Giant virus diversity and host interactions through global metagenomics.

Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.

Tau-typing: a Nextflow pipeline for finding the best phylogenetic markers in the genome for molecular typing of microbial species.

SUMMARY: Tau-typing is an integrated analysis pipeline for identifying genes or genomic segments whose phylogenetic resolving power most closely resembles the genome-wide resolving power of an input collection of genomes using the Kendall Tau rank correlation statistic. The pipeline is implemented in Nextflow and uses Docker and Singularity containers to ensure reliable scalability and reproducibility of results. This pipeline is particularly suitable for organisms for which whole-genome sequencing remains unaffordable or unscalable for routine applications, such as protozoan parasites which are not amenable to laboratory culture-based methods. AVAILABILITY AND IMPLEMENTATION: Tau-typing is freely available at https://github.com/hseabolt/tautyping. The pipeline is implemented in Nextflow with Singularity support.

Multi-omics analysis of the gut microbiome and metabolites associated with the psychoneurological symptom cluster in children with cancer receiving chemotherapy.

BACKGROUND: Children with cancer receiving chemotherapy commonly report a cluster of psychoneurological symptoms (PNS), including pain, fatigue, anxiety, depression, and cognitive dysfunction. The role of the gut microbiome and its functional metabolites in PNS is rarely studied among children with cancer. This study investigated the associations between the gut microbiome-metabolome pathways and PNS in children with cancer across chemotherapy as compared to healthy children. METHODS: A case-control study was conducted. Cancer cases were recruited from Children's Healthcare of Atlanta and healthy controls were recruited via flyers. Participants reported PNS using the Pediatric Patient-Reported Outcomes Measurement Information System. Data for cases were collected pre-cycle two chemotherapy (T0) and post-chemotherapy (T1), whereas data for healthy controls were collected once. Gut microbiome and its metabolites were measured using fecal specimens. Gut microbiome profiling was performed using 16S rRNA V4 sequencing, and metabolome was performed using an untargeted liquid chromatography-mass spectrometry approach. A multi-omics network integration program analyzed microbiome-metabolome pathways of PNS. RESULTS: Cases (n = 21) and controls (n = 14) had mean ages of 13.2 and 13.1 years. For cases at T0, PNS were significantly associated with microbial genera (e.g., Ruminococcus, Megasphaera, and Prevotella), which were linked with carnitine shuttle (p = 0.0003), fatty acid metabolism (p = 0.001) and activation (p = 0.001), and tryptophan metabolism (p = 0.008). Megasphaera, clustered with aspartate and asparagine metabolism (p = 0.034), carnitine shuttle (p = 0.002), and tryptophan (p = 0.019), was associated with PNS for cases at T1. Gut bacteria with potential probiotic functions, along with fatty acid metabolism, tryptophan, and carnitine shuttle, were more clustered in cancer cases than the control network and this linkage with PNS needs further studies. CONCLUSIONS: Using multi-omics approaches, this study indicated specific microbiome-metabolome pathways linked with PNS in children with cancer across chemotherapy. Due to limitations such as antibiotic use in cancer cases, these findings need to be further confirmed in a larger cohort.

Judicial Opinion 129.

Opinion 129 addresses the status of Firmicutes corrig. Gibbons and Murray 1978 (Approved Lists 1980). The name has the category 'division' and was included in the Approved Lists of Bacterial Names, although that category had previously been removed from the International Code of Nomenclature of Bacteria (1975 revision onwards). When the category 'phylum' was introduced into the International Code of Nomenclature of Prokaryotes (ICNP) in 2021, equivalence between 'phylum' and 'division' was not stipulated. Since the definition of the taxonomic categories and their relative order is one of the principal tasks of every code of nomenclature, the inclusion of Firmicutes corrig. Gibbons and Murray 1978 in the Approved Lists was an error. The name is either not validly published or illegitimate because its category is not covered by the ICNP. If Firmicutes corrig. Gibbons and Murray 1978 (Approved Lists 1980) was a validly published phylum name, it would be illegitimate because it would contravene Rule 8, which does not permit any deviation from the requirement to derive a phylum name from the name of the type genus. Since Firmicutes corrig. Gibbons and Murray 1978 is also part of a 'misfitting megaclassification' recognized in Opinion 128, the name is rejected, without any pre-emption regarding a hypothetically validly published name Firmicutes at the rank of phylum. Gracilicutes Gibbons and Murray 1978 (Approved Lists 1980) and Anoxyphotobacteriae Gibbons and Murray 1978 (Approved Lists 1980) are also rejected. The validly published phylum names have a variety of advantages over their not validly published counterparts and cannot be replaced with ad hoc names suggested in the literature. To ease the transition, it is recommended to mention the not validly published phylum names which strongly deviate in spelling from their validly published counterparts along with the latter in publications during the next years.

