MerCat2: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from omics data.

Motivation: MerCat2 ("Mer-Catenate2") is a versatile, parallel, scalable and modular property software package for robustly analyzing features in omics data. Using massively parallel sequencing raw reads, assembled contigs, and protein sequences from any platform as input, MerCat2 performs k-mer counting of any length k, resulting in feature abundance counts tables, quality control reports, protein feature metrics, and graphical representation (i.e. principal component analysis (PCA)). Results: MerCat2 allows for direct analysis of data properties in a database-independent manner that initializes all data, which other profilers and assembly-based methods cannot perform. MerCat2 represents an integrated tool to illuminate omics data within a sample for rapid cross-examination and comparisons. Availability and implementation: MerCat2 is written in Python and distributed under a BSD-3 license. The source code of MerCat2 is freely available at https://github.com/raw-lab/mercat2. MerCat2 is compatible with Python 3 on Mac OS X and Linux. MerCat2 can also be easily installed using bioconda: mamba create -n mercat2 -c conda-forge -c bioconda mercat2.

DeGenPrime provides robust primer design and optimization unlocking the biosphere.

Motivation: Polymerase chain reaction (PCR) is the world's most important molecular diagnostic with applications ranging from medicine to ecology. PCR can fail because of poor primer design. The nearest-neighbor thermodynamic properties, picking conserved regions, and filtration via penalty of oligonucleotides form the basis for good primer design. Results: DeGenPrime is a console-based high-quality PCR primer design tool that can utilize MSA formats and degenerate bases expanding the target range for a single primer set. Our software utilizes thermodynamic properties, filtration metrics, penalty scoring, and conserved region finding of any proposed primer. It has degeneracy, repeated k-mers, relative GC content, and temperature range filters. Minimal penalty scoring is included according to secondary structure self-dimerization metrics, GC clamping, tri- and tetra-loop hairpins, and internal repetition. We compared PrimerDesign-M, DegePrime, ConsensusPrimer, and DeGenPrime on acceptable primer yield. PrimerDesign-M, DegePrime, and ConsensusPrimer provided 0%, 11%, and 17% yield, respectively, for the alternative iron nitrogenase (anfD) gene target. DeGenPrime successfully identified quality primers within the conserved regions of the T4-like phage major capsid protein (g23), conserved regions of molybdenum-based nitrogenase (nif), and its alternatives vanadium (vnf) and iron (anf) nitrogenase. DeGenPrime provides a universal and scalable primer design tool for the entire tree of life. Availability and implementation: DeGenPrime is written in C++ and distributed under a BSD-3-Clause license. The source code for DeGenPrime is freely available on www.github.com/raw-lab/degenprime.

Viral enumeration using cost-effective wet-mount epifluorescence microscopy for aquatic ecosystems and modern microbialites.

IMPORTANCE: Low-cost and robust viral enumeration is a critical first step toward understanding the global virome. Our method is a deep drive integration providing a window into viral dark matter within aquatic ecosystems. We enumerated the viruses within Green Lake and Great Salt Lake microbialites, EPS, and water column. The entire weight of all the viruses in Green Lake and Great Salt Lake are ~598 g and ~2.2 kg, respectively.

Meeting report: The first soil viral workshop 2022.

Soil viral ecology is a growing research field; however, the state of knowledge still lags behind that of aquatic systems. Therefore, to facilitate progress, the first Soil Viral Workshop was held to encourage international scientific discussion and collaboration, suggest guidelines for future research, and establish soil viral research as a concrete research area. The workshop took place at Søminestationen, Denmark, between 15 and 17th of June 2022. The meeting was primarily held in person, but the sessions were also streamed online. The workshop was attended by 23 researchers from ten different countries and from a wide range of subfields and career stages. Eleven talks were presented, followed by discussions revolving around three major topics: viral genomics, virus-host interactions, and viruses in the soil food web. The main take-home messages and suggestions from the discussions are summarized in this report.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

The Future of Virology is Synthetic.

The virosphere (i.e., global virome) represents a vast library of unknown genes on the planet. Synthetic biology through engineering principles could be the key to unlocking this massive global gene repository. Synthetic viruses may also be used as tools to understand "the rules of life" in diverse microbial ecosystems. Such insights may be crucial for understanding the assembly, diversity, structure, and scale of virus-mediated function. Viruses directly affect resilience, stability, and microbial community selection via death resistance cycles. Interpreting and clarifying these effects is essential for predicting the system's ecology, evolution, and ecosystem stability in an increasingly unstable global climate. A "silent looming pandemic" due to multidrug-resistant microbes will directly impact the global economy, and synthetic virology could provide a future strategy of treatment using targeted viral therapy. This commentary will discuss current techniques for manipulating viruses synthetically, contributing to improved human health and sustainable agriculture.

