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Brachypodium distachyon is an excellent model system for the grasses and has been adopted as a research organism by many laboratories around the world. It has all of the biological traits required for a model system, including small stature, short life cycle, small genome, simple growth requirements, and a close relationship to major crop plants (cereals). In addition, numerous resources have been developed for working with this species, including genome sequences for many lines, sequenced mutant collections, and a large, freely available germplasm collection. Fortunately, among grasses B. distachyon is one of the most easily transformed species, an absolute necessity for a model system. Agrobacterium-mediated transformation is the preferred method to transform plants because it usually results in simple insertions of target DNA. In this article, we describe a method for Agrobacterium-mediated transformation of the inbred B. distachyon lines Bd21 and Bd21-3. Embryogenic callus induced from immature embryos is co-cultivated with Agrobacterium tumefaciens strain AGL1 or Agrobacterium rhizogenes strain 18r12v. Hygromycin and paromomycin are used as selective agents, with comparable transformation efficiencies (defined as the percentage of co-cultivated callus that produce transgenic plants) of 40% to 70%. It takes 20 to 30 weeks to obtain T 1 seeds starting from the initial step of dissecting out immature embryos. This protocol has been shown to be efficient and facile in several studies that resulted in the creation of over 22,000 T-DNA mutants. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Microbes drive critical ecosystem functions and affect global nutrient cycling along with human health and disease. They do so under strong constraints exerted by viruses, which shape microbial communities' structure and shift host cell metabolism during infection. While the majority of viruses and their associated impacts remain poorly characterized, a number of mechanisms by which viruses alter microbial cells and ecosystems have already been revealed. Here I outline how a comprehensive host-resolved mapping of viral sequence space will enable a thorough characterization of virus-encoded mechanisms for microbial manipulation. With soon-to-be millions of virus genomes obtained from metagenomes, one of the major challenges resides in the development of methods for high-throughput and high-resolution virus-host pairing, before multi-omics approaches can be leveraged to fully decipher virus-host dynamics in nature. Beyond novel fundamental biological knowledge, these studies will likely provide new molecular tools enabling a precise engineering of microbial cells and communities., Competing Interests: Conflict of Interest Disclosures: S.R. has nothing to disclose., (Copyright © 2019 Roux.)

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla ( Calditrichota , SM32-31, AABM5-125-24, and KSB1) within the broader FCB ( Fibrobacteres-Chlorobi-Bacteroidetes ) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies. IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines., (Copyright © 2019 American Society for Microbiology.)

Calcifying microbial mats in hypersaline environments are important model systems for the study of the earliest ecosystems on Earth that started to appear more than three billion years ago and have been preserved in the fossil record as laminated lithified structures known as stromatolites. It is believed that sulfate-reducing bacteria play a pivotal role in the lithification process by increasing the saturation index of calcium minerals within the mat. Strain L21-Syr-AB T was isolated from anoxic samples of a several centimeters-thick microbialite-forming cyanobacterial mat of a hypersaline lake on the Kiritimati Atoll (Kiribati, Central Pacific). The novel isolate was assigned to the family Desulfovibrionaceae within the Deltaproteobacteria . Available 16S rRNA-based population surveys obtained from discrete layers of the mat indicate that the occurrence of a species-level clade represented by strain L21-Syr-AB T is restricted to a specific layer of the suboxic zone, which is characterized by the presence of aragonitic spherulites. To elucidate a possible function of this sulfate-reducing bacterium in the mineral formation within the mat a comprehensive phenotypic characterization was combined with the results of a comparative genome analysis. Among the determined traits of strain L21-Syr-AB T , several features were identified that could play a role in the precipitation of calcium carbonate: (i) the potential deacetylation of polysaccharides and consumption of substrates such as lactate and sulfate could mobilize free calcium; (ii) under conditions that favor the utilization of formate and hydrogen, the alkalinity engine within the mat is stimulated, thereby increasing the availability of carbonate; (iii) the production of extracellular polysaccharides could provide nucleation sites for calcium mineralization. In addition, our data suggest the proposal of the novel species and genus Desulfohalovibrio reitneri represented by the type strain L21-Syr-AB T (=DSM 26903 T = JCM 18662 T ).

Following publication of the original article.

Asinibacterium sp. strains OR43 and OR53 belong to the phylum Bacteroidetes and were isolated from subsurface sediments in Oak Ridge, TN. Both strains grow at elevated levels of heavy metals. Here, we present the closed genome sequence of Asinibacterium sp. strain OR53 and the draft genome sequence of Asinibacterium sp. strain OR43., (Copyright © 2019 Brzoska et al.)

In previous work (D. R. Harris et al., J Bacteriol 191:5240-5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident. IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent., (Copyright © 2019 American Society for Microbiology.)

