Bacteria and Archaea display a variety of phenotypic traits and can adapt to diverse ecological niches. However, systematic annotation of prokaryotic phenotypes is lacking. We have therefore developed ProTraits, a resource containing ∼545 000 novel phenotype inferences, spanning 424 traits assigned to 3046 bacterial and archaeal species. These annotations were assigned by a computational pipeline that associates microbes with phenotypes by text-mining the scientific literature and the broader World Wide Web, while also being able to define novel concepts from unstructured text. Moreover, the ProTraits pipeline assigns phenotypes by drawing extensively on comparative genomics, capturing patterns in gene repertoires, codon usage biases, proteome composition and co-occurrence in metagenomes. Notably, we find that gene synteny is highly predictive of many phenotypes, and highlight examples of gene neighborhoods associated with spore-forming ability. A global analysis of trait interrelatedness outlined clusters in the microbial phenotype network, suggesting common genetic underpinnings. Our extended set of phenotype annotations allows detection of 57 088 high confidence gene-trait links, which recover many known associations involving sporulation, flagella, catalase activity, aerobicity, photosynthesis and other traits. Over 99% of the commonly occurring gene families are involved in genetic interactions conditional on at least one phenotype, suggesting that epistasis has a major role in shaping microbial gene content.

Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. However, assessing the impact of homologous recombination on accessory genes which are only present in a subset of strains of a given species remains challenging due to their complex phylogenetic relationships. Quantifying homologous recombination for accessory genes (which are important for niche-specific adaptations) in comparison to core genes (which are present in all strains and have essential functions) is critical to understanding how selection acts on variation to shape species diversity and genome structures of bacteria. Here, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome using >100,000 whole genome sequences from Streptococcus pneumoniae and several additional species. By analyzing diverse sets of sequence clusters, we show that core genes often have higher recombination rates than accessory genes, and for some bacterial species the associated effect sizes for these differences are pronounced. In a subset of species, we find that gene frequency and homologous recombination rate are positively correlated. For S. pneumoniae and several additional species, we find that while the recombination rate is higher for the core genome, the mutational divergence is lower, indicating that divergence-based homologous recombination barriers could contribute to differences in recombination rates between the core and accessory genome. Homologous recombination may therefore play a key role in increasing the efficiency of selection in the most conserved parts of the genome.

Analysis of any newly sequenced bacterial genome starts with the identification of protein-coding genes. Despite the accumulation of multiple complete genome sequences, which provide useful comparisons with close relatives among other organisms during the annotation process, accurate gene prediction remains quite difficult. A major reason for this situation is that genes are tightly packed in prokaryotes, resulting in frequent overlap. Thus, detection of translation initiation sites and/or selection of the correct coding regions remain difficult unless appropriate biological knowledge (about the structure of a gene) is imbedded in the approach.

The decades-long global trend of urbanization has led to a population that spends increasing amounts of time indoors. Exposure to microbes in buildings, and specifically in dust, is thus also increasing, and has been linked to various health outcomes and to antibiotic resistance genes (ARGs). These are most efficiently screened using DNA sequencing, but this method does not determine which microbes are viable, nor does it reveal whether their ARGs can actually disseminate to other microbes. We have thus performed the first study to: 1) examine the potential for ARG dissemination in indoor dust microbial communities, and 2) validate the presence of detected mobile ARGs in viable dust bacteria. Specifically, we integrated 166 dust metagenomes from 43 different buildings. Sequences were assembled, annotated, and screened for potential integrons, transposons, plasmids, and associated ARGs. The same dust samples were further investigated using cultivation and isolate genome and plasmid sequencing. Potential ARGs were detected in dust isolate genomes, and we confirmed their placement on mobile genetic elements using long-read sequencing. We found 183 ARGs, of which 52 were potentially mobile (associated with a putative plasmid, transposon or integron). One dust isolate related to Staphylococcus equorum proved to contain a plasmid carrying an ARG that was detected metagenomically and confirmed through whole genome and plasmid sequencing. This study thus highlights the power of combining cultivation with metagenomics to assess the risk of potentially mobile ARGs for public health.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming "swede midge" Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

The widespread nature of plastic pollution has given rise to wide scientific and social concern regarding the capacity of these materials to serve as vectors for pathogenic bacteria and reservoirs for Antimicrobial Resistance Genes (ARG). In- and ex-situ incubations were used to characterise the riverine plastisphere taxonomically and functionally in order to determine whether antibiotics within the water influenced the ARG profiles in these microbiomes and how these compared to those on natural surfaces such as wood and their planktonic counterparts.

The uncomposted faeces of dairy cow are usually stacked on cow breeding farms, dried under natural conditions and then used as cow bedding material or they may be continuously piled up. However, no information is available to evaluate variations in the human and animal pathogen genes and antibiotic resistance during the accumulation of fresh faeces of dairy cow to manure. Here, we present the metagenomic analysis of fresh faeces and manure from a dairy farm in Ning Xia, showing a unique enrichment of human and animal pathogen genes and antibiotic resistance genes (ARGs) in manure. We found that manure accumulation could significantly increase the diversity and abundance of the pathogenic constituents. Furthermore, pathogens from manure could spread to the plant environment and enphytotic pathogens could affect the yield and quality of crops during the use of manure as a fertilizer. Levels of virulence genes and ARGs increased with the enrichment of microbes and pathogens when faeces accumulated to manure. Accumulated manure was also the transfer station of ARGs to enrich the ARGs in the environment, indicating the ubiquitous presence of environmental antibiotic resistance genes. Our results demonstrate that manure accumulation and usage without effective manure management is an unreasonable approach that could enrich pathogenic microorganisms and ARGs in the environment. The manure metagenome structure allows us to appreciate the overall influence and interaction of animal waste on water, soil and other areas impacted by faecal accumulation and the factors that influence pathogen occurrence in products from dairy cows.

In this study, compound microbial inoculants, including three Bacillus strains and one Yeast strain, were inoculated into swine manure composting to explore the effects on antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), microbial community structure, and pathogenic bacteria. The results indicated that the abundances of the detected ARGs ranged from 3.6 × 103 to 1.13 × 108 copies/g. The ARGs with the highest abundance was sul2, and the lowest was blaCTX. Composting removes most of the ARGs and MGEs by 22.8-99.7%. These ARGs were significantly reduced during the thermophilic phase of compost. The removal rate of ARGs at the different layers of compost pile was different as follows: middle layer > upper layer > lower layer. But some ARGs proliferated significantly in the maturation phase of compost, especially the sulfonamide resistance genes. Compound microbial inoculants increased the temperature of compost, accelerated water loss, nitrogen fixation, and increased the removal rate of β-lactamase resistance genes, the transposon gene tn916 and part of tetracycline resistance genes by 3.7-23.8% in compost. Compound microbial inoculants changed the community structure and increased the Bacillus abundance in the thermophilic phase of compost. And it was helpful for removing pathogens during composting. The addition of compound microbial inoculants causes the decrease of Firmicutes and the increase of Bacteroidetes, which may be related to the removal and proliferation of ARGs.

The genders of Drosophila melanogaster vary in their sensitivities to microbial pathogens. While many of the immunity-related genes are located on the X chromosome, the polymorphisms within the Y chromosome were also shown to affect the immunity of flies. In this study, we investigated the necessity of individual genes on the Y chromosome (Y-genes) for male sensitivity to microbes. We identified several Y-genes whose genetic inactivation either increases or decreases the sensitivity of males to gastrointestinal infections with fungal Saccharomyces cerevisiae and bacterial Serratia liquefaciens. Specifically, the loss of function mutations in fly kl-5 and Ppr-Y Y-genes lead to increased and decreased sensitivity of males to fungal challenge, respectively, compared to female sensitivity. In contrast, mutations in Drosophila Pp1-Y1, kl-5, kl-3, Ppr-Y, CCY, and FDY Y-genes lead to increased sensitivity of males to bacterial infection, compared to females. Moreover, while these Y-genes are necessary, the Y chromosome is not sufficient for the sensitivity of males to microbes, since the sensitivity of XXY females to fungal and bacterial challenges was not different from the sensitivity of wild-type female flies, compared to males. This study assigns a new immunity-related function to numerous Y-genes in D. melanogaster.