Gestational diabetes mellitus is associated with the neonatal gut microbiota and metabolome.

BACKGROUND: Gestational diabetes mellitus (GDM) is a metabolic disease that occurs in pregnant women and increases the risk for the development of diabetes. The relationship between GDM and meconium microbiota and metabolome remains incompletely understood. METHODS: Four hundred eighteen mothers (147 women with GDM and 271 normal pregnant women) and their neonates from the GDM Mother and Child Study were included in this study. Meconium microbiota were profiled by 16S rRNA gene sequencing. Meconium and maternal serum metabolome were examined by UPLC-QE. RESULTS: Microbial communities in meconium were significantly altered in neonates from the GDM mothers. A reduction in alpha diversity was observed in neonates of GDM mothers. At the phylum level, the abundance of Firmicutes and Proteobacteria changed significantly in neonates of GDM mothers. Metabolomic analysis of meconium showed that metabolic pathways including taurine and hypotaurine metabolism, pyrimidine metabolism, beta-alanine metabolism, and bile acid biosynthesis were altered in GDM subjects. Several changed metabolites varying by the similar trend across the maternal serum and neonatal meconium were observed. CONCLUSION: Altogether, these findings suggest that GDM could alter the serum metabolome and is associated with the neonatal meconium microbiota and metabolome, highlighting the importance of maternal factors on early-life metabolism.

Homogeneous Cytochrome 579 Is an Octamer That Reacts Too Slowly With Soluble Iron to Be the Initial Iron Oxidase in the Respiratory Chain of Leptospirillum ferriphilum.

The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c-type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane.

Multiple 'omics'-analysis reveals the role of prostaglandin E2 in Hirschsprung's disease.

The etiology and pathogenesis of Hirschsprung's disease (HSCR) remain largely unknown. We examined colon tissues from three independent populations with a combined analysis of metabolomics, transcriptomics and proteomics to understand HSCR pathogenesis, according to which mouse model was used to examine prostaglandin E2 (PGE2) induced clinical presentation of HSCR. SH-SY5Y and SK-N-BE(2) cell lines were studied for PGE2 inhibited cell migration through EP2. Our integrated multiple 'omics'-analysis suggests that the levels of PGE2, the expression of the gene encoding PGE2 receptor (EP2), and PGE2 synthesis enzyme genes (PTGS1 and PTGES) increased in HSCR colon tissues, together with a decreased synthesis of PGE2-related byproducts. In vivo, the pregnant mice treated with PGE2 gave birth to offspring with the decrease of ganglion cells in their colon and gut function. In in vitro study, when EP2 was blocked, the PGE2-inhibited cell migration was recovered. Our study identified a novel pathway highlighting the link between expression of PTGS1 and PTGES, levels of PGE2, expression of PTGER2, and neural crest cell migration in HSCR, providing a novel strategy for future diagnosis and prevention of HSCR.

Fundamentals of genomic epidemiology, lessons learned from the coronavirus disease 2019 (COVID-19) pandemic, and new directions.

The coronavirus disease 2019 (COVID-19) pandemic was one of the significant causes of death worldwide in 2020. The disease is caused by severe acute coronavirus syndrome (SARS) coronavirus 2 (SARS-CoV-2), an RNA virus of the subfamily Orthocoronavirinae related to 2 other clinically relevant coronaviruses, SARS-CoV and MERS-CoV. Like other coronaviruses and several other viruses, SARS-CoV-2 originated in bats. However, unlike other coronaviruses, SARS-CoV-2 resulted in a devastating pandemic. The SARS-CoV-2 pandemic rages on due to viral evolution that leads to more transmissible and immune evasive variants. Technology such as genomic sequencing has driven the shift from syndromic to molecular epidemiology and promises better understanding of variants. The COVID-19 pandemic has exposed critical impediments that must be addressed to develop the science of pandemics. Much of the progress is being applied in the developed world. However, barriers to the use of molecular epidemiology in low- and middle-income countries (LMICs) remain, including lack of logistics for equipment and reagents and lack of training in analysis. We review the molecular epidemiology literature to understand its origins from the SARS epidemic (2002-2003) through influenza events and the current COVID-19 pandemic. We advocate for improved genomic surveillance of SARS-CoV and understanding the pathogen diversity in potential zoonotic hosts. This work will require training in phylogenetic and high-performance computing to improve analyses of the origin and spread of pathogens. The overarching goals are to understand and abate zoonosis risk through interdisciplinary collaboration and lowering logistical barriers.