Guidelines for interpreting the International Code of Nomenclature of Prokaryotes and for preparing a Request for an Opinion.

In this paper the Judicial Commission provides general guidance for interpreting the International Code of Nomenclature of Prokaryotes (ICNP) and specific assistance to authors, reviewers and editors of a Request for an Opinion, or of other suggestions related to the ICNP. The role of the Judicial Commission is recapitulated, particularly with respect to the processing of such Requests. Selected kinds of nomenclature-related proposals are discussed that are unsuitable as the basis for a Request. Particular emphasis is put on Requests for placing names or epithets on the list of nomina rejicienda, and a dichotomous identification key is provided to guide potential authors of a Request that targets the name of a species or subspecies because of issues with its type strain. To this end, the criteria for the valid publication of such names under the ICNP are revisited. Aspects of other kinds of Requests are also addressed. The study is based on a comprehensive review of all Judicial Opinions issued since the publication of the Approved Lists in 1980. One goal of this paper is to assist potential authors in deciding whether their concern should be the subject of a Request, and if so, in composing it with the greatest chance of success. It is also clarified how to obtain additional help regarding nomenclature-related issues.

Judicial Opinion 128.

Judicial Opinion 128 addresses nomenclatural issues related to the names of classes validly published under the International Code of Nomenclature of Prokaryotes. It is confirmed that the common ending -proteobacteria of some class names is not indicative of a joint taxonomic or phylogenetic placement; that the nomenclatural type of Mollicutes Edward and Freundt 1967 (Approved Lists 1980) is Mycoplasmatales Freundt 1955 (Approved Lists 1980); and that the placement of a name on the list of rejected names does not imply that another name with the same spelling but a distinct rank is also placed on that list. The names at the rank of class Anoxyphotobacteria (Gibbons and Murray 1978) Murray 1988, Archaeobacteria Murray 1988, Bacteria Haeckel 1894 (Approved Lists 1980), Firmibacteria Murray 1988, Microtatobiotes Philip 1956 (Approved Lists 1980), Oxyphotobacteria (ex Gibbons and Murray 1978) Murray 1988, Photobacteria Gibbons and Murray 1978 (Approved Lists 1980), Proteobacteria Stackebrandt et al. 1988, Schizomycetes Nägeli 1857 (Approved Lists 1980), Scotobacteria Gibbons and Murray 1978 (Approved Lists 1980) are placed on the list of rejected names. For three common nominative singular suffixes of genus names their genitive singular and nominative plural forms are confirmed: -bacter (-bacteris, -bacteres); -fex (-ficis, -fices); and -genes (-genis, -genes). The class names Aquificae Reysenbach 2002, Chrysiogenetes Garrity and Holt 2002, Chthonomonadetes Lee et al. 2011, Gemmatimonadetes Zhang et al. 2003, Opitutae Choo et al. 2007 and Verrucomicrobiae Hedlund et al. 1998 are orthographically corrected to Aquificia, Chrysiogenia, Chthonomonadia, Gemmatimonadia, Opitutia and Verrucomicrobiia, respectively.

The Great Oxidation Event expanded the genetic repertoire of arsenic metabolism and cycling.

