MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects.IMPORTANCEWe created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Annotated whole-genome sequences of fermentative and spoilage associated Bacilli and proteobacteria autochthonous to commercial cucumber fermentation.

We report 36 whole-genome sequences, along with annotations, of fermentative (n = 12) and spoilage associated (n = 6) lactic acid bacteria, Lysinibacillus (n = 3), Streptococcus (n = 1), and Proteobacteria (n = 14) isolated from commercial cucumber fermentations. Fifty-three percent of the genome sequence assemblies consist of 1-4 contigs, and the remainder have fewer than 16.

IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy.

Background: Oral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants. Objective: Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials. Methods: Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides. Results: Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered. Conclusion: At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut.

RNA virus discoveries in the electric ant, Wasmannia auropunctata.

Despite being one of the most destructive invasive species of ants, only two natural enemies are known currently for Wasmannia auropunctata, commonly known as the electric ant or little fire ant. Because viruses can be effective biological control agents against many insect pests, including ants, a metagenomics/next-generation sequencing approach was used to facilitate discovery of virus sequences from the transcriptomes of W. auropunctata. Five new and complete positive sense, single-stranded RNA virus genomes, and one new negative sense, single-stranded RNA virus genome were identified, sequenced, and characterized from W. auropunctata collected in Argentina by this approach, including a dicistrovirus (Electric ant dicistrovirus), two polycipiviruses (Electric ant polycipivirus 1; Electric ant polycipivirus 2), a solinvivirus (Electric ant solinvivirus), a divergent genome with similarity to an unclassified group in the Picornavirales (Electric ant virus 1), and a rhabdovirus (Electric ant rhabdovirus). An additional virus genome was detected that is likely Solenopsis invicta virus 10 (MH727527). The virus genome sequences were absent from the transcriptomes of W. auropunctata collected in the USA (Hawaii and Florida). Additional limited field surveys corroborated the absence of these viruses in regions where the electric ant is invasive (the USA and Australia). The replicative genome strand of four of the viruses (Electric ant polycipivirus 2, Electric ant solinvivirus, Electric ant virus 1, and Solenopsis invicta virus 10 (in the electric ant) was detected in Argentinean-collected W. auropunctata indicating that the ant is a host for these viruses. These are the first virus discoveries to be made from W. auropunctata.

Graph-based pangenomics maximizes genotyping density and reveals structural impacts on fungal resistance in melon.

The genomic sequences segregating in experimental populations are often highly divergent from the community reference and from one another. Such divergence is problematic under various short-read-based genotyping strategies. In addition, large structural differences are often invisible despite being strong candidates for causal variation. These issues are exacerbated in specialty crop breeding programs with fewer, lower-quality sequence resources. Here, we examine the benefits of complete genomic information, based on long-read assemblies, in a biparental mapping experiment segregating at numerous disease resistance loci in the non-model crop, melon (Cucumis melo). We find that a graph-based approach, which uses both parental genomes, results in 19% more variants callable across the population and raw allele calls with a 2 to 3-fold error-rate reduction, even relative to single reference approaches using a parent genome. We show that structural variation has played a substantial role in shaping two Fusarium wilt resistance loci with known causal genes. We also report on the genetics of powdery mildew resistance, where copy number variation and local recombination suppression are directly interpretable via parental genome alignments. Benefits observed, even in this low-resolution biparental experiment, will inevitably be amplified in more complex populations.

GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes.

BACKGROUND: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens. FINDINGS: GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1-2 minutes. CONCLUSIONS: GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

Gross Morphology of Diseased Tissues in Nezara viridula (Hemiptera: Pentatomidae) and Molecular Characterization of an Associated Microsporidian.

