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Abstract Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.

Abstract The North Temperate Lakes Long-Term Ecological Research (NTL-LTER) program has been extensively used to improve understanding of how aquatic ecosystems respond to environmental stressors, climate fluctuations, and human activities. Here, we report on the metagenomes of samples collected between 2000 and 2019 from Lake Mendota, a freshwater eutrophic lake within the NTL-LTER site. We utilized the distributed metagenome assembler MetaHipMer to coassemble over 10 terabases (Tbp) of data from 471 individual Illumina-sequenced metagenomes. A total of 95,523,664 contigs were assembled and binned to generate 1,894 non-redundant metagenome-assembled genomes (MAGs) with ≥50% completeness and ≤10% contamination. Phylogenomic analysis revealed that the MAGs were nearly exclusively bacterial, dominated by Pseudomonadota (Proteobacteria, N = 623) and Bacteroidota (N = 321). Nine eukaryotic MAGs were identified by eukCC with six assigned to the phylum Chlorophyta. Additionally, 6,350 high-quality viral sequences were identified by geNomad with the majority classified in the phylum Uroviricota. This expansive coassembled metagenomic dataset provides an unprecedented foundation to advance understanding of microbial communities in freshwater ecosystems and explore temporal ecosystem dynamics.

Abstract Soil microbiomes are heterogeneous, complex microbial communities. Metagenomic analysis is generating vast amounts of data, creating immense challenges in sequence assembly and analysis. Although advances in technology have resulted in the ability to easily collect large amounts of sequence data, soil samples containing thousands of unique taxa are often poorly characterized. These challenges reduce the usefulness of genome-resolved metagenomic (GRM) analysis seen in other fields of microbiology, such as the creation of high quality metagenomic assembled genomes and the adoption of genome scale modeling approaches. The absence of these resources restricts the scale of future research, limiting hypothesis generation and the predictive modeling of microbial communities. Creating publicly available databases of soil MAGs, similar to databases produced for other microbiomes, has the potential to transform scientific insights about soil microbiomes without requiring the computational resources and domain expertise for assembly and binning.

Abstract Bridging molecular information to ecosystem-level processes would provide the capacity to understand system vulnerability and, potentially, a means for assessing ecosystem health. Here, we present an integrated dataset containing environmental and metagenomic information from plant-associated microbial communities, plant transcriptomics, plant and soil metabolomics, and soil chemistry and activity characterization measurements derived from the model tree species Populus trichocarpa. Soil, rhizosphere, root endosphere, and leaf samples were collected from 27 different P. trichocarpa genotypes grown in two different environments leading to an integrated dataset of 318 metagenomes, 98 plant transcriptomes, and 314 metabolomic profiles that are supported by diverse soil measurements. This expansive dataset will provide insights into causal linkages that relate genomic features and molecular level events to system-level properties and their environmental influences.

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus so far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.

ABSTRACT Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, “who is eating what?” and “who is active?” within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism’s taxonomic identity and genome composition while providing quantitative estimates of the microorganism’s isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

ABSTRACT Petabases of environmental metagenomic data are publicly available, presenting an opportunity to characterize complex environments and discover novel lineages of life. Metagenome coassembly, in which many metagenomic samples from an environment are simultaneously analyzed to infer the underlying genomes’ sequences, is an essential tool for achieving this goal. We applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 terabases (Tbp) of metagenome data from a tropical soil in the Luquillo Experimental Forest (LEF), Puerto Rico. The resulting coassembly yielded 39 high-quality (>90% complete,

Plant roots host diverse fungal communities that affect plant health. Here, Mesny et al. use comparative genomics and transcriptomics of fungal isolates from the Arabidopsis thaliana root mycobiota, together with root colonization assays, to identify genetic determinants of endophytism.

Marc Van Goethem et al. combine short- and long-read sequencing from biocrust samples to report nearly 3,000 biosynthetic gene clusters (BGCs) encoding microbial secondary metabolites. Their results demonstrate the advantage of integrated metatranscriptomic and long-read metagenomic sequencing in analyzing BGCs and provides insight into the role of secondary metabolites in maintaining phylogenetic niches in biocrusts.