The rise of oxygen on the early Earth about 2.4 billion years ago reorganized the redox cycle of harmful metal(loids), including that of arsenic, which doubtlessly imposed substantial barriers to the physiology and diversification of life. Evaluating the adaptive biological responses to these environmental challenges is inherently difficult because of the paucity of fossil records. Here we applied molecular clock analyses to 13 gene families participating in principal pathways of arsenic resistance and cycling, to explore the nature of early arsenic biogeocycles and decipher feedbacks associated with planetary oxygenation. Our results reveal the advent of nascent arsenic resistance systems under the anoxic environment predating the Great Oxidation Event (GOE), with the primary function of detoxifying reduced arsenic compounds that were abundant in Archean environments. To cope with the increased toxicity of oxidized arsenic species that occurred as oxygen built up in Earth's atmosphere, we found that parts of preexisting detoxification systems for trivalent arsenicals were merged with newly emerged pathways that originated via convergent evolution. Further expansion of arsenic resistance systems was made feasible by incorporation of oxygen-dependent enzymatic pathways into the detoxification network. These genetic innovations, together with adaptive responses to other redox-sensitive metals, provided organisms with novel mechanisms for adaption to changes in global biogeocycles that emerged as a consequence of the GOE.

Rapid changes in coastal ocean microbiomes uncoupled with shifts in environmental variables.

Disturbances, here defined as events that directly alter microbial community composition, are commonly studied in host-associated and engineered systems. In spite of global change both altering environmental averages and increasing extreme events, there has been relatively little research into the causes, persistence and population-level impacts of disturbance in the dynamic coastal ocean. Here, we utilize 3 years of observations from a coastal time series to identify disturbances based on the largest week-over-week changes in the microbiome (i.e. identifying disturbance as events that alter the community composition). In general, these microbiome disturbances were not clearly linked to specific environmental factors and responsive taxa largely differed, aside from SAR11, which generally declined. However, several disturbance metagenomes identified increased phage-associated genes, suggesting that unexplained community shifts might be caused by increased mortality. Furthermore, a category 1 hurricane, the only event that would likely be classified a priori as an environmental disturbance, was not an outlier in microbiome composition, but did enhance a bloom in seasonally abundant phytoplankton. Thus, as extreme environmental changes intensify, assumptions of what constitutes a disturbance should be re-examined in the context of ecological history and microbiome responses.

Microbially enhanced methane uptake under warming enlarges ecosystem carbon sink in a Tibetan alpine grassland.

The alpine grasslands of the Tibetan Plateau store 23.2 Pg soil organic carbon, which becomes susceptible to microbial degradation with climate warming. However, accurate prediction of how the soil carbon stock changes under future climate warming is hampered by our limited understanding of belowground complex microbial communities. Here, we show that 4 years of warming strongly stimulated methane (CH4 ) uptake by 93.8% and aerobic respiration (CO2 ) by 11.3% in the soils of alpine grassland ecosystem. Due to no significant effects of warming on net ecosystem CO2 exchange (NEE), the warming-stimulated CH4 uptake enlarged the carbon sink capacity of whole ecosystem. Furthermore, precipitation alternation did not alter such warming effects, despite the significant effects of precipitation on NEE and soil CH4 fluxes were observed. Metagenomic sequencing revealed that warming led to significant shifts in the overall microbial community structure and the abundances of functional genes, which contrasted to no detectable changes after 2 years of warming. Carbohydrate utilization genes were significantly increased by warming, corresponding with significant increases in soil aerobic respiration. Increased methanotrophic genes and decreased methanogenic genes were observed under warming, which significantly (R2  = .59, p < .001) correlated with warming-enhanced CH4 uptakes. Furthermore, 212 metagenome-assembled genomes were recovered, including many populations involved in the degradation of various organic matter and a highly abundant methylotrophic population of the Methyloceanibacter genus. Collectively, our results provide compelling evidence that specific microbial functional traits for CH4 and CO2 cycling processes respond to climate warming with differential effects on soil greenhouse gas emissions. Alpine grasslands may play huge roles in mitigating climate warming through such microbially enhanced CH4 uptake.

Judicial Opinions 103-111.