The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.

Amplicon sequencing of 16S, ITS, and 18S regions of microbial genomes is a commonly used first step toward understanding microbial communities of interest for human health, agriculture, and the environment. Correlation network analysis is an emerging tool for investigating the interactions within these microbial communities. However, when data from different habitats (e.g., sampling sites, host genotype, etc.) are combined into one analysis, habitat filtering (co-occurrence of microbes due to habitat sampled rather than biological interactions) can induce apparent correlations, resulting in a network dominated by habitat effects and masking correlations of biological interest. We developed an algorithm to correct for habitat filtering effects in microbial correlation network analysis in order to reveal the true underlying microbial correlations. This algorithm was tested on simulated data that was constructed to exhibit habitat filtering. Our algorithm significantly improved correlation detection accuracy for these data compared to Spearman and Pearson correlations. We then used our algorithm to analyze a two real data sets of 16S variable region amplicon sequences that were expected to exhibit habitat filtering. Our algorithm was found to effectively reduce habitat effects, enabling the construction of consensus correlation networks from data sets combining multiple related sample habitats.

Some glycoside hydrolases have broad specificity for hydrolysis of glycosidic bonds, potentially increasing their functional utility and flexibility in physiological and industrial applications. To deepen the understanding of the structural and evolutionary driving forces underlying specificity patterns in glycoside hydrolase family 5, we quantitatively screened the activity of the catalytic core domains from subfamily 4 (GH5_4) and closely related enzymes on four substrates: lichenan, xylan, mannan, and xyloglucan. Phylogenetic analysis revealed that GH5_4 consists of three major clades, and one of these clades, referred to here as clade 3, displayed average specific activities of 4.2 and 1.2 U/mg on lichenan and xylan, approximately 1 order of magnitude larger than the average for active enzymes in clades 1 and 2. Enzymes in clade 3 also more consistently met assay detection thresholds for reaction with all four substrates. We also identified a subfamily-wide positive correlation between lichenase and xylanase activities, as well as a weaker relationship between lichenase and xyloglucanase. To connect these results to structural features, we used the structure of CelE from Hungateiclostridium thermocellum (PDB 4IM4) as an example clade 3 enzyme with activities on all four substrates. Comparison of the sequence and structure of this enzyme with others throughout GH5_4 and neighboring subfamilies reveals at least two residues (H149 and W203) that are linked to strong activity across the substrates. Placing GH5_4 in context with other related subfamilies, we highlight several possibilities for the ongoing evolutionary specialization of GH5_4 enzymes., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Transcription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after whole genome duplication (WGD) event, transcription factors are retained at a significantly higher rate. Little is known about how TF duplicates have diverged in their expression and regulation, the answer to which may contribute to a better understanding of the elevated retention rate among TFs., Results: Here we assessed what features may explain differences in the retention of TF duplicates and other genes using Arabidopsis thaliana as a model. We integrated 34 expression, sequence, and conservation features to build a linear model for predicting the extent of duplicate retention following WGD events among TFs and 19 groups of genes with other functions. We found that TFs was the least well predicted, demonstrating the features of TFs are substantially deviated from duplicate genes in other function groups. Consistent with this, the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates: one "ancestral" TF duplicate retains most ancestral expression and cis-regulatory sites, while the "non-ancestral" duplicate is enriched for novel regulatory sites. By modeling the retention of ancestral expression and cis-regulatory states in duplicate pairs using a system of differential equations, we found that TF duplicate pairs in a partitioned state are preferentially maintained., Conclusions: These TF duplicates with asymmetrically partitioned ancestral states are likely maintained because one copy retains ancestral functions while the other, at least in some cases, acquires novel cis-regulatory sites that may be important for novel, adaptive traits.