Latitudinal ecosystem boundaries in the global upper ocean may be driven by many factors. Here the authors investigate pole-to-pole eukaryotic phytoplankton metatranscriptomes, gene co-expression networks, and beta diversity, finding that geographic patterns are best explained by temperature gradients.

Multiomics approaches need to be applied in the central Arctic Ocean to benchmark biodiversity change and to identify novel species and their genes. As part of MOSAiC, EcoOmics will therefore be essential for conservation and sustainable bioprospecting in one of the least explored ecosystems on Earth.

ABSTRACT Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Mycorrhizal symbioses have evolved repeatedly in diverse fungal lineages. A large phylogenomic analysis sheds light on genomic changes associated with transitions from saprotrophy to symbiosis, including divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Cowpea is one of the major legumes cultivated in arid and semiarid regions of the world. Four soil-microbial samples (SS-1 through SS-4) collected from semiarid soils in Punjab, Pakistan were planted with cowpea (Vigna unguiculata) crops, which were grown under salinity stress to analyze bacterial composition in the rhizosphere and within nodules using cultivation-dependent and -independent methods. Two varieties, 603 and the salt-tolerant CB 46, were each inoculated with or without the four different native soil samples or grown in medium either N-deficient (−N) or supplemented with N (+N). Plants inoculated with soil samples SS-2 and SS-4 grew better than plants inoculated with SS-1- and SS-3 and grew comparably with the +N controls. Environmental DNA (eDNA) was isolated from SS-1 and SS-4, and, by 16S ribosomal RNA sequencing, the soil microbiomes consisted mainly of Actinobacteria, Firmicutes, Proteobacteria, and other nonproteobacterial genera. However, analysis of eDNA isolated from cowpea nodules established by the trap plants showed that the nodule microbiome consisted almost exclusively of proteobacterial sequences, particularly species of Bradyrhizobium. Bacteria were isolated from both soils and nodules, and 34 of the 51 isolates tested positive for plant-growth-promoting rhizobacteria traits in plate assays. Many could serve as future inocula for crops in arid soils. The discrepancy between the types of bacteria isolated by culturing bacteria isolated from surface-sterilized cowpea nodules (proteobacteria and nonproteobacteria) versus those detected by sequencing DNA isolated from the nodules (proteobacteria) from cowpea nodules (proteobacteria and nonproteobacteria) versus those detected in the nodule microbiome (proteobacteria) needs further study.

Aspergillus fungi classified within the section Flavi include harmful and beneficial species. Here, Kjærbølling et al. analyse the genomes of 23 Flavi species, showing high genetic diversity and potential for synthesis of over 13,700 CAZymes and 1600 secondary metabolites.

Abstract Background Currently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction). Methods Shotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen. Results The three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration. Conclusion This Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades.

The DOE JGI Metagenome Workflow is designed for processing metagenomic data sets starting from Illumina fastq files. It performs data preprocessing, error correction, assembly, structural and functional annotation, and binning.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Summary: Single-cell sequencing of environmental microorganisms is an essential component of the microbial ecology toolkit. However, large-scale targeted single-cell sequencing for the whole-genome recovery of uncultivated eukaryotes is lagging. The key challenges are low abundance in environmental communities, large complex genomes, and cell walls that are difficult to break. We describe a pipeline composed of state-of-the art single-cell genomics tools and protocols optimized for poorly studied and uncultivated eukaryotic microorganisms that are found at low abundance. This pipeline consists of seven distinct steps, beginning with sample collection and ending with genome annotation, each equipped with quality review steps to ensure high genome quality at low cost. We tested and evaluated each step on environmental samples and cultures of early-diverging lineages of fungi and Chromista/SAR. We show that genomes produced using this pipeline are almost as good as complete reference genomes for functional and comparative genomics for environmental microbial eukaryotes.

ABSTRACT Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria. IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

Abstract Paraburkholderia caballeronis is a plant-associated bacterium. Strain TNe-841T was isolated from the rhizosphere of tomato (Solanum lycopersicum L. var. lycopersicum) growing in Nepantla Mexico State. Initially this bacterium was found to effectively nodulate Phaseolus vulgaris L. However, from an analysis of the genome of strain TNe-841T and from repeat inoculation experiments, we found that this strain did not nodulate bean and also lacked nodulation genes, suggesting that the genes were lost. The genome consists of 7,115,141 bp with a G + C content of 67.01%. The sequence includes 6251 protein-coding genes and 87 RNA genes.