In Opinion 103, the request to place the name Spirillum volutans Ehrenberg 1832 (Approved Lists 1980) on the list of rejected names is denied because a neotype may be designated. Similarly, because a neotype may be designated, in Opinion 104 the request to place the name Beijerinckia fluminensis Döbereiner and Ruschel 1958 (Approved Lists 1980) on the list of rejected names is denied. In Opinion 105, it is emphasized that the name Rhodoligotrophos Fukuda et al. 2012 does not contravene the Code. The request to orthographically correct Rhodoligotrophos Fukuda et al. 2012 to Rhodoligotrophus corrig. Fukuda et al. 2012 is denied. Opinion 106 addresses two Requests for an Opinion and results in the placement of the epithet hoagii in Corynebacterium hoagii (Morse 1912) Eberson 1918 (Approved Lists 1980) and Rhodococcus hoagii (Morse 1912) Kämpfer et al. 2014 on the list of rejected specific and subspecific epithets. Since this removes all known available earlier synonyms of Rhodococcus equi (Magnusson 1923) Goodfellow and Alderson 1977 (Approved Lists 1980), the request to conserve the epithet equi in this name is denied. In Opinion 107, Thermomicrobium fosteri Phillips and Perry 1976 (Approved Lists 1980) is placed on the list of rejected names as a nomen dubium et confusum. Opinion 108 denies the request to place Hyphomonas rosenbergii Weiner et al. 2000 on the list of rejected names because the information provided to the Judicial Commission is not sufficient to draw a conclusion on this matter. In Opinion 109, which addresses three Requests for an Opinion, the Judicial Commission denies the requests to place the names Bacillus aerius Shivaji et al. 2006, Bacillus aerophilus Shivaji et al. 2006 and Bacillus stratosphericus Shivaji et al. 2006 on the list of rejected names. Instead, it is concluded that these three names had not met the requirements for valid publication. Likewise, the Judicial Commission concludes in Opinion 110 that the name Actinobaculum massiliense corrig. Greub and Raoult 2006 had not met the requirements for valid publication. The Judicial Commission reaffirms in Opinion 111 that Methanocorpusculum parvum Zellner et al. 1988 is the nomenclatural type of Methanocorpusculum Zellner et al. 1988 and further emphasizes that the species was not in danger of losing this status. These Opinions were ratified by the voting members of the International Committee on Systematics of Prokaryotes.

Judicial Opinions 112-122.

Opinion 112 denies the request to place Seliberia Aristovskaya and Parinkina 1963 (Approved Lists 1980) on the list of rejected names because the information provided is insufficient. For the same reason, Opinion 113 denies the request to reject Shewanella irciniae Lee et al. 2006 and Opinion 114 denies the request to reject the name Enterobacter siamensis Khunthongpan et al. 2014. Opinion 115 rejects the epithet of Moorella thermoautotrophica (Wiegel et al. 1981) Collins et al. 1994, which is regarded as a nomen confusum. To assess the consequences of Rule 8, Opinion 116 revisits names of taxa above the rank of genus which should comprise the stem of the name of a nomenclatural type and a category-specific ending but fail to do so. Such names should be orthographically corrected if the sole error is the inadvertent usage of an incorrect stem or be regarded as illegitimate if otherwise. The necessary corrections are made for a number of names. In Opinion 117, the request to designate Methylothermus subterraneus Hirayama et al. 2011 as the type species of the genus Methylothermus is denied because an equivalent action compatible with the Code was already conducted. In Opinion 118, the possible orthographical correction of the name Flaviaesturariibacter is treated, as are the analogous cases of Fredinandcohnia and Hydrogeniiclostidium. The genus names are corrected to Flaviaestuariibacter, Ferdinandcohnia and Hydrogeniiclostridium, respectively. Opinion 119 concludes that assigning Actinomycetales Buchanan 1917 (Approved Lists 1980) as nomenclatural type of the class Actinobacteria Stackebrandt et al. 1997 would not render that name legitimate if Rule 8 remained retroactive. The request is granted but Actinomycetales is also assigned as type of Actinomycetes Krassilnikov 1949 (Approved Lists 1980). In Opinion 120, the possible orthographical correction of the name Amycolatopsis albidoflavus is treated. It is grammatically corrected to Amycolatopsis albidoflava. Six names which could according to Rule 61 be grammatically corrected by anyone are also corrected. Opinion 121 denies the request to revise Opinion 69 and notes that Opinion 69 does not have the undesirable consequences emphasized in the request. In Opinion 122, the request to reject various taxon names of Mollicutes proposed in 2018 is denied because it is based on misinterpretations of the Code, which are clarified. Alternative ways to solve the perceived problems are outlined. These Opinions were ratified by the voting members of the International Committee on Systematics of Prokaryotes.

