant organisms; however, its role in resistance gene distribution and dissemination remains poorly understood. This study integrates metagenomic and metatranscriptomic analyses, elucidating the dynamics of antimicrobial resistance in hospital wastewater. Integrated metagenomic and metatranscriptomic sequencing were used to identify actively expressed antimicrobial-resistant genes and antimicrobial-resistant bacteria, offering comprehensive insights into antimicrobial resistance dynamics in hospital wastewater. Liquid chromatography–tandem mass spectrometry analysis revealed the presence of ampicillin, sulbactam, levofloxacin, sulfamethoxazole, and trimethoprim in the sample, which could apply selective pressure on antimicrobial resistance gene expression. While multidrug resistance genes were the most prevalent sequences in both metagenome-assembled genomes and plasmids, plasmid-derived sequences showed a high mRNA/DNA ratio, emphasizing the presence of functionally expressed antimicrobial resistance genes on plasmids rather than on chromosomes. The metagenomic and metatranscriptomic analyses revealed Serratia nevei MAG14 with high mRNA levels of antimicrobial resistance genes; moreover, multidrug-resistant Serratia sp., genetically related to MAG14, was isolated from the wastewater, supporting the phenotypic characterization of crucial antimicrobial-resistant bacteria and validating the genome analysis results. The findings underscore key genes and bacteria as targets for antimicrobial resistance surveillance in hospital wastewater to protect public and environmental health. Full article

Bacterial keratitis caused by Pseudomonas aeruginosa is indeed a serious concern due to its potential to cause blindness and its resistance to antibiotics, partly attributed to biofilm formation and cytotoxicity to the cornea. The present study uses a meta-analysis of a transcriptomics dataset to identify important genes and pathways in biofilm formation of P. aeruginosa induced keratitis. By combining data from several studies, meta-analysis can enhance statistical power and robustness, enabling the identification of 83 differentially expressed candidate genes, including fis that could serve as therapeutic targets. The approach of combining meta-analysis with virtual screening and in vitro methods provides a comprehensive strategy for identifying potential target genes and pathways crucial for bacterial biofilm formation and development anti-biofilm medications against P. aeruginosa infections. The study identified 83 candidate genes that exhibited differential expression in the biofilm state, with fis proposed as an ideal target for therapy for P. aeruginosa biofilm formation. These techniques, meta-analysis, virtual screening, and invitro methods were used in combination to diagnostically identify these genes, which play a significant role in biofilms. This finding has highlighted a hallmark target list for P. aeruginosa anti-biofilm potential treatments. Full article

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts. Full article

Comparative metatranscriptomics of the bacterial and yeast communities of two milk kefir beverages (MKAA1 and MKAA2) was carried out. They were obtained by fermentation with two different frozen stocks of the kefir grain CIDCA AGK1, differing in rheological features and production of organic acids. We hypothesised that the differences in their physicochemical and rheological properties might be due to the microbial activity in each product. The dominance of lactic acid bacteria, yeast, and a marginal amount of acetic acid bacteria characterised the microbiome. The bacterial families Lactobacillaceae and Streptococcaceae accounted for almost all of the bacterial gene transcripts, with Lactobacillus helveticus, L. kefiranofaciens, L. gallinarum, and Lactococcus lactis being most frequent in the microbiome of the MKAA1 beverage and L. kefiranofaciens, Lc. Lactis, and Leuconostoc mesenteroides being the most prevalent in MKAA2. Dipodascaceae and Saccharomycetaceae were the leading yeast families, represented by Yarrowia lipolytica, Saccharomyces unisporus, and Kluyveromyces marxianus. MKAA1 and MKAA2 shared >75% KEGG Ortologs (KOs) in their bacteria and yeast libraries. The considerable decreases in total expressed genes (KEGG Ortologs) assigned to Lactobacillus helveticus and L. gallinarum might be related to the variations in the rheological features of the beverages, probably by compromising the interrelations with L. kefiranofaciens, which might explain the variations in the rheological features of the beverages. Full article

Bacteria from the genus Sulfitobacter are distributed across various marine habitats and play a significant role in sulfur cycling. However, the metabolic features of Sulfitobacter inhabiting marine biofilms are still not well understood. Here, complete genomes and paired metatranscriptomes of eight Sulfitobacter strains, isolated from biofilms on subtidal stones, have been analyzed to explore their central energy metabolism and potential of secondary metabolite biosynthesis. Based on average nucleotide identity and phylogenetic analysis, the eight strains were classified into six novel species and two novel strains. The reconstruction of the metabolic pathways indicated that all strains had a complete Entner–Doudoroff pathway, pentose phosphate pathway, and diverse pathways for amino acid metabolism, suggesting the presence of an optimized central carbon metabolism. Pangenome analysis further revealed the differences between the gene cluster distribution patterns among the eight strains, suggesting significant functional variation. Moreover, a total of 47 biosynthetic gene clusters were discovered, which were further classified into 37 gene cluster families that showed low similarity with previously documented clusters. Furthermore, metatranscriptomic analysis revealed the expressions of key functional genes involved in the biosynthesis of ribosomal peptides in in situ marine biofilms. Overall, this study sheds new light on the metabolic features, adaptive strategies, and value of genome mining in this group of biofilm-associated Sulfitobacter bacteria. Full article

