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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

KBase Narrative - Supplemental Data for "Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes"

Here we provide the complete set of circular elements produced by SCAPP from metagenomic data that may represent mobile genetic elements (MGEs). For the publication, these sequences were de-replicated within the high [U] and low [U] sample sets. Additionally, 98 of the circular elements were removed for downstream analysis due to high suspicion of being (1) chloroplast or mitochondrial sequences or (2) erroneously circularized long repeat regions. The complete list of circular elements used for the analysis along with metadata can be found in Tables S5 and S6.

Goff, Jennifer↗
DOE OSTI2020

Single-Cell Genomics of Novel Actinobacteria With the Wood–Ljungdahl Pathway Discovered in a Serpentinizing System

Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (~50°C; pH~10.5–11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprises Nitrospirae (47%), “Parcubacteria” (19%), Deinococcus-Thermus (16%), and Actinobacteria (9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98–1.00; estimated completeness: 33–93%; estimated genome size: ~2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO 2 fixation via the Wood–Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for the Actinobacteria phylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H + /Na + pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within the Actinobacteria phylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group “OPB41.” We propose to name this bacterium ‘Candidatus Hakubanella thermoalkaliphilus.'

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Portable Rabies Virus Sequencing in Canine Rabies Endemic Countries Using the Oxford Nanopore MinION

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION’s potential for low-cost, high volume sequencing of pathogens in locations with limited resources.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1990

Kalman filter based range estimation for autonomous navigation using imaging sensors

Rotorcraft operating in high-threat environments fly close to the surface of the earth to utilize surrounding terrain, vegetation, or man-made objects to minimize the risk of being detected by the enemy. Two basic requirements for obstacle avoidance are detection and range estimation of the object from the current rotorcraft position. There are many approaches to the estimation of range using a sequence of images. The approach used in this analysis differes from previous methods in two significant ways: an attempt is not made to estimate the rotorcraft's motion from the images; and the interest lies in recursive algorithms. The rotorcraft parameters are assumed to be computed using an onboard inertial navigation system. Given a sequence of images, using image-object differential equations, a Kalman filter (Sridhar and Phatak, 1988) can be used to estimate both the relative coordinates and the earth coordinates of the objects on the ground. The Kalman filter can also be used in a predictive mode to track features in the images, leading to a significant reduction of search effort in the feature extraction step of the algorithm. The purpose is to summarize early results obtained in extending the Kalman filter for use with actual image sequences. The experience gained from the application of this algorithm to real images is very valuable and is a necessary step before proceeding to the estimation of range during low-altitude curvilinear flight. A simple recursive method is presented to estimate range to objects using a sequence of images. The method produces good range estimates using real images in a laboratory set up and needs to be evaluated further using several different image sequences to test its robustness. The feature generation part of the algorithm requires further refinement on the strategies to limit the number of features (Sridhar and Phatak, 1989). The extension of the work reported here to curvilinear flight may require the use of the extended Kalman filter.

Sridhar, Banavar↗
DOE OSTI2023

Unraveling the functional dark matter through global metagenomics

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Phylogeny of Bacterial and Archaeal Genomes Using Conserved Genes: Supertrees and Supermatrices

Over 3000 microbial (bacterial and archaeal) genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML) tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA), as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a ‘‘primary concordance’’ tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy) tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Comparative Analysis of 35 Basidiomycete Genomes Reveals Diversity and Uniqueness of the Phylum

Fungi of the phylum Basidiomycota (basidiomycetes), make up some 37percent of the described fungi, and are important in forestry, agriculture, medicine, and bioenergy. This diverse phylum includes symbionts, pathogens, and saprobes including wood decaying fungi. To better understand the diversity of this phylum we compared the genomes of 35 basidiomycete fungi including 6 newly sequenced genomes. The genomes of basidiomycetes span extremes of genome size, gene number, and repeat content. A phylogenetic tree of Basidiomycota was generated using the Phyldog software, which uses all available protein sequence data to simultaneously infer gene and species trees. Analysis of core genes reveals that some 48percent of basidiomycete proteins are unique to the phylum with nearly half of those (22percent) comprising proteins found in only one organism. Phylogenetic patterns of plant biomass-degrading genes suggest a continuum rather than a sharp dichotomy between the white rot and brown rot modes of wood decay among the members of Agaricomycotina subphylum. There is a correlation of the profile of certain gene families to nutritional mode in Agaricomycotina. Based on phylogenetically-informed PCA analysis of such profiles, we predict that that Botryobasidium botryosum and Jaapia argillacea have properties similar to white rot species, although neither has liginolytic class II fungal peroxidases. Furthermore, we find that both fungi exhibit wood decay with white rot-like characteristics in growth assays. Analysis of the rate of discovery of proteins with no or few homologs suggests the high value of continued sequencing of basidiomycete fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