Judicial Opinions 123-127.

Opinion 123 places the epithet of the name Aeromonas punctata on the list of rejected epithets and clarifies the citation of authors of selected names within the genus Aeromonas. Opinion 124 denies the request to place Borreliella on the list of rejected names because the request is based on a misinterpretation of the Code, which is clarified. There are alternative ways to solve the perceived problem. Opinion 125 denies the request to place Lactobacillus fornicalis on the list of rejected names because the provided information does not yield a reason for rejection. Opinion 126 denies the request to place Prolinoborus and Prolinoborus fasciculus on the list of rejected names because a relevant type strain deposit was not examined. Opinion 127 grants the request to assign the strain deposited as ATCC 4720 as the type strain of Agrobacterium tumefaciens, thereby correcting the Approved Lists. These Opinions were ratified by the voting members of the International Committee on Systematics of Prokaryotes.

Quinone Moieties Link the Microbial Respiration of Natural Organic Matter to the Chemical Reduction of Diverse Nitroaromatic Compounds.

Insensitive munitions compounds (IMCs) are emerging nitroaromatic contaminants developed by the military as safer-to-handle alternatives to conventional explosives. Biotransformation of nitroaromatics via microbial respiration has only been reported for a limited number of substrates. Important soil microorganisms can respire natural organic matter (NOM) by reducing its quinone moieties to hydroquinones. Thus, we investigated the NOM respiration combined with the abiotic reduction of nitroaromatics by the hydroquinones formed. First, we established nitroaromatic concentration ranges that were nontoxic to the quinone respiration. Then, an enrichment culture dominated by Geobacter anodireducens could indirectly reduce a broad array of nitroaromatics by first respiring NOM components or the NOM surrogate anthraquinone-2,6-disulfonate (AQDS). Without quinones, no nitroaromatic tested was reduced except for the IMC 3-nitro-1,2,4-triazol-5-one (NTO). Thus, the quinone respiration expanded the spectrum of nitroaromatics susceptible to transformation. The system functioned with very low quinone concentrations because NOM was recycled by the nitroaromatic reduction. A metatranscriptomic analysis demonstrated that the microorganisms obtained energy from quinone or NTO reduction since respiratory genes were upregulated when AQDS or NTO was the electron acceptor. The results indicated microbial NOM respiration sustained by the nitroaromatic-dependent cycling of quinones. This process can be applied as a nitroaromatic remediation strategy, provided that a quinone pool is available for microorganisms.

SAR11 bacteria linked to ocean anoxia and nitrogen loss.

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world's largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth's most abundant organismal group.

Responses of tundra soil microbial communities to half a decade of experimental warming at two critical depths.

Northern-latitude tundra soils harbor substantial carbon (C) stocks that are highly susceptible to microbial degradation with rising global temperatures. Understanding the magnitude and direction (e.g., C release or sequestration) of the microbial responses to warming is necessary to accurately model climate change. In this study, Alaskan tundra soils were subjected to experimental in situ warming by ∼1.1 °C above ambient temperature, and the microbial communities were evaluated using metagenomics after 4.5 years, at 2 depths: 15 to 25 cm (active layer at outset of the experiment) and 45 to 55 cm (transition zone at the permafrost/active layer boundary at the outset of the experiment). In contrast to small or insignificant shifts after 1.5 years of warming, 4.5 years of warming resulted in significant changes to the abundances of functional traits and the corresponding taxa relative to control plots (no warming), and microbial shifts differed qualitatively between the two soil depths. At 15 to 25 cm, increased abundances of carbohydrate utilization genes were observed that correlated with (increased) measured ecosystem carbon respiration. At the 45- to 55-cm layer, increased methanogenesis potential was observed, which corresponded with a 3-fold increase in abundance of a single archaeal clade of the Methanosarcinales order, increased annual thaw duration (45.3 vs. 79.3 days), and increased CH4 emissions. Collectively, these data demonstrate that the microbial responses to warming in tundra soil are rapid and markedly different between the 2 critical soil layers evaluated, and identify potential biomarkers for the corresponding microbial processes that could be important in modeling.