Between a Rock and a Soft Place: The Role of Viruses in Lithification of Modern Microbial Mats.

Stromatolites are geobiological systems formed by complex microbial communities, and fossilized stromatolites provide a record of some of the oldest life on Earth. Microbial mats are precursors of extant stromatolites; however, the mechanisms of transition from mat to stromatolite are controversial and are still not well understood. To fully recognize the profound impact that these ecosystems have had on the evolution of the biosphere requires an understanding of modern lithification mechanisms and how they relate to the geological record. We propose here viral mechanisms in carbonate precipitation, leading to stromatolite formation, whereby viruses directly or indirectly impact microbial metabolisms that govern the transition from microbial mat to stromatolite. Finding a tangible link between host-virus interactions and changes in biogeochemical processes will provide tools to interpret mineral biosignatures through geologic time, including those on Earth and beyond.

Functional Gene Expression in Shark Bay Hypersaline Microbial Mats: Adaptive Responses.

Microbial mat communities possess extensive taxonomic and functional diversity, which drive high metabolic rates and rapid cycling of major elements. Modern microbial mats occurring in hypersaline environments are considered as analogs to extinct geobiological formations dating back to ∼ 3.5 Gyr ago. Despite efforts to understand the diversity and metabolic potential of hypersaline microbial mats in Shark Bay, Western Australia, there has yet to be molecular analyses at the transcriptional level in these microbial communities. In this study, we generated metatranscriptomes for the first time from actively growing mats comparing the type of mat, as well as the influence of diel and seasonal cycles. We observed that the overall gene transcription is strongly influenced by microbial community structure and seasonality. The most transcribed genes were associated with tackling the low nutrient conditions by the uptake of fatty acids, phosphorus, iron, and nickel from the environment as well as with protective mechanisms against elevated salinity conditions and to prevent build-up of ammonium produced by nitrate reducing microorganisms. A range of pathways involved in carbon, nitrogen, and sulfur cycles were identified in mat metatranscriptomes, with anoxygenic photosynthesis and chemoautotrophy using the Arnon-Buchanan cycle inferred as major pathways involved in the carbon cycle. Furthermore, enrichment of active anaerobic pathways (e.g., sulfate reduction, methanogenesis, Wood-Ljungdahl) in smooth mats corroborates previous metagenomic studies and further advocates the potential of these communities as modern analogs of ancient microbialites.

Microbial dark matter filling the niche in hypersaline microbial mats.

BACKGROUND: Shark Bay, Australia, harbours one of the most extensive and diverse systems of living microbial mats that are proposed to be analogs of some of the earliest ecosystems on Earth. These ecosystems have been shown to possess a substantial abundance of uncultivable microorganisms. These enigmatic microbes, jointly coined as 'microbial dark matter' (MDM), are hypothesised to play key roles in modern microbial mats. RESULTS: We reconstructed 115 metagenome-assembled genomes (MAGs) affiliated to MDM, spanning 42 phyla. This study reports for the first time novel microorganisms (Zixibacterial order GN15) putatively taking part in dissimilatory sulfate reduction in surface hypersaline settings, as well as novel eukaryote signature proteins in the Asgard archaea. Despite possessing reduced-size genomes, the MDM MAGs are capable of fermenting and degrading organic carbon, suggesting a role in recycling organic carbon. Several forms of RuBisCo were identified, allowing putative CO2 incorporation into nucleotide salvaging pathways, which may act as an alternative carbon and phosphorus source. High capacity of hydrogen production was found among Shark Bay MDM. Putative schizorhodopsins were also identified in Parcubacteria, Asgard archaea, DPANN archaea, and Bathyarchaeota, allowing these members to potentially capture light energy. Diversity-generating retroelements were prominent in DPANN archaea that likely facilitate the adaptation to a dynamic, host-dependent lifestyle. CONCLUSIONS: This is the first study to reconstruct and describe in detail metagenome-assembled genomes (MAGs) affiliated with microbial dark matter in hypersaline microbial mats. Our data suggests that these microbial groups are major players in these systems. In light of our findings, we propose H2, ribose and CO/CO2 as the main energy currencies of the MDM community in these mat systems. Video Abstract.