germs-lab/LAMPS-miscanthus-microbiome

Data and analysis to accompany our submitted paper characterizing the LAMPS Miscanthus Microbiome comparing 16S rRNA gene DNA sequencing of Miscanthus in a staggered-start experiment. Authors for this analysis include Fernando Igne Rocha, Lanying Ma, and Adina Howe.

Igne Rocha, Fernando↗
NASA NTRS1992

Search for evidence of a clock related to the solar 154 day complex of periodicities

Evidence that has recently been compiled (Bai and Sturrock 1991) indicates that the enigmatic 154-day periodicity in solar activity may be viewed as part of a complex of periodicities that are approximate multiples of 25.8 days, suggesting that the Sun contains a 'clock' with frequency in the range 440 to 463 nano Hz. The clock may comprise either an oscillator or a rotator, each of which may be either real or virtual. We have reconsidered a previous spectrum analysis of the Zurich sunspot-number sequence by Knight, Schatten, and Sturrock (1979) which revealed a sharp, persistent and significant periodicity with a period of 12.072 days, corresponding to a frequency of about 958.8 nano Hz. This periodicity may be regarded as the (second) upper sideband of the second harmonic (2nu(sub R) + 2nu(sub E)) of a fundamental frequency of 447.7 nano Hz that is clearly within the search band. In this expression, nu(sub R) is the sidereal frequency of the hypothetical rotator and nu(sub E) is the frequency (31.69 nano Hz) of the Earth in its orbital motion around the Sun. In analyzing sunspot area data derived from the Greenwich data set, and on noting that any frequency is defined only to within the Nyquist frequency, we find clear evidence not only for the upper sideband of the second harmonic, but also for the second harmonic (2nu(sub R)) and the lower sideband of the second harmonic (2nu(sub R) - 2nu(sub E)). There is no strong peak at the fundamental frequency in the Greenwich data, but there is in the Zurich sunspot data. The effect of a linear oscillator is, to the lowest order in the amplitude, the same as the combined effect of two rotators of opposite polarities. A rotator that has arbitrary orientation with respect to the ecliptic may influence the outer layers of the Sun and thereby modulate the occurrence of solar activity such as sunspots. By analyzing a simple model, we find that such a rotator would influence surface activity in such a way that the spectrum of a 'signal' (such as the record of sunspots), as seen from the Earth, would contain components with frequencies that are certain integral combinations of nu(sub R) and nu(sub E). The amplitudes of the various components depend sensitively on theta, the angle between the axis of the rotator and the axis of the Earth's orbital motion. This simple model therefore offers a kinematical (but not dynamical) interpretation of the sunspot spectrum. The present analysis, while offering support of our conjectures that the Sun contains a clock that regulates the 154-day complex of periodicities, cannot distinguish between an osillator or a rotator (that might be a traveling wave), nor between a real rotator or a virtual rotator (that might be an apparent traveling wave due to the aliasing effect of an oscillator in a rotating system). Further analysis of sunspot and other data sets will be required to confirm the existence of such clock and (if it is real) to determine its physical nature.

Sturrock, P. A.↗
NASA NTRS1972

Sequence data - Magnitude and implications of some ambiguities.

A stochastic model is applied to the divergence of the horse-pig lineage from a common ansestor in terms of the alpha and beta chains of hemoglobin and fibrinopeptides. The results are compared with those based on the minimum mutation distance model of Fitch (1972). Buckwheat and cauliflower cytochrome c sequences are analyzed to demonstrate their ambiguities. A comparative analysis of evolutionary rates for various proteins of horses and pigs shows that errors of considerable magnitude are introduced by Glx and Asx ambiguities into evolutionary conclusions drawn from sequences of incompletely analyzed proteins.