Perinereis species are essential benthonic animals in coastal ecosystems and have significant roles as live feed in aquaculture, owing to their high-protein and low-fat nutritional profile. Despite their ecological importance, the viral communities associated with these organisms need to be better understood. In this study, we generated 2.6 × 10⁸ reads using meta-transcriptomic sequencing and de novo assembled 5.3 × 10³ virus-associated contigs. We identified 12 novel RNA viruses from two species, Perinereis aibuhitensis and P. wilsoni, which were classified into four major viral groups: Picobirnaviridae, Marnaviridae, unclassified Picornavirales, and unclassified Bunyavirales. Our findings revealed the hidden diversity of viruses and genome structures in Perinereis, enriching the RNA virosphere and expanding the host range of Picobirnaviridae, Marnaviridae, and Bunyavirales. This study also highlighted the potential biosecurity risk of the novel viruses carried by Perinereis to aquaculture. Full article

Macrofungi play important roles in the soil elemental cycle of terrestrial ecosystems. Fungal viruses are common in filamentous fungi, and some of them can affect the growth and development of hosts. However, the composition and evolution of macrofungal viruses are understudied. In this study, ninety strains of Trametes versicolor, Coprinellus micaceus, Amanita strobiliformis, and Trametes hirsuta were collected in China. Four mixed pools were generated by combining equal quantities of total RNA from each strain, according to the fungal species, and then subjected to RNA sequencing. The sequences were assembled, annotated, and then used for phylogenetic analysis. Twenty novel viruses or viral fragments were characterized from the four species of macrofungi. Based on the phylogenetic analysis, most of the viral contigs were classified into ten viral families or orders: Barnaviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Fusariviridae, Hypoviridae, Totiviridae, Mitoviridae, Mymonaviridae, and Bunyavirales. Of these, ambi-like viruses with circular genomes were widely distributed among the studied species. Furthermore, the number and overall abundance of viruses in these four species of macrofungi (Basidiomycota) were found to be much lower than those in broad-host phytopathogenic fungi (Ascomycota: Sclerotinia sclerotiorum, and Botrytis cinerea). By employing metatranscriptomic analysis in this study, for the first time, we demonstrated the presence of multiple mycoviruses in Amanita strobiliformis, Coprinellus micaceus, Trametes hirsute, and Trametes versicolor, significantly contributing to research on mycoviruses in macrofungi. Full article

The skin is a complex ecosystem colonized by millions of microorganisms, the skin microbiota, which are crucial in regulating not only the physiological functions of the skin but also the metabolic changes underlying the onset of skin diseases. The high microbial colonization together with a low diversity at the phylum level and a high diversity at the species level of the skin is very similar to that of the gastrointestinal tract. Moreover, there is an important communication pathway along the gut–brain–skin axis, especially associated with the modulation of neurotransmitters by the microbiota. Therefore, it is evident that the high complexity of the skin system, due not only to the genetics of the host but also to the interaction of the host with resident microbes and between microbe and microbe, requires a multi-omics approach to be deeply understood. Therefore, an integrated analysis, with high-throughput technologies, of the consequences of microbial interaction with the host through the study of gene expression (genomics and metagenomics), transcription (transcriptomics and meta-transcriptomics), and protein production (proteomics and meta-proteomics) and metabolite formation (metabolomics and lipidomics) would be useful. Although to date very few studies have integrated skin metabolomics data with at least one other ‘omics’ technology, in the future, this approach will be able to provide simple and fast tests that can be routinely applied in both clinical and cosmetic settings for the identification of numerous skin diseases and conditions. It will also be possible to create large archives of multi-omics data that can predict individual responses to pharmacological treatments and the efficacy of different cosmetic products on individual subjects by means of specific allotypes, with a view to increasingly tailor-made medicine. In this review, after analyzing the complexity of the skin ecosystem, we have highlighted the usefulness of this emerging integrated omics approach for the analysis of skin problems, starting with one of the latest ‘omics’ sciences, metabolomics, which can photograph the expression of the genome during its interaction with the environment. Full article

The mutualistic symbiosis relationship between the gut microbiome and their insect hosts has attracted much scientific attention. The native woodwasp, Sirex nitobei, and the invasive European woodwasp, Sirex noctilio, are two pests that infest pines in northeastern China. Following its encounter with the native species, however, there is a lack of research on whether the gut microbiome of S. noctilio changed, what causes contributed to these alterations, and whether these changes were more conducive to invasive colonization. We used high-throughput and metatranscriptomic sequencing to investigate S. noctilio larval gut and frass from four sites where only S. noctilio and both two Sirex species and investigated the effects of environmental factors, biological interactions, and ecological processes on S. noctilio gut microbial community assembly. Amplicon sequencing of two Sirex species revealed differential patterns of bacterial and fungal composition and functional prediction. S. noctilio larval gut bacterial and fungal diversity was essentially higher in coexistence sites than in separate existence sites, and most of the larval gut bacterial and fungal community functional predictions were significantly different as well. Moreover, temperature and precipitation positively correlate with most of the highly abundant bacterial and fungal genera. Source-tracking analysis showed that S. noctilio larvae at coexistence sites remain dependent on adult gut transmission (vertical transmission) or recruitment to frass (horizontal transmission). Meanwhile, stochastic processes of drift and dispersal limitation also have important impacts on the assembly of S. noctilio larval gut microbiome, especially at coexistence sites. In summary, our results reveal the potential role of changes in S. noctilio larval gut microbiome in the successful colonization and better adaptation of the environment. Full article