Integrated network modeling approach defines key metabolic responses of soil microbiomes to perturbations.

The soil environment is constantly changing due to shifts in soil moisture, nutrient availability and other conditions. To contend with these changes, soil microorganisms have evolved a variety of ways to adapt to environmental perturbations, including regulation of gene expression. However, it is challenging to untangle the complex phenotypic response of the soil to environmental change, partly due to the absence of predictive modeling frameworks that can mechanistically link molecular-level changes in soil microorganisms to a community's functional phenotypes (or metaphenome). Towards filling this gap, we performed a combined analysis of metabolic and gene co-expression networks to explore how the soil microbiome responded to changes in soil moisture and nutrient conditions and to determine which genes were expressed under a given condition. Our integrated modeling approach revealed previously unknown, but critically important aspects of the soil microbiomes' response to environmental perturbations. Incorporation of metabolomic and transcriptomic data into metabolic reaction networks identified condition-specific signature genes that are uniquely associated with dry, wet, and glycine-amended conditions. A subsequent gene co-expression network analysis revealed that drought-associated genes occupied more central positions in a network model of the soil community, compared to the genes associated with wet, and glycine-amended conditions. These results indicate the occurrence of system-wide metabolic coordination when soil microbiomes cope with moisture or nutrient perturbations. Importantly, the approach that we demonstrate here to analyze large-scale multi-omics data from a natural soil environment is applicable to other microbiome systems for which multi-omics data are available.

A Genome Resource for the Apple Powdery Mildew Pathogen Podosphaera leucotricha.

Powdery mildew, caused by Podosphaera leucotricha, is an economically important disease of apple and pear trees. A single monoconidial strain (PuE-3) of this biotrophic fungus was used to extract DNA for Illumina sequencing. Data were assembled to form a draft genome of 43.8 Mb consisting of 8,921 contigs, 9,372 predicted genes, and 96.1% of complete benchmarking universal single copy orthologs (BUSCOs). This is the first reported genome sequence of P. leucotricha that will enable studies of the population biology, epidemiology, and fungicide resistance of this pathogen. Furthermore, this resource will be fundamental to uncover the genetic and molecular mechanisms of the apple-powdery mildew interaction, and support future pome fruit breeding efforts.

Biosignatures Associated with Freshwater Microbialites.

Freshwater microbialites (i.e., lithifying microbial mats) are quite rare in northern latitudes of the North American continent, with two lakes (Pavilion and Kelly Lakes) of southeastern BC containing a morphological variety of such structures. We investigated Kelly Lake microbialites using carbon isotope systematics, phospholipid fatty acids (PLFAs) and quantitative PCR to obtain biosignatures associated with microbial metabolism. δ13CDIC values (mean δ13CDIC -4.9 ± 1.1‱, n = 8) were not in isotopic equilibrium with the atmosphere; however, they do indicate 13C-depleted inorganic carbon into Kelly Lake. The values of carbonates on microbialite surfaces (δ13C) fell within the range predicted for equilibrium precipitation from ambient lake water δ13CDIC (-2.2 to -5.3‱). Deep microbialites (26 m) had an enriched δ13Ccarb value of -0.3 ± 0.5‱, which is a signature of photoautotrophy. The deeper microbialites (>20 m) had higher biomass estimates (via PLFAs), and a greater relative abundance of cyanobacteria (measured by 16S copies via qPCR). The majority of PLFAs constituted monounsaturated and saturated PLFAs, which is consistent with gram-negative bacteria, including cyanobacteria. The central PLFA δ13C values were highly depleted (-9.3 to -15.7‱) relative to δ13C values of bulk organic matter, suggesting a predominance of photoautotrophy. A heterotrophic signature was also detected via the depleted iso- and anteiso-15:0 lipids (-3.2 to -5.2‱). Based on our carbonate isotopic biosignatures, PLFA, and qPCR measurements, photoautotrophy is enriched in the microbialites of Kelly Lake. This photoautotrophy enrichment is consistent with the microbialites of neighboring Pavilion Lake. This indication of photoautotrophy within Kelly Lake at its deepest depths raises new insights into the limits of measurable carbonate isotopic biosignatures under light and nutrient limitations.

Deciphering the microbiome shift during fermentation of medicinal plants.