Abstract The genetic platforms of Deinococcus species remain the only systems in which massive ionizing radiation (IR)-induced genome damage can be investigated in vivo at exposures commensurate with cellular survival. We report the whole genome sequence of the extremely IR-resistant rod-shaped bacterium Deinococcus ficus KS 0460 and its phenotypic characterization. Deinococcus ficus KS 0460 has been studied since 1987, first under the name Deinobacter grandis, then Deinococcus grandis. The D. ficus KS 0460 genome consists of a 4.019 Mbp sequence (69.7% GC content and 3894 predicted genes) divided into six genome partitions, five of which are confirmed to be circular. Circularity was determined manually by mate pair linkage. Approximately 76% of the predicted proteins contained identifiable Pfam domains and 72% were assigned to COGs. Of all D. ficus KS 0460 proteins, 79% and 70% had homologues in Deinococcus radiodurans ATCC BAA-816 and Deinococcus geothermalis DSM 11300, respectively. The most striking differences between D. ficus KS 0460 and D. radiodurans BAA-816 identified by the comparison of the KEGG pathways were as follows: (i) D. ficus lacks nine enzymes of purine degradation present in D. radiodurans, and (ii) D. ficus contains eight enzymes involved in nitrogen metabolism, including nitrate and nitrite reductases, that D. radiodurans lacks. Moreover, genes previously considered to be important to IR resistance are missing in D. ficus KS 0460, namely, for the Mn-transporter nramp, and proteins DdrF, DdrJ and DdrK, all of which are also missing in Deinococcus deserti. Otherwise, D. ficus KS 0460 exemplifies the Deinococcus lineage.

Abstract Chryseobacterium bovis DSM 19482T (Hantsis-Zacharov et al., Int J Syst Evol Microbiol 58:1024-1028, 2008) is a Gram-negative, rod shaped, non-motile, facultative anaerobe, chemoorganotroph bacterium. C. bovis is a member of the Flavobacteriaceae, a family within the phylum Bacteroidetes. It was isolated when psychrotolerant bacterial communities in raw milk and their proteolytic and lipolytic traits were studied. Here we describe the features of this organism, together with the draft genome sequence and annotation. The DNA G + C content is 38.19%. The chromosome length is 3,346,045 bp. It encodes 3236 proteins and 105 RNA genes. The C. bovis genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes study.

The ascomycete Cenococcum geophilum is a beneficial mycorrhizal symbiont found frequently on tree roots. Here the authors use comparative genomics and transcriptomics to define genomic signatures that differentiate the beneficial C. geophilumfrom its saprotrophic and pathogenic relatives.

Although the taxonomy of Burkholderia has been extensively scrutinized, significant uncertainty remains regarding the generic boundaries and composition of this large and heterogeneous taxon. Here we used the amino acid and nucleotide sequences of 106 conserved proteins from 92 species to infer robust maximum likelihood phylogenies with which to investigate the generic structure of Burkholderia sensu lato. These data unambiguously supported five distinct lineages, of which four correspond to Burkholderia sensu stricto and the newly introduced genera Paraburkholderia, Caballeronia, and Robbsia. The fifth lineage was represented by P. rhizoxinica. Based on these findings, we propose 13 new combinations for those species previously described as members of Burkholderia but that form part of Caballeronia. These findings also suggest revision of the taxonomic status of P. rhizoxinica as it is does not form part of any of the genera currently recognized in Burkholderia sensu lato. From a phylogenetic point of view, Burkholderia sensu stricto has a sister relationship with the Caballeronia+Paraburkholderia clade. Also, the lineages represented by P. rhizoxinica and R. andropogonis, respectively, emerged prior to the radiation of the Burkholderia sensu stricto+Caballeronia+Paraburkholderia clade. Our findings therefore constitute a solid framework, not only for supporting current and future taxonomic decisions, but also for studying the evolution of this assemblage of medically, industrially and agriculturally important species.

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

BackgroundThe emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation.Methodology/principal findingsIn this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis.ConclusionThese data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.