Gulf of Mexico sediments harbor numerous hydrocarbon seeps associated with high sedimentation rates and thermal maturation of organic matter. These ecosystems host abundant and diverse microbial communities that directly or indirectly metabolize components of the emitted fluid. To investigate microbial function and activities in these ecosystems, metabolic potential (metagenomic) and gene expression (metatranscriptomic) analyses of two cold seep areas of the Gulf of Mexico were carried out. Seeps emitting biogenic methane harbored microbial communities dominated by archaeal anaerobic methane oxidizers of phylogenetic group 1 (ANME-1), whereas seeps producing fluids containing a complex mixture of thermogenic hydrocarbons were dominated by ANME-2 lineages. Metatranscriptome measurements in both communities indicated high levels of expression of genes for methane metabolism despite their distinct microbial communities and hydrocarbon composition. In contrast, the transcription level of sulfur cycle genes was quite different. In the thermogenic seep community, high levels of transcripts indicative of syntrophic anaerobic oxidation of methane (AOM) coupled to sulfate reduction were detected. This syntrophic partnership between the dominant ANME-2 and sulfate reducers potentially involves direct electron transfer through multiheme cytochromes. In the biogenic methane seep, genes from an ANME-1 lineage that are potentially involved in polysulfide reduction were highly expressed, suggesting a novel bacterium-independent anaerobic methane oxidation pathway coupled to polysulfide reduction. The observed divergence in AOM activities provides a new model for bacterium-independent AOM and emphasizes the variation that exists in AOM pathways between different ANME lineages. IMPORTANCE Cold seep sediments are complex and widespread marine ecosystems emitting large amounts of methane, a potent greenhouse gas, and other hydrocarbons. Within these sediments, microbial communities play crucial roles in production and degradation of hydrocarbons, modulating oil and gas emissions to seawater. Despite this ecological importance, our understanding of microbial functions and methane oxidation pathways in cold seep ecosystems is poor. Based on gene expression profiling of environmental seep sediment samples, the present work showed that (i) the composition of the emitted fluids shapes the microbial community in general and the anaerobic methanotroph community specifically and (ii) AOM by ANME-2 in this seep may be coupled to sulfate reduction by Deltaproteobacteria by electron transfer through multiheme cytochromes, whereas AOM by ANME-1 lineages in this seep may involve a different, bacterium-independent pathway, coupling methane oxidation to elemental sulfur/polysulfide reduction.

Microbial genome web portals have a broad range of capabilities that address a number of information-finding and analysis needs for scientists. This article compares the capabilities of the major microbial genome web portals to aid researchers in determining which portal(s) are best suited to their needs. We assessed both the bioinformatics tools and the data content of BioCyc, KEGG, Ensembl Bacteria, KBase, IMG, and PATRIC. For each portal, our assessment compared and tallied the available capabilities. The strengths of BioCyc include its genomic and metabolic tools, multi-search capabilities, table-based analysis tools, regulatory network tools and data, omics data analysis tools, breadth of data content, and large amount of curated data. The strengths of KEGG include its genomic and metabolic tools. The strengths of Ensembl Bacteria include its genomic tools and large number of genomes. The strengths of KBase include its genomic tools and metabolic models. The strengths of IMG include its genomic tools, multi-search capabilities, large number of genomes, table-based analysis tools, and breadth of data content. The strengths of PATRIC include its large number of genomes, table-based analysis tools, metabolic models, and breadth of data content.

Using genome-wide mutant fitness assays in diverse bacteria, we identified novel oxidative pathways for the catabolism of 2-deoxy-d-ribose and 2-deoxy-d-ribonate. We propose that deoxyribose is oxidized to deoxyribonate, oxidized to ketodeoxyribonate, and cleaved to acetyl coenzyme A (acetyl-CoA) and glyceryl-CoA. We have genetic evidence for this pathway in three genera of bacteria, and we confirmed the oxidation of deoxyribose to ketodeoxyribonate in vitro . In Pseudomonas simiae, the expression of enzymes in the pathway is induced by deoxyribose or deoxyribonate, while in Paraburkholderia bryophila and in Burkholderia phytofirmans, the pathway proceeds in parallel with the known deoxyribose 5-phosphate aldolase pathway. We identified another oxidative pathway for the catabolism of deoxyribonate, with acyl-CoA intermediates, in Klebsiella michiganensis. Of these four bacteria, only P. simiae relies entirely on an oxidative pathway to consume deoxyribose. The deoxyribose dehydrogenase of P. simiae is either nonspecific or evolved recently, as this enzyme is very similar to a novel vanillin dehydrogenase from Pseudomonas putida that we identified. So, we propose that these oxidative pathways evolved primarily to consume deoxyribonate, which is a waste product of metabolism. IMPORTANCE Deoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not contain any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants' abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of metabolism. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.

The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O 2 under certain conditions via a putative novel type of heme copper O 2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H 2 metabolism were central metabolic features. The ability to use O 2 as a terminal electron acceptor must have been subsequently acquired by these lineages.

The Genomes Online Database (GOLD) (https://gold.jgi.doe.gov) is an open online resource, which maintains an up-to-date catalog of genome and metagenome projects in the context of a comprehensive list of associated metadata. Information in GOLD is organized into four levels: Study, Biosample/Organism, Sequencing Project and Analysis Project. Currently GOLD hosts information on 33 415 Studies, 49 826 Biosamples, 313 324 Organisms, 215 881 Sequencing Projects and 174 454 Analysis Projects with a total of 541 metadata fields, of which 80 are based on controlled vocabulary (CV) terms. GOLD provides a user-friendly web interface to browse sequencing projects and launch advanced search tools across four classification levels. Users submit metadata on a wide range of Sequencing and Analysis Projects in GOLD before depositing sequence data to the Integrated Microbial Genomes (IMG) system for analysis. GOLD conforms with and supports the rules set by the Genomic Standards Consortium (GSC) Minimum Information standards. The current version of GOLD (v.7) has seen the number of projects and associated metadata increase exponentially over the years. This paper provides an update on the current status of GOLD and highlights the new features added over the last two years.