The importance of the human-microbiome relationship for positive health outcomes has become more apparent over the last decade. Influencing the gut microbiome via modification of diet represents a possibility of maintaining a healthy gut flora. Fermented food and lactic acid bacteria (LAB) display a preventive way to inhibit microbial dysbioses and diseases, but their ecology on plants is poorly understood. We characterized the microbiome of medicinal plants (Matricaria chamomilla L. and Calendula officinalis L.) using 16S rRNA gene profiling from leaves that were fermented over a six-week time course. The unfermented samples were characterized by a distinct phyllosphere microbiome, while the endosphere revealed a high similarity. During fermentation, significant microbial shifts were observed, whereby LAB were enhanced in all approaches but never numerically dominated. Among the LAB, Enterococcaceae were identified as the most dominant family in both plants. M. chamomilla community had higher relative abundances of Lactobacillaceae and Carnobacteriaceae, while C. officinalis showed a higher presence of Leuconostocaceae and Streptococcaceae. The natural leaf microbiome and the indigenous LAB communities of field-grown Asteraceae medicinal plants are plant-specific and habitat-specific and are subjected to significant shifts during fermentation. Leaf surfaces as well as leaf endospheres were identified as sources for biopreservative LAB.

Revealing the Draft Genome Sequence of Bradyrhizobium sp. Strain USDA 3458, an Effective Symbiotic Diazotroph Isolated from Cowpea (Vigna unguiculata) Genotype IT82E-16.

Pairing plants with plant growth-promoting bacteria is critical to the future of agriculture. Bradyrhizobium sp. strain USDA 3458 isolated from Vigna unguiculata (cowpea) paired with cowpea genotype IT82E-16 represents a novel combination in arid regions. Here, we report the draft genome sequence of strain USDA 3458.

Elucidation of the Genome of Bradyrhizobium sp. Strain USDA 3456, a Historic Agricultural Diazotroph from Cowpea (Vigna unguiculata).

Bradyrhizobium sp. strain USDA 3456 is a historic strain from the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) National Rhizobium Germplasm Collection isolated from Vigna unguiculata (cowpea) in 1966. Strain USDA 3456 has been utilized in global agricultural applications, including improving soil nitrogen fertility. The draft genome sequence here provides a genetic reference of a novel diazotroph.

Metaphenomic Responses of a Native Prairie Soil Microbiome to Moisture Perturbations.

Climate change is causing shifts in precipitation patterns in the central grasslands of the United States, with largely unknown consequences on the collective physiological responses of the soil microbial community, i.e., the metaphenome. Here, we used an untargeted omics approach to determine the soil microbial community's metaphenomic response to soil moisture and to define specific metabolic signatures of the response. Specifically, we aimed to develop the technical approaches and metabolic mapping framework necessary for future systematic ecological studies. We collected soil from three locations at the Konza Long-Term Ecological Research (LTER) field station in Kansas, and the soils were incubated for 15 days under dry or wet conditions and compared to field-moist controls. The microbiome response to wetting or drying was determined by 16S rRNA amplicon sequencing, metatranscriptomics, and metabolomics, and the resulting shifts in taxa, gene expression, and metabolites were assessed. Soil drying resulted in significant shifts in both the composition and function of the soil microbiome. In contrast, there were few changes following wetting. The combined metabolic and metatranscriptomic data were used to generate reaction networks to determine the metaphenomic response to soil moisture transitions. Site location was a strong determinant of the response of the soil microbiome to moisture perturbations. However, some specific metabolic pathways changed consistently across sites, including an increase in pathways and metabolites for production of sugars and other osmolytes as a response to drying. Using this approach, we demonstrate that despite the high complexity of the soil habitat, it is possible to generate insight into the effect of environmental change on the soil microbiome and its physiology and functions, thus laying the groundwork for future, targeted studies.IMPORTANCE Climate change is predicted to result in increased drought extent and intensity in the highly productive, former tallgrass prairie region of the continental United States. These soils store large reserves of carbon. The decrease in soil moisture due to drought has largely unknown consequences on soil carbon cycling and other key biogeochemical cycles carried out by soil microbiomes. In this study, we found that soil drying had a significant impact on the structure and function of soil microbial communities, including shifts in expression of specific metabolic pathways, such as those leading toward production of osmoprotectant compounds. This study demonstrates the application of an untargeted multi-omics approach to decipher details of the soil microbial community's metaphenotypic response to environmental perturbations and should be applicable to studies of other complex microbial systems as well.