Holmquist, R.↗
NASA NTRS1999

Reclassification of Methanogenium tationis and Methanogenium liminatans as Methanofollis tationis gen. nov., comb. nov. and Methanofollis liminatans comb. nov. and description of a new strain of Methanofollis liminatans

Sequencing of 16S rRNA genes and phylogenetic analysis of Methanogenium tationis DSM 2702T (OCM 43T) (T = type strain) and Methanogenium liminatans GKZPZT (= DSM 4140T) as well as other members of the family Methanomicrobiaceae revealed that both species belong to a separate line of descent within this family. In addition, a new strain of Methanogenium liminatans, strain BM1 (= DSM 10196), was isolated from a butyrate-degrading, fluidized bed reactor and characterized. Cells of both species are mesophilic, highly irregular cocci that use H2/CO2 and formate for growth and methanogenesis. In addition, Methanogenium liminatans strains GKZPZT and BM1 used 2-propanol/CO2, 2-butanol/CO2 and cyclopentanol/CO2. Both species contained diether and tetraether lipids. The polar lipids comprised amino-phosphopentanetetrol derivatives, which appear to be characteristic lipids within the family Methanomicrobiaceae. The pattern of glycolipids, phosphoglycolipids and amino-phosphoglycolipids was consistent with the assignment of these two species to a taxon within the family Methanomicrobiaceae, but also permitted them to be distinguished from other higher taxa within this family. The G+C contents of the DNA of Methanogenium tationis and Methanogenium liminatans were 54 and 60 mol% (Tm and HPLC), respectively. On the basis of the data presented, the transfer of Methanogenium tationis and Methanogenium liminatans to the genus Methanofollis gen. nov. as Methanofollis tationis comb. nov. and Methanofollis liminatans comb. nov., respectively, is proposed, with Methanofollis tationis as the type species.

NASA Discipline Exobiology↗
DOE OSTI2022

RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery

Accurate genome annotations are essential in modern biology and biotechnology, yet they are still largely based on genome sequencing and comparative analyses. We show that the Clostridium acetobutylicum genome annotation can be markedly improved by integrating bioinformatic predictions with RNA sequencing (RNAseq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth conditions. Analysis of an initial assembly revealed errors due to background signals and limitations of assembly algorithms. Hurdles for RNAseq transcriptome mapping include optimizing library complexity and sequencing depth, yet most studies report low sequencing depth and ignore the effect of ribosomal RNA abundance. An integrative analysis was developed to combine motif predictions, single-nucleotide resolution sequencing depth, and library complexity to resolve difficulties in assembly curation. This minimized false positive error and determined gene boundaries, in some cases, to the exact base-pair of prior studies. This will be the first strand-specific transcriptome assembly in a Clostridium organism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Isolation and characterization of a novel mycovirus from Penicillium digitatum

A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211 bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed spherical particles ~35 nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. - Highlights: • A novel victorivirus designated Penicillium digitatum virus 1 (PdV1) was isolated fromP. digitatum. • The dsRNA genome of PdV1 are 5211 bp long, two ORFs encoding CP and RdRp, and are encased in virions of ~35 nm in diameter. • PdV1 infection led to hypovirulent effect on P. digitatum. PdV1 may potentially be used for citrus green mold biocontrol. • Our study provides a research basis for establishing a model system for the study of P. digitatum–mycovirus interactions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Riemann solutions for counterflow combustion in light porous foam

The paper is motivated by a model for the injection of air into a porous medium that contains a solid fuel. In previous work, a system of three evolutionary partial differential equations that models combustion of light porous foam under air injection was considered. The existence and uniqueness of traveling waves were studied and the wave sequences appearing in Riemann solutions were identified. This analysis was done under assumption that the combustion wave velocity is positive. In the present work, this hypothesis was neglected and the results generalized including the case of negative combustion wave speed. The existence of such waves was proved and the uniqueness investigated for some particular cases using Melnikov’s integral. The wave sequences appearing in Riemann solutions were identified and the numerical examples using finite difference scheme were presented.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2016