MicrobeAnnotator: a user-friendly, comprehensive functional annotation pipeline for microbial genomes.

BACKGROUND: High-throughput sequencing has increased the number of available microbial genomes recovered from isolates, single cells, and metagenomes. Accordingly, fast and comprehensive functional gene annotation pipelines are needed to analyze and compare these genomes. Although several approaches exist for genome annotation, these are typically not designed for easy incorporation into analysis pipelines, do not combine results from different annotation databases or offer easy-to-use summaries of metabolic reconstructions, and typically require large amounts of computing power for high-throughput analysis not available to the average user. RESULTS: Here, we introduce MicrobeAnnotator, a fully automated, easy-to-use pipeline for the comprehensive functional annotation of microbial genomes that combines results from several reference protein databases and returns the matching annotations together with key metadata such as the interlinked identifiers of matching reference proteins from multiple databases [KEGG Orthology (KO), Enzyme Commission (E.C.), Gene Ontology (GO), Pfam, and InterPro]. Further, the functional annotations are summarized into Kyoto Encyclopedia of Genes and Genomes (KEGG) modules as part of a graphical output (heatmap) that allows the user to quickly detect differences among (multiple) query genomes and cluster the genomes based on their metabolic similarity. MicrobeAnnotator is implemented in Python 3 and is freely available under an open-source Artistic License 2.0 from https://github.com/cruizperez/MicrobeAnnotator . CONCLUSIONS: We demonstrated the capabilities of MicrobeAnnotator by annotating 100 Escherichia coli and 78 environmental Candidate Phyla Radiation (CPR) bacterial genomes and comparing the results to those of other popular tools. We showed that the use of multiple annotation databases allows MicrobeAnnotator to recover more annotations per genome compared to faster tools that use reduced databases and is computationally efficient for use in personal computers. The output of MicrobeAnnotator can be easily incorporated into other analysis pipelines while the results of other annotation tools can be seemingly incorporated into MicrobeAnnotator to generate summary plots.

The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

Hidden Diversity within Common Protozoan Parasites as Revealed by a Novel Genomotyping Scheme.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is the causative agent of giardiasis, one of the most common diarrheal infections in humans. Evolutionary relationships among G. duodenalis genotypes (or subtypes) of assemblage B, one of two genetic assemblages causing the majority of human infections, remain unclear due to poor phylogenetic resolution of current typing methods. In this study, we devised a methodology to identify new markers for a streamlined multilocus sequence typing (MLST) scheme based on comparisons of all core genes against the phylogeny of whole-genome sequences (WGS). Our analysis identified three markers with resolution comparable to that of WGS data. Using newly designed PCR primers for our novel MLST loci, we typed an additional 68 strains of assemblage B. Analyses of these strains and previously determined genome sequences showed that genomes of this assemblage can be assigned to 16 clonal complexes, each with unique gene content that is apparently tuned to differential virulence and ecology. Obtaining new genomes of Giardia spp. and other eukaryotic microbial pathogens remains challenging due to difficulties in culturing the parasites in the laboratory. Hence, the methods described here are expected to be widely applicable to other pathogens of interest and advance our understanding of their ecology and evolution.IMPORTANCEGiardia duodenalis assemblage B is a major waterborne pathogen and the most commonly identified genotype causing human giardiasis worldwide. The lack of morphological characters for classification requires the use of molecular techniques for strain differentiation; however, the absence of scalable and affordable next-generation sequencing (NGS)-based typing methods has prevented meaningful advancements in high-resolution molecular typing for further understanding of the evolution and epidemiology of assemblage B. Prior studies have reported high sequence diversity but low phylogenetic resolution at standard loci in assemblage B, highlighting the necessity of identifying new markers for accurate and robust molecular typing. Data from comparative analyses of available genomes in this study identified three loci that together form a novel high-resolution typing scheme with high concordance to whole-genome-based phylogenomics and which should aid in future public health endeavors related to this parasite. In addition, data from newly characterized strains suggest evidence of biogeographic and ecologic endemism.

The Reliability of Metagenome-Assembled Genomes (MAGs) in Representing Natural Populations: Insights from Comparing MAGs against Isolate Genomes Derived from the Same Fecal Sample.