Antimicrobial resistance (AMR) is a global public health threat. Environmental microbial communities act as reservoirs for AMR, containing genes associated with resistance, their precursors, and the selective pressures to encourage their persistence. Genomic surveillance could provide insight into how these reservoirs are changing and their impact on public health. The ability to enrich for AMR genomic signatures in complex microbial communities would strengthen surveillance efforts and reduce time-to-answer. Here, we test the ability of nanopore sequencing and adaptive sampling to enrich for AMR genes in a mock community of environmental origin. Our setup implemented the MinION mk1B, an NVIDIA Jetson Xavier GPU, and flongle flow cells. We observed consistent enrichment by composition when using adaptive sampling. On average, adaptive sampling resulted in a target composition that was 4x higher than a treatment without adaptive sampling. Despite a decrease in total sequencing output, the use of adaptive sampling increased target yield in most replicates.

Antimicrobial resistance (AMR) is a global public health threat. Environmental microbial communities act as reservoirs for AMR, containing genes associated with resistance, their precursors, and the selective pressures promoting their persistence. Genomic surveillance could provide insights into how these reservoirs change and impact public health. Enriching for AMR genomic signatures in complex microbial communities would strengthen surveillance efforts and reduce time-to-answer. Here, we tested the ability of nanopore sequencing and adaptive sampling to enrich for AMR genes in a mock community of environmental origin. Our setup implemented the MinION mk1B, an NVIDIA Jetson Xavier GPU, and Flongle flow cells. Using adaptive sampling, we observed consistent enrichment by composition. On average, adaptive sampling resulted in a target composition 4× higher than without adaptive sampling. Despite a decrease in total sequencing output, adaptive sampling increased target yield in most replicates. We also demonstrate enrichment in a diverse community using an environmental sample. This method enables rapid and flexible genomic surveillance.

Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming.

Pathogens resistant to antimicrobials form a significant threat to public health worldwide. Tackling multidrug-resistant pathogens via screening metagenomic libraries has become a common approach for the discovery of new antibiotics from uncultured microorganisms. This study focuses on capturing nonribosomal peptide synthase (NRPS) gene clusters implicated in the synthesis of many natural compounds of industrial relevance. A NRPS PCR assay was used to screen 2976 Escherichia coli clones in a soil metagenomic library to target NRPS genes. DNA extracts from 4 clones were sequenced and subjected to bioinformatic analysis to identify NRPS domains, their phylogeny, and substrate specificity.Successfully, 17 NRPS-positive hits with a biosynthetic potential were identified. DNA sequencing and BLAST analysis confirmed that NRPS protein sequences shared similarities with members of the genus Delftia in the Proteobacteria taxonomic position. Multiple alignment and phylogenetic analysis demonstrated that clones no. 15cd35 and 15cd37 shared low bootstrap values (54%) and were distantly far from close phylogenetic neighbors. Additionally, NRPS domain substrate specificity has no hits with the known ones; hence, they are more likely to use different substrates to produce new diverse antimicrobials. Further analysis confirmed that the NRPS hits resemble several transposon elements from other bacterial taxa, confirming its diversity. We confirmed that the analyses of the soil metagenomic library revealed a diverse set of NRPS related to the genus Delftia. An in-depth understanding of those positive NRPS hits is a crucial step for genetic manipulation of NRPS, shedding light on alternative novel antimicrobial compounds that can be used in drug discovery and hence supports the pharmaceutical sector.