Genomic analysis of three Bifidobacterium species isolated from the calf gastrointestinal tract

Ruminant animals contribute significantly to the global value of agriculture and rely on a complex microbial community for efficient digestion. However, little is known of how this microbial-host relationship develops and is maintained. To begin to address this, we have determined the ability of three Bifidobacterium species isolated from the faeces of newborn calves to grow on carbohydrates typical of a newborn ruminant diet. Genome sequences have been determined for these bacteria with analysis of the genomes providing insights into the host association and identification of several genes that may mediate interactions with the ruminant gastrointestinal tract. The present study provides a starting point from which we can define the role of potential beneficial microbes in the nutrition of young ruminants and begin to influence the interactions between the microbiota and the host. The differences observed in genomic content hint at niche partitioning among the bifidobacterial species analysed and the different strategies they employ to successfully adapt to this habitat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Volatile Memory Extraction- Based Approach for Level 0-1 CPS Forensics

Most security analyzers operate on system state that is far removed from end-point components in cyber-physical systems (CPS) identified as level 0-1 in the Purdue Architecture Reference Architecture (PERA) [1]. For example, many operate on system logs and other data dumps to disks. Tremendous value that can be gained in cyber security forensics if low level details such as dynamic changes to volatile memory can be extracted and provided to more sophisticated analysis tools. However, obtaining detailed and dynamic system state at the level of volatile memory is extremely challenging [2]. Here, we attempt to apply IT memory forensic mechanisms to CPS end-point devices and statistically evaluate them. Our focus is to extract volatile and dynamically changing internal information form CPS 0-1 level devices, and design preliminary schemes to exploit that extracted information. This new capability of generating a sequence of volatile memory snapshots for offline, detailed and sophisticated analysis opens a new class of cyber security schemes for CPS forensic analysis. As a case study for our ongoing research, we apply the proposed methodology to Modicon PLC using Modbus protocol. We extract the memory layout and subject the device to read operations at the most critical regions of memory. Similarly, write operations are initiated to carefully determine memory locations (for example, bytes that hold the firmware version number). This capability of generating a sequence of volatile memory snapshots for offline, detailed and sophisticated analysis opens a new class of cyber security schemes for CPS forensic analysis. Also, the ability to dynamically make controlled modifications to specific memory locations opens the potential for new mechanisms such as taint analysis and watermarking.

Asmar Awad, Rima↗
DOE OSTI2020

Phaseolus vulgaris SUT1.1 is a high affinity sucrose–proton co–transporter

Plant sucrose transporters are required for phloem loading, and therefore are essential for plant growth and development. In common beans (Phaseolus vulgaris) there are only two sucrose transporters functionally characterized. Through a previous RNA-seq study, we identified a putative sucrose transporter in common bean, which we hypothesize to function in import of sucrose into plant cells. In silico analysis revealed that PvSUT1.1 is a putative sucrose-proton co-transporter distinct from other characterized sucrose transporters in common bean indicating that this is a previously undescribed transporter protein in beans. Further analysis revealed that PvSUT1.1 shares high protein sequence homology to the phloem loader Arabidopsis SUC2; both have 12 transmembrane domains, a typical characteristic of plant sucrose transporters. Heterologous expression in yeast further showed PvSUT1.1 to be functional and it imported sucrose into yeast cells with a K m of 0.7 mM sucrose. Import of sucrose through PvSUT1.1 is also pH-dependent with highest uptake at pH 4.0, and activity is lost in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Consistent with identification of PvSUT1.1 as a Type I transporter, PvSUT1.1 also transports esculin. Finally, PvSUT1.1 showed expression in multiple tissues and the protein was localized to the plasma membrane. The results show that PvSUT1.1 is a sucrose transporter that is probably involved in the uptake of sucrose into source and sink cells. The potential role of PvSUT1.1 in leaf phloem loading of sucrose in common beans and its importance in heat tolerance of reproductive tissues are further discussed.

59 BASIC BIOLOGICAL SCIENCES↗