Marine algae convert a substantial fraction of fixed carbon dioxide into various polysaccharides. Flavobacteriia that are specialized on algal polysaccharide degradation feature genomic clusters termed polysaccharide utilization loci (PULs). As knowledge on extant PUL diversity is sparse, we sequenced the genomes of 53 North Sea Flavobacteriia and obtained 400 PULs. Bioinformatic PUL annotations suggest usage of a large array of polysaccharides, including laminarin, α-glucans, and alginate as well as mannose-, fucose-, and xylose-rich substrates. Many of the PULs exhibit new genetic architectures and suggest substrates rarely described for marine environments. The isolates' PUL repertoires often differed considerably within genera, corroborating ecological niche-associated glycan partitioning. Polysaccharide uptake in Flavobacteriia is mediated by SusCD-like transporter complexes. Respective protein trees revealed clustering according to polysaccharide specificities predicted by PUL annotations. Using the trees, we analyzed expression of SusC/D homologs in multiyear phytoplankton bloom-associated metaproteomes and found indications for profound changes in microbial utilization of laminarin, α-glucans, β-mannan, and sulfated xylan. We hence suggest the suitability of SusC/D-like transporter protein expression within heterotrophic bacteria as a proxy for the temporal utilization of discrete polysaccharides.

The molecular composition of soil organic matter (SOM) sets the foundation for terrestrial microbial community structures and carbon cycling dynamics. However, the specific chemical constituents of SOM are underexplored. In this chapter we present a protocol for the extraction of small molecule metabolites from soil followed by compound detection and identification using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. There are options within the protocol to assess either the extracellular pool of metabolites or the total pool (including intracellular) and either polar or nonpolar metabolites, depending on the reader's research interests. These methods can be followed individually for a more targeted analysis or all methods can be combined to obtain a more comprehensive understanding of SOM metabolite composition (such as amino acids, nucleobases, organic acids, fatty acids, carbohydrates, secondary metabolites, and antibiotics).

Rationale: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D 2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids., Methods: Lipid extracts of D 2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D 2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections., Results: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day., Conclusions: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D 2 O., (© 2018 John Wiley & Sons, Ltd.)

The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolytica msn2 (Yl msn2 ) ortholog is required for hyphal growth and found that overexpression of Yl msn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G 1 -to-S phase progression. We found that overexpression of either the Yl mbp1 or Yl swi6 homologs decreased hyphal growth and that deletion of either Yl mbp1 or Yl swi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Yl chk1 and Yl nik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Yl ssk2 , Yl pbs2 , and Yl hog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways. IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Yl msn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Yl chk1 and Yl nik1 as well as the MAP kinases of the HOG pathway (Yl ssk2 , Yl pbs2 , and Yl hog1 transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response., (Copyright © 2018 Pomraning et al.)

Lentinus tigrinus is a species of wood-decaying fungi (Polyporales) that has an agaricoid form (a gilled mushroom) and a secotioid form (puffball-like, with enclosed spore-bearing structures). Previous studies suggested that the secotioid form is conferred by a recessive allele of a single locus. We sequenced the genomes of one agaricoid (Aga) strain and one secotioid (Sec) strain (39.53-39.88 Mb, with 15,581-15,380 genes, respectively). We mated the Sec and Aga monokaryons, genotyped the progeny, and performed bulked segregant analysis (BSA). We also fruited three Sec/Sec and three Aga/Aga dikaryons, and sampled transcriptomes at four developmental stages. Using BSA, we identified 105 top candidate genes with nonsynonymous SNPs that cosegregate with fruiting body phenotype. Transcriptome analyses of Sec/Sec versus Aga/Aga dikaryons identified 907 differentially expressed genes (DEGs) along four developmental stages. On the basis of BSA and DEGs, the top 25 candidate genes related to fruiting body development span 1.5 Mb (4% of the genome), possibly on a single chromosome, although the precise locus that controls the secotioid phenotype is unresolved. The top candidates include genes encoding a cytochrome P450 and an ATP-dependent RNA helicase, which may play a role in development, based on studies in other fungi.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

The number and proportion of genes with no known function are growing rapidly. To quantify this phenomenon and provide criteria for prioritizing genes for functional characterization, we developed a bioinformatics pipeline that identifies robustly defined protein families with no annotated domains, ranks these with respect to phylogenetic breadth, and identifies them in metagenomics data. We applied this approach to 271 965 protein families from the SFams database and discovered many with no functional annotation, including >118 000 families lacking any known protein domain. From these, we prioritized 6 668 conserved protein families with at least three sequences from organisms in at least two distinct classes. These Function Unknown Families (FUnkFams) are present in Tara Oceans Expedition and Human Microbiome Project metagenomes, with distributions associated with sampling environment. Our findings highlight the extent of functional novelty in sequence databases and establish an approach for creating a "most wanted" list of genes to prioritize for further characterization., Competing Interests: The authors have declared that no competing interests exist.