Secretion of antimicrobial peptides (AMPep) is a central defense mechanism used by invertebrates to combat infections. In Drosophila the synthesis of these peptides is a highly regulated process allowing their rapid release in the hemolymph upon contact with pathogens and the arrest of their production after pathogen clearance. We observed that AMPep genes have either a transient or sustained expression profile in S2 Drosophila cells treated with peptidoglycan. Moreover, AMPep genes containing AU-rich elements (ARE) in their 3'-untranslated region (UTR) are subject to a post-transcriptional control affecting mRNA stability, thereby contributing to their transient expression profile. Cecropin A1 (CecA1) constitutes the prototype of this latter class of AMPeps. CecA1 mRNA bears in its 3'-UTR an ARE similar to class II AREs found in several short-lived mammalian mRNAs. In response to immune deficiency cascade signaling activated by Gram-negative peptidoglycans, CecA1 mRNA is transiently stabilized and subsequently submitted to deadenylation and decay mediated by the ARE present in its 3'-UTR. The functionality of CecA1 ARE relies on its ability to recruit TIS11 protein, which accelerates CecA1 mRNA deadenylation and decay. Moreover, we observed that CecA1 mRNA deadenylation is a biphasic process. Whereas early deadenylation is independent of TIS11, the later deadenylation phase depends on TIS11 and is mediated by CAF1 deadenylase. We also report that in contrast to tristetraprolin, its mammalian homolog, TIS11, is constitutively expressed in S2 cells and accumulates in cytoplasmic foci distinct from processing bodies, suggesting that the Drosophila ARE-mediated mRNA deadenylation and decay mechanism is markedly different in invertebrates and mammals.

Soil degradation is a serious global problem, but little is known about how soil microbial communities respond to soil degradation as well as their feedback to ecosystem functioning. In this study, we found the microbial community composition, structure and functional potential significantly altered in the degraded soils with bacterial wilt (termed as degraded soils). Compared with healthy soils, OTU richness of beneficial microorganisms were significantly decreased, but OTU richness of pathogenic microorganisms were significantly increased in the degraded soils. Functional gene array (GeoChip 5.0) analysis showed the functional metabolic potential of genes involved in stress, virulence, sulfur cycle, metal resistance, degradation of plant cell wall was significantly increased in the degraded soils. Increased functional metabolic potential of these genes may be related to the acidification and severe plant disease of degraded soils. Biological activity of degraded soils was obviously decreased with weakened soil enzyme activities when compared to the healthy soils. Soil pH and enzyme activities were negatively correlated with the abundance of genes involved in sulfur cycle, virulence, and stress responses. This study provides new insights into our understanding of soil microbial community responses to soil degradation.

Lateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.

Microbial communities underpin the Earth's biological and geochemical processes, but their complexity hampers understanding. Motivated by the challenge of diversity and the need to forge ways of capturing dynamical behaviour connecting genes to function, biologically independent experimental communities comprising hundreds of microbial genera were established from garden compost and propagated on nitrogen-limited minimal medium with cellulose (paper) as sole carbon source. After 1 year of bi-weekly transfer, communities retained hundreds of genera. To connect genes to function, we used a simple experimental manipulation that involved the periodic collection of selfish genetic elements (SGEs) from separate communities, followed by pooling and redistribution across communities. The treatment was predicted to promote amplification and dissemination of SGEs and thus horizontal gene transfer. Confirmation came from comparative metagenomics, which showed the substantive movement of ecologically significant genes whose dynamic across space and time could be followed. Enrichment of genes implicated in nitrogen metabolism, and particularly ammonification, prompted biochemical assays that revealed a measurable impact on community function. Our simple experimental strategy offers a conceptually new approach for unravelling dynamical processes affecting microbial community function. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.