Nezara viridula (L.) (Hemiptera: Pentatomidae), commonly known in the U.S. as the southern green stink bug (SGSB), is a cosmopolitan, highly polyphagous feeder that causes severe damage to a wide range of agronomically important crops such as fruit, vegetable, grain, tobacco, and cotton, throughout much of the United States, and is a global pest of considerable ecological, agricultural, and economical interest. During dissection of female Nz. viridula, conspicuous black and brown spots or lesions were observed on various internal organs. To determine the cause of these spots or lesions, tissues of fat body, spermatheca, ovaries, and ovulated eggs were collected from healthy and infected individuals. The gross morphology of the spots was characterized, and the microorganisms associated with the infection were identified by amplicon sequencing of the V4 region of the small subunit rRNA gene. The presence of a microsporidian pathogen Nosema maddoxi, Becnel, Solter, Hajek, Huang, Sanscrainte, & Estep (Microsporidia: Nosematidae) which has been observed on other species of stink bug, was evidenced for the first time. The characterization of the gross morphology of this associated microsporidian may enable more rapid determination of microsporidia infection in stink bug colonies and field populations.

Intrinsic variation in the vertically transmitted core virome of the mosquito Aedes aegypti.

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level.

AT Homopolymer Strings in Salmonella enterica Subspecies I Contribute to Speciation and Serovar Diversity.

Adenine and thymine homopolymer strings of at least 8 nucleotides (AT 8+mers) were characterized in Salmonella enterica subspecies I. The motif differed between other taxonomic classes but not between Salmonella enterica serovars. The motif in plasmids was possibly associated with serovar. Approximately 12.3% of the S. enterica motif loci had mutations. Mutability of AT 8+mers suggests that genomes undergo frequent repair to maintain optimal gene content, and that the motif facilitates self-recognition; in addition, serovar diversity is associated with plasmid content. A theory that genome regeneration accounts for both persistence of predominant Salmonella serovars and serovar diversity provides a new framework for investigating root causes of foodborne illness.

Advancing Equity and Inclusion in Microbiome Research and Training.

This article proposes ways to improve inclusion and training in microbiome science and advocates for resource expansion to improve scientific capacity across institutions and countries. Specifically, we urge mentors, collaborators, and decision-makers to commit to inclusive and accessible research and training that improves the quality of microbiome science and begins to rectify long-standing inequities imposed by wealth disparities and racism that stall scientific progress.

Under-the-Radar Dengue Virus Infections in Natural Populations of Aedes aegypti Mosquitoes.

The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort concerning the dengue mosquito vector, Aedes aegypti, and its microbiome, which has been shown to influence virus transmission success. We examined the "metavirome" of four populations of Aedes aegypti mosquitoes collected in 2016 to 2017 in Manatee County, FL. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a 2-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, and explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of the DENV4 infections in A. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak.IMPORTANCE Since 1999, dengue outbreaks in the continental United States involving local transmission have occurred only episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from countries where the disease is endemic. To date, the U.S. public health response to dengue outbreaks has been largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of DENV4 transmission to and maintenance within a local mosquito vector population in the continental United States in the absence of a human case during two consecutive years. Our data suggest that molecular surveillance of mosquito populations in high-risk, high-tourism areas of the United States may enable proactive, targeted vector control before potential arbovirus outbreaks.

Genomic Signatures for Sedimentary Microbial Utilization of Phytoplankton Detritus in a Fast-Flowing Estuary.

In fast-flowing, river-dominated estuaries, "hotspots" of microbial biogeochemical cycling can be found within areas of extended water retention. Lateral bays located off of the North and South channels of the Columbia River estuary are proposed to be such hotspots. Previous metagenomic studies on water samples indicated that these regions function both as sources and sinks of biogenic particles, with potential to impact organic matter fluxes in the estuary. To extend this work, we analyzed 11 sediment metagenomes from three disparate bays: the freshwater Cathlamet Bay, and the brackish Youngs Bay and more saline Baker Bay located nearer the mouth to the south and north of the main channel, respectively. Samples were collected from upper layers of sediments in August of 2011 and 2013 for DNA extraction and metagenome sequencing. All metagenomes were dominated by bacterial sequences, although diatom sequences as high as 26% of the total annotated sequences were observed in the higher salinity samples. Unsupervised 2D hierarchical clustering analysis resulted in the eleven metagenome samples clustered into four groups by microbial taxonomic composition, with Bacteroides, diatom, and phage levels driving most of the grouping. Results of functional gene clustering further indicated that diatom bloom degradation stage (early vs. late) was an important factor. While the Flavobacteriia and Cytophagia classes were well represented in metagenomes containing abundant diatoms, taxa from the Bacteroidia class, along with certain members of the Sphingobacteriia class, were particularly abundant in metagenomes representing later stages of diatom decomposition. In contrast, the sediment metagenomes with low relative abundance of diatom and Bacteroidetes sequences appeared to have a metabolic potential biased toward microbial growth under nutrient limitation. While differences in water salinity clearly also influenced the microbial community composition and metabolic potential, our results highlight a central role for allochthonous labile organic matter (i.e., diatom detritus), in shaping bacterial taxonomic and functional properties in the Columbia River estuary lateral bay sediments. These results suggest that in fast-flowing, river-dominated estuaries, sediment microbial communities in areas of extended water retention, such as the lateral bays, may contribute disproportionately to estuarine organic matter degradation and recycling.