There is a growing appreciation for the important roles microorganisms play in association with plants. Microorganisms are drawn to distinct plant surfaces by the nutrient-rich microenvironment, and in turn some of these colonizing microbes provide mutualistic benefits to their host. The development of plant probiotics to increase crop yield and provide plant resistance against biotic and abiotic stresses, while minimizing chemical inputs, would benefit from a deeper mechanistic understanding of plant-microbe interaction. Technological advances in molecular biology and high-throughput -omics provide stepping stones to the elucidation of critical microbiome gene functions that aid in improving plant performance. Here, we review -omics-based approaches that are propelling forward the current understanding of plant-associated bacterial gene functions, and describe how these technologies have helped unravel key bacterial genes and pathways that mediate pathogenic, beneficial, and commensal host interactions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Grasses are essential plants for ecosystem functioning. Quantifying the selective pressures that act on natural variation in grass species is therefore essential regarding biodiversity maintenance. In this study, we investigate the selection pressures that act on two distinct populations of the grass model Brachypodium distachyon without prior knowledge about the traits under selection. We took advantage of whole-genome sequencing data produced for 44 natural accessions of B. distachyon and used complementary genome-wide selection scans (GWSS) methods to detect genomic regions under balancing and positive selection. We show that selection is shaping genetic diversity at multiple temporal and spatial scales in this species, and affects different genomic regions across the two populations. Gene ontology annotation of candidate genes reveals that pathogens may constitute important factors of positive and balancing selection in B. distachyon. We eventually cross-validated our results with quantitative trait locus data available for leaf-rust resistance in this species and demonstrate that, when paired with classical trait mapping, GWSS can help pinpointing candidate genes for further molecular validation. Thanks to a near base-perfect reference genome and the large collection of freely available natural accessions collected across its natural range, B. distachyon appears as a prime system for studies in ecology, population genomics and evolutionary biology., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Draft genome sequences of 11 bacteria belonging to the family Microbacteriaceae were obtained using Illumina technology. The genomes of these strains have sizes from 3.14 to 4.30 Mb with their genomic DNA characterized as having high G+C contents (above 65%). These genomic data will be useful for natural taxonomy and comparative genomic studies of bacterial strains of the family Microbacteriaceae.

Background: Nanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity., Results: From 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs., Conclusions: This population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

Vermifiltration is a biological treatment process during which earthworms (e.g., Eisenia fetida ) and microorganisms reduce the organic load of wastewater. To infer microbial pathways responsible for nutrient conversion, past studies characterized the microbiota in vermifilters and suggested that nitrifying and denitrifying bacteria play a significant role during this wastewater treatment process. In contrast to previous studies, which were limited by low-resolution sequencing methods, the work presented here utilized next generation sequencing to survey in greater detail the microbiota of wastewater from a commercial dairy during various stages of vermifiltration. To complement sequence analysis, nitrogenous compounds in and gaseous emissions from the wastewater were measured. Analysis of 16S rRNA gene profiles from untreated wastewater, vermifilter influent, and vermifilter effluent suggested that members of Comamonadaceae , a family of the Betaproteobacteria involved in denitrification, increased in abundance during the vermifiltration process. Subsequent functional gene analysis indicated an increased abundance of nitrification genes in the effluent and suggested that the nitrogen removal during vermifiltration is due to the microbial conversion of ammonia, a finding that was also supported by the water chemistry and emission data. This study demonstrates that microbial communities are the main drivers behind reducing the nitrogen load of dairy wastewater during vermifiltration, providing a valuable knowledge framework for more sustainable and economical wastewater management strategies for commercial dairies.

Background: Crassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO 2 uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown., Results: Using an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C 3 , C 4 and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C 3 , C 4 and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules., Conclusions: This work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C 3 ancestors in Agave.

Repeated evolutionary events imply underlying genetic constraints that can make evolutionary mechanisms predictable. Morphological traits are thought to evolve frequently through cis-regulatory changes because these mechanisms bypass constraints in pleiotropic genes that are reused during development. In contrast, the constraints acting on metabolic traits during evolution are less well studied. Here we show how a metabolic bottleneck gene has repeatedly adopted similar cis-regulatory solutions during evolution, likely due to its pleiotropic role integrating flux from multiple metabolic pathways. Specifically, the genes encoding phosphoglucomutase activity (PGM1/PGM2), which connect GALactose catabolism to glycolysis, have gained and lost direct regulation by the transcription factor Gal4 several times during yeast evolution. Through targeted mutations of predicted Gal4-binding sites in yeast genomes, we show this galactose-mediated regulation of PGM1/2 supports vigorous growth on galactose in multiple yeast species, including Saccharomyces uvarum and Lachancea kluyveri. Furthermore, the addition of galactose-inducible PGM1 alone is sufficient to improve the growth on galactose of multiple species that lack this regulation, including Saccharomyces cerevisiae. The strong association between regulation of PGM1/2 by Gal4 even enables remarkably accurate predictions of galactose growth phenotypes between closely related species. This repeated mode of evolution suggests that this specific cis-regulatory connection is a common way that diverse yeasts can govern flux through the pathway, likely due to the constraints imposed by this pleiotropic bottleneck gene. Since metabolic pathways are highly interconnected, we argue that cis-regulatory evolution might be widespread at pleiotropic genes that control metabolic bottlenecks and intersections.

Sulfite-reducing and sulfate-reducing microorganisms (SRM) play important roles in anoxic environments, linking the sulfur and carbon cycles. With climate warming, the distribution of anoxic habitats conductive to dissimilatory SRM is expanding. Consequently, we hypothesize that novel SRM are likely to emerge from the rare biosphere triggered by environmental changes. Using the dsrB gene as a  molecular marker of sulfite-reducers and sulfate-reducers, we analyzed the diversity, community composition, and abundance of SRM in 200 samples representing 14 different ecosystems, including marine and freshwater environments, oil reservoirs, and engineered infrastructure. Up to 167,397 species-level OTUs affiliated with 47 different families were identified. Up to 96% of these can be considered as "rare biosphere SRM". One  third of the dsrB genes identified belonged to uncharacterized lineages. The dsrB sequences exhibited a  strong pattern of selection in different ecosystems. These results expand our knowledge of the biodiversity and distribution of SRM, with implications for carbon and sulfur cycling in anoxic ecosystems.

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Burkholderia sensu lato is a large and complex group, containing pathogenic, phytopathogenic, symbiotic and non-symbiotic strains from a very wide range of environmental (soil, water, plants, fungi) and clinical (animal, human) habitats. Its taxonomy has been evaluated several times through the analysis of 16S rRNA sequences, concantenated 4⁻7 housekeeping gene sequences, and lately by genome sequences. Currently, the division of this group into Burkholderia , Caballeronia, Paraburkholderia , and Robbsia is strongly supported by genome analysis. These new genera broadly correspond to the various habitats/lifestyles of Burkholderia s.l., e.g., all the plant beneficial and environmental (PBE) strains are included in Paraburkholderia (which also includes all the N₂-fixing legume symbionts) and Caballeronia , while most of the human and animal pathogens are retained in Burkholderia sensu stricto. However, none of these genera can accommodate two important groups of species. One of these includes the closely related Paraburkholderia rhizoxinica and Paraburkholderia endofungorum , which are both symbionts of the fungal phytopathogen Rhizopus microsporus . The second group comprises the Mimosa -nodulating bacterium Paraburkholderia symbiotica , the phytopathogen Paraburkholderia caryophylli , and the soil bacteria Burkholderia dabaoshanensis and Paraburkholderia soli . In order to clarify their positions within Burkholderia sensu lato, a phylogenomic approach based on a maximum likelihood analysis of conserved genes from more than 100 Burkholderia sensu lato species was carried out. Additionally, the average nucleotide identity (ANI) and amino acid identity (AAI) were calculated. The data strongly supported the existence of two distinct and unique clades, which in fact sustain the description of two novel genera Mycetohabitans gen. nov. and Trinickia gen. nov. The newly proposed combinations are Mycetohabitans endofungorum comb. nov., Mycetohabitans rhizoxinica comb. nov., Trinickia caryophylli comb. nov., Trinickia dabaoshanensis comb. nov., Trinickia soli comb. nov., and Trinickia symbiotica comb. nov. Given that the division between the genera that comprise Burkholderia s.l. in terms of their lifestyles is often complex, differential characteristics of the genomes of these new combinations were investigated. In addition, two important lifestyle-determining traits-diazotrophy and/or symbiotic nodulation, and pathogenesis-were analyzed in depth i.e., the phylogenetic positions of nitrogen fixation and nodulation genes in Trinickia via-à-vis other Burkholderiaceae were determined, and the possibility of pathogenesis in Mycetohabitans and Trinickia was tested by performing infection experiments on plants and the nematode Caenorhabditis elegans . It is concluded that (1) T. symbiotica nif and nod genes fit within the wider Mimosa -nodulating Burkholderiaceae but appear in separate clades and that T. caryophylli nif genes are basal to the free-living Burkholderia s.l. strains, while with regard to pathogenesis (2) none of the Mycetohabitans and Trinickia strains tested are likely to be pathogenic, except for the known phytopathogen T. caryophylli .

Deforestation in the Brazilian Amazon occurs at an alarming rate, which has broad effects on global greenhouse gas emissions, carbon storage, and biogeochemical cycles. In this study, soil metagenomes and metagenome-assembled genomes (MAGs) were analyzed for alterations to microbial community composition, functional groups, and putative physiology as it related to land-use change and tropical soil. A total of 28 MAGs were assembled encompassing 10 phyla, including both dominant and rare biosphere lineages. Amazon Acidobacteria subdivision 3, Melainabacteria, Microgenomates, and Parcubacteria were found exclusively in pasture soil samples, while Candidatus Rokubacteria was predominant in the adjacent rainforest soil. These shifts in relative abundance between land-use types were supported by the different putative physiologies and life strategies employed by the taxa. This research provides unique biological insights into candidate phyla in tropical soil and how deforestation may impact the carbon cycle and affect climate change.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

Background: Advances in genomic technologies have expanded our ability to accurately and exhaustively detect natural genomic variants that can be applied in crop improvement and to increase our knowledge of plant evolution and adaptation. Switchgrass (Panicum virgatum L.), an allotetraploid (2n = 4× = 36) perennial C4 grass (Poaceae family) native to North America and a feedstock crop for cellulosic biofuel production, has a large potential for genetic improvement due to its high genotypic and phenotypic variation. In this study, we analyzed single nucleotide polymorphism (SNP) variation in 372 switchgrass genotypes belonging to 36 accessions for 12 genes putatively involved in biomass production to investigate signatures of selection that could have led to ecotype differentiation and to population adaptation to geographic zones., Results: A total of 11,682 SNPs were mined from ~ 15 Gb of sequence data, out of which 251 SNPs were retained after filtering. Population structure analysis largely grouped upland accessions into one subpopulation and lowland accessions into two additional subpopulations. The most frequent SNPs were in homozygous state within accessions. Sixty percent of the exonic SNPs were non-synonymous and, of these, 45% led to non-conservative amino acid changes. The non-conservative SNPs were largely in linkage disequilibrium with one haplotype being predominantly present in upland accessions while the other haplotype was commonly present in lowland accessions. Tajima's test of neutrality indicated that PHYB, a gene involved in photoperiod response, was under positive selection in the switchgrass population. PHYB carried a SNP leading to a non-conservative amino acid change in the PAS domain, a region that acts as a sensor for light and oxygen in signal transduction., Conclusions: Several non-conservative SNPs in genes potentially involved in plant architecture and adaptation have been identified and led to population structure and genetic differentiation of ecotypes in switchgrass. We suggest here that PHYB is a key gene involved in switchgrass natural selection. Further analyses are needed to determine whether any of the non-conservative SNPs identified play a role in the differential adaptation of upland and lowland switchgrass.

Few pan-genomic studies have been conducted in plants, and none of them have focused on the intraspecific diversity and evolution of their plastid genomes. We address this issue in Brachypodium distachyon and its close relatives B. stacei and B. hybridum, for which a large genomic data set has been compiled. We analyze inter- and intraspecific plastid comparative genomics and phylogenomic relationships within a family-wide framework. Major indel differences were detected between Brachypodium plastomes. Within B. distachyon, we detected two main lineages, a mostly Extremely Delayed Flowering (EDF+) clade and a mostly Spanish (S+) - Turkish (T+) clade, plus nine chloroplast capture and two plastid DNA (ptDNA) introgression and micro-recombination events. Early Oligocene (30.9 million yr ago (Ma)) and Late Miocene (10.1 Ma) divergence times were inferred for the respective stem and crown nodes of Brachypodium and a very recent Mid-Pleistocene (0.9 Ma) time for the B. distachyon split. Flowering time variation is a main factor driving rapid intraspecific divergence in B. distachyon, although it is counterbalanced by repeated introgression between previously isolated lineages. Swapping of plastomes between the three different genomic groups, EDF+, T+, S+, probably resulted from random backcrossing followed by stabilization through selection pressure., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Stenotrophomonas acidaminiphila is an aerobic, glucose non-fermentative, Gram-negative bacterium that been isolated from various environmental sources, particularly aquatic ecosystems. Although resistance to multiple antimicrobial agents has been reported in S. acidaminiphila , the mechanisms are largely unknown. Here, for the first time, we report the complete genome and antimicrobial resistome analysis of a clinical isolate S. acidaminiphila SUNEO which is resistant to sulfamethoxazole. Comparative analysis among closely related strains identified common and strain-specific genes. In particular, comparison with a sulfamethoxazole-sensitive strain identified a mutation within the sulfonamide-binding site of folP in SUNEO, which may reduce the binding affinity of sulfamethoxazole. Selection pressure analysis indicated folP in SUNEO is under purifying selection, which may be owing to long-term administration of sulfonamide against Stenotrophomonas .

