Microbiome Metadata Standards: Report of the National Microbiome Data Collaborative's Workshop and Follow-On Activities.

Microbiome samples are inherently defined by the environment in which they are found. Therefore, data that provide context and enable interpretation of measurements produced from biological samples, often referred to as metadata, are critical. Important contributions have been made in the development of community-driven metadata standards; however, these standards have not been uniformly embraced by the microbiome research community. To understand how these standards are being adopted, or the barriers to adoption, across research domains, institutions, and funding agencies, the National Microbiome Data Collaborative (NMDC) hosted a workshop in October 2019. This report provides a summary of discussions that took place throughout the workshop, as well as outcomes of the working groups initiated at the workshop.

Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass.

BACKGROUND: The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms. RESULTS: C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was prepared for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNA-seq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5% false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared. CONCLUSIONS: Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.

Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis.

Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins present in plant cell walls. AGPs are characterized by arabinose-/galactose-rich side chains, which define their interactive molecular surface. Fucose residues are found in some dicotyledon AGPs, and AGP fucosylation is developmentally regulated. We previously identified Arabidopsis thaliana FUT4 and FUT6 genes as AGP-specific fucosyltransferases (FUTs) based on their enzymatic activities when heterologously expressed in tobacco (Nicotiana tabacum) BY2 suspension-cultured cells. Here, the functions of FUT4 and FUT6 and the physiological roles of fucosylated AGPs were further investigated using Arabidopsis fut4, fut6, and fut4/fut6 mutant plants. All mutant plants showed no phenotypic differences compared to wild-type plants under physiological conditions, but showed reduced root growth in the presence of elevated NaCl. However, roots of wild-type and fut4 mutant plants contained terminal fucose epitopes, which were absent in fut6 and fut4/fut6 mutant plants as indicated by eel lectin staining. Monosaccharide analysis showed fucose was present in wild-type leaf and root AGPs, but absent in fut4 leaf AGPs and in fut4/fut6 double mutant leaf and root AGPs, indicating that FUT4 was required for fucosylation of leaf AGPs while both FUT4 and FUT6 contributed to fucosylation of root AGPs. Glycome profiling of cell wall fractions from mutant roots and leaves showed distinct glycome profiles compared to wild-type plants, indicating that fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and other wall components. The current work exemplifies the possibilities of refinement of cell wall structures by manipulation of a single or a few cell wall biosynthetic genes.

New insight into the structure of RNA in red clover necrotic mosaic virus and the role of divalent cations revealed by small-angle neutron scattering.

Red clover necrotic mosaic virus (RCNMV) is a 36-nm-diameter, T = 3 icosahedral plant virus with a genome that is split between two single-stranded RNA molecules of approximately 3.9 kb and 1.5 kb, as well as a 400-nucleotide degradation product. The structure of the virus capsid and its response to removing Ca(2+) and Mg(2+) was previously studied by cryo-electron microscopy (cryo-EM) (Sherman et al. J Virol 80:10395-10406, 2006) but the structure of the RNA was only partially resolved in that study. To better understand the organization of the RNA and conformational changes resulting from the removal of divalent cations, small-angle neutron scattering with contrast variation experiments were performed. The results expand upon the cryo-EM results by clearly showing that virtually all of the RNA is contained in a thin shell that is in contact with the interior domains of the viral capsid protein, and they provide new insight into changes in the RNA packing that result from removal of divalent cations.

Twenty-one genome sequences from Pseudomonas species and 19 genome sequences from diverse bacteria isolated from the rhizosphere and endosphere of Populus deltoides.

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.

Draft genome sequence of the thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1.

The genes and molecular machines that allow for a thermoalkaliphilic lifestyle have not been defined. To address this goal, we report on the improved high-quality draft genome sequence of Caldalkalibacillus thermarum strain TA2.A1, an obligately aerobic bacterium that grows optimally at pH 9.5 and 65 to 70°C on a wide variety of carbon and energy sources.

Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus.

Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Šresolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Šin the analysis presented here.

Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae.

The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na(+)) tolerance and not acetate (Ac(-)) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na(+) and Ac(-) ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.

Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations.

BACKGROUND: Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly. RESULTS: In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point. Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation. CONCLUSION: High oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.

Involvement of Shewanella oneidensis MR-1 LuxS in biofilm development and sulfur metabolism.

The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. These results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.

BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea.

Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization.

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea.

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.