The recovery of metagenome-assembled genomes (MAGs) from metagenomic data has recently become a common task for microbial studies. The strengths and limitations of the underlying bioinformatics algorithms are well appreciated by now based on performance tests with mock data sets of known composition. However, these mock data sets do not capture the complexity and diversity often observed within natural populations, since their construction typically relies on only a single genome of a given organism. Further, it remains unclear if MAGs can recover population-variable genes (those shared by >10% but <90% of the members of the population) as efficiently as core genes (those shared by >90% of the members). To address these issues, we compared the gene variabilities of pathogenic Escherichia coli isolates from eight diarrheal samples, for which the isolate was the causative agent, against their corresponding MAGs recovered from the companion metagenomic data set. Our analysis revealed that MAGs with completeness estimates near 95% captured only 77% of the population core genes and 50% of the variable genes, on average. Further, about 5% of the genes of these MAGs were conservatively identified as missing in the isolate and were of different (non-Enterobacteriaceae) taxonomic origin, suggesting errors at the genome-binning step, even though contamination estimates based on commonly used pipelines were only 1.5%. Therefore, the quality of MAGs may often be worse than estimated, and we offer examples of how to recognize and improve such MAGs to sufficient quality by (for instance) employing only contigs longer than 1,000 bp for binning.IMPORTANCE Metagenome assembly and the recovery of metagenome-assembled genomes (MAGs) have recently become common tasks for microbiome studies across environmental and clinical settings. However, the extent to which MAGs can capture the genes of the population they represent remains speculative. Current approaches to evaluating MAG quality are limited to the recovery and copy number of universal housekeeping genes, which represent a small fraction of the total genome, leaving the majority of the genome essentially inaccessible. If MAG quality in reality is lower than these approaches would estimate, this could have dramatic consequences for all downstream analyses and interpretations. In this study, we evaluated this issue using an approach that employed comparisons of the gene contents of MAGs to the gene contents of isolate genomes derived from the same sample. Further, our samples originated from a diarrhea case-control study, and thus, our results are relevant for recovering the virulence factors of pathogens from metagenomic data sets.

High Arsenic Levels Increase Activity Rather than Diversity or Abundance of Arsenic Metabolism Genes in Paddy Soils.

Arsenic (As) metabolism genes are generally present in soils, but their diversity, relative abundance, and transcriptional activity in response to different As concentrations remain unclear, limiting our understanding of the microbial activities that control the fate of an important environmental pollutant. To address this issue, we applied metagenomics and metatranscriptomics to paddy soils showing a gradient of As concentrations to investigate As resistance genes (ars) including arsR, acr3, arsB, arsC, arsM, arsI, arsP, and arsH as well as energy-generating As respiratory oxidation (aioA) and reduction (arrA) genes. Somewhat unexpectedly, the relative DNA abundances and diversities of ars, aioA, and arrA genes were not significantly different between low and high (∼10 versus ∼100 mg kg-1) As soils. Compared to available metagenomes from other soils, geographic distance rather than As levels drove the different compositions of microbial communities. Arsenic significantly increased ars gene abundance only when its concentration was higher than 410 mg kg-1. In contrast, metatranscriptomics revealed that relative to low-As soils, high-As soils showed a significant increase in transcription of ars and aioA genes, which are induced by arsenite, the dominant As species in paddy soils, but not arrA genes, which are induced by arsenate. These patterns appeared to be community wide as opposed to taxon specific. Collectively, our findings advance understanding of how microbes respond to high As levels and the diversity of As metabolism genes in paddy soils and indicated that future studies of As metabolism in soil or other environments should include the function (transcriptome) level. IMPORTANCE Arsenic (As) is a toxic metalloid pervasively present in the environment. Microorganisms have evolved the capacity to metabolize As, and As metabolism genes are ubiquitously present in the environment even in the absence of high concentrations of As. However, these previous studies were carried out at the DNA level; thus, the activity of the As metabolism genes detected remains essentially speculative. Here, we show that the high As levels in paddy soils increased the transcriptional activity rather than the relative DNA abundance and diversity of As metabolism genes. These findings advance our understanding of how microbes respond to and cope with high As levels and have implications for better monitoring and managing an important toxic metalloid in agricultural soils and possibly other ecosystems.