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

Dark septate endophytes (DSE) are a form-group of root endophytic fungi with elusive functions. Here, the genomes of two common DSE of semiarid areas, Cadophora sp. and Periconia macrospinosa were sequenced and analyzed with another 32 ascomycetes of different lifestyles. Cadophora sp. (Helotiales) and P. macrospinosa (Pleosporales) have genomes of 70.46 Mb and 54.99 Mb with 22,766 and 18,750 gene models, respectively. The majority of DSE-specific protein clusters lack functional annotation with no similarity to characterized proteins, implying that they have evolved unique genetic innovations. Both DSE possess an expanded number of carbohydrate active enzymes (CAZymes), including plant cell wall degrading enzymes (PCWDEs). Those were similar in three other DSE, and contributed a signal for the separation of root endophytes in principal component analyses of CAZymes, indicating shared genomic traits of DSE fungi. Number of secreted proteases and lipases, aquaporins, and genes linked to melanin synthesis were also relatively high in our fungi. In spite of certain similarities between our two DSE, we observed low levels of convergence in their gene family evolution. This suggests that, despite originating from the same habitat, these two fungi evolved along different evolutionary trajectories and display considerable functional differences within the endophytic lifestyle.

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii ), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis ), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii ), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis , but there was also evidence for other thermophilic fermentative anaerobes ( Caldanaerobacter , Fervidobacterium , Caloramator , and Clostridium ). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator , had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes. IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic., (Copyright © 2018 American Society for Microbiology.)

Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year's JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain., (© 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.)

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Here, we report the draft genome sequence of Monaibacterium marinum C7 T , a strain that represents a new member of the Roseobacter clade of the family Rhodobacteraceae ( Alphaproteobacteria ). The genome size of Monaibacterium marinum C7 T is 3.7 Mb (3,734,267 bp), with a G+C content of 58.86%., (Copyright © 2018 Chernikova et al.)

There is a need to clarify relationships within the actinobacterial genus Micromonospora, the type genus of the family Micromonosporaceae, given its biotechnological and ecological importance. Here, draft genomes of 40 Micromonospora type strains and two non-type strains are made available through the Genomic Encyclopedia of Bacteria and Archaea project and used to generate a phylogenomic tree which showed they could be assigned to well supported phyletic lines that were not evident in corresponding trees based on single and concatenated sequences of conserved genes. DNA G+C ratios derived from genome sequences showed that corresponding data from species descriptions were imprecise. Emended descriptions include precise base composition data and approximate genome sizes of the type strains. antiSMASH analyses of the draft genomes show that micromonosporae have a previously unrealised potential to synthesize novel specialized metabolites. Close to one thousand biosynthetic gene clusters were detected, including NRPS, PKS, terpenes and siderophores clusters that were discontinuously distributed thereby opening up the prospect of prioritising gifted strains for natural product discovery. The distribution of key stress related genes provide an insight into how micromonosporae adapt to key environmental variables. Genes associated with plant interactions highlight the potential use of micromonosporae in agriculture and biotechnology.

Thermoanaerobacterium sp. strain RBIITD was isolated from contaminated rich growth medium at 55°C in an anaerobic chamber. It primarily produces butyrate as a fermentation product from plant biomass-derived sugars. The whole-genome sequence of the strain is 3.4 Mbp, with 3,444 genes and 32.48% GC content., (Copyright © 2018 Biswas et al.)

The ever-increasing metagenomic data necessitate appropriate cataloguing in a way that facilitates the comparison and better contextualization of the underlying investigations. To this extent, information associated with the sequencing data as well as the original sample and the environment where it was obtained from is crucial. To date, there are not any publicly available repositories able to capture environmental metadata pertaining to hydrocarbon-rich environments. As such, contextualization and comparative analysis among sequencing datasets derived from these environments is to a certain degree hindered or cannot be fully evaluated. The metagenomics data management system for hydrocarbon resources (MetaHCRs) enables the capturing of marker gene and whole metagenome sequencing data as well as over 300 contextual attributes associated with samples, organisms, environments and geological properties, among others. Moreover, MetaHCR implements the Minimum Information about any Sequence-hydrocarbon resource specification from the Genomic Standards Consortium; it integrates a user-friendly web interface and relational database model, and it enables the generation of complex custom search. MetaHCR has been tested with 36 publicly available metagenomic studies, and its modular architecture can be easily customized for other types of environmental and metagenomics studies.