Nine new RNA viruses associated with the fire ant Solenopsis invicta from its native range.

The red imported fire ant (Solenopsis invicta) escaped its natural enemies when it was introduced into North America in the 1930s from South America. US efforts have focused on discovery of natural enemies, like viruses, to provide sustainable control of the ant. Nine new virus genomes were sequenced from the invasive fire ant Solenopsis invicta using metagenomic RNA sequencing. The virus genomes were verified by Sanger sequencing and random amplification of cDNA ends reactions. In addition to the nine new virus genomes, the previously described Solenopsis viruses were also detected, including Solenopsis invicta virus 1 (SINV-1), SINV-2, SINV-3, SINV-4, SINV-5, and Solenopsis invicta densovirus. The virus sequences came from S. invicta workers, larvae, pupae, and dead workers taken from midden piles collected from across the ant's native range in Formosa, Argentina. One of the new virus genomes (Solenopsis invicta virus 6) was also detected in populations of North American S. invicta. Phylogenetic analysis of the RNA dependent RNA polymerase, the entire nonstructural polyprotein, and genome characteristics were used to tentatively taxonomically place these new virus genome sequences; these include four new species of Dicistroviridae, one Polycipiviridae, one Iflaviridae, one Totiviridae, and two genome sequences that were too taxonomically divergent to be placed with certainty. The S. invicta virome is the best characterized from any ant species and includes 13 positive-sense, single-stranded RNA viruses (Solenopsis invicta virus 1 to Solenopsis invicta virus 13), one double-stranded RNA virus (Solenopsis midden virus), and one double-stranded DNA virus (Solenopsis invicta densovirus). These new additions to the S. invicta virome offer potentially new classical biological control agents for S. invicta.

ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis.

The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU's) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.

Patterns and drivers of fungal community depth stratification in Sphagnum peat.

Peatlands store an immense pool of soil carbon vulnerable to microbial oxidation due to drought and intentional draining. We used amplicon sequencing and quantitative PCR to (i) examine how fungi are influenced by depth in the peat profile, water table and plant functional group at the onset of a multiyear mesocosm experiment, and (ii) test if fungi are correlated with abiotic variables of peat and pore water. We hypothesized that each factor influenced fungi, but that depth would have the strongest effect early in the experiment. We found that (i) communities were strongly depth stratified; fungi were four times more abundant in the upper (10-20 cm) than the lower (30-40 cm) depth, and dominance shifted from ericoid mycorrhizal fungi to saprotrophs and endophytes with increasing depth; (ii) the influence of plant functional group was depth dependent, with Ericaceae structuring the community in the upper peat only; (iii) water table had minor influences; and (iv) communities strongly covaried with abiotic variables, including indices of peat and pore water carbon quality. Our results highlight the importance of vertical stratification to peatland fungi, and the depth dependency of plant functional group effects, which must be considered when elucidating the role of fungi in peatland carbon dynamics.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses.

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

Experimental Identification of Small Non-Coding RNAs in the Model Marine Bacterium Ruegeria pomeroyi DSS-3.

In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon (C) and nitrogen (N) limitation. RNAseq analysis identified 99 putative sRNAs. Sixty-nine were cis-encoded and located antisense to a presumed target gene. Thirty were trans-encoded and initial target prediction was performed computationally. The most prevalent functional roles of genes anti-sense to the cis-sRNAs were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Most sRNAs were transcribed equally under both C and N limitation, and may be involved in a general stress response. However, 14 were regulated differentially between the C and N treatments and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi cis-sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome.