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Amplification-based gene characterization allows for sensitive and specific quantification of functional genes. There is often a large diversity of genes represented for functional gene groups, and multiple primers may be necessary to target associated genes.
Abstract Viruses impact microbial systems through killing hosts, horizontal gene transfer, and altering cellular metabolism, consequently impacting nutrient cycles. A virus-infected cell, a “virocell,” is distinct from its uninfected sister cell as the virus commandeers cellular machinery to produce viruses rather than replicate cells. Problematically, virocell responses to the nutrient-limited conditions that abound in nature are poorly understood. Here we used a systems biology approach to investigate virocell metabolic reprogramming under nutrient limitation. Using transcriptomics, proteomics, lipidomics, and endo- and exo-metabolomics, we assessed how low phosphate (low-P) conditions impacted virocells of a marine Pseudoalteromonas host when independently infected by two unrelated phages (HP1 and HS2). With the combined stresses of infection and nutrient limitation, a set of nested responses were observed. First, low-P imposed common cellular responses on all cells (virocells and uninfected cells), including activating the canonical P-stress response, and decreasing transcription, translation, and extracellular organic matter consumption. Second, low-P imposed infection-specific responses (for both virocells), including enhancing nitrogen assimilation and fatty acid degradation, and decreasing extracellular lipid relative abundance. Third, low-P suggested virocell-specific strategies. Specifically, HS2-virocells regulated gene expression by increasing transcription and ribosomal protein production, whereas HP1-virocells accumulated host proteins, decreased extracellular peptide relative abundance, and invested in broader energy and resource acquisition. These results suggest that although environmental conditions shape metabolism in common ways regardless of infection, virocell-specific strategies exist to support viral replication during nutrient limitation, and a framework now exists for identifying metabolic strategies of nutrient-limited virocells in nature.
In recent years, methods for estimating atmospheric latent heating vertical structure from both passive and active microwave remote sensing have matured to the point where quantitative evaluation of these methods is the next logical step. Two approaches for heating algorithm evaluation are proposed: First, application of heating algorithms to synthetic data, based upon cloud-resolving model simulations, can be used to test the internal consistency of heating estimates in the absence of systematic errors in physical assumptions. Second, comparisons of satellite-retrieved vertical heating structures to independent ground-based estimates, such as rawinsonde-derived analyses of heating, provide an additional test. The two approaches are complementary, since systematic errors in heating indicated by the second approach may be confirmed by the first. A passive microwave and combined passive/active microwave heating retrieval algorithm are evaluated using the described approaches. In general, the passive microwave algorithm heating profile estimates are subject to biases due to the limited vertical heating structure information contained in the passive microwave observations. These biases may be partly overcome by including more environment-specific a priori information into the algorithm s database of candidate solution profiles. The combined passive/active microwave algorithm utilizes the much higher-resolution vertical structure information provided by spaceborne radar data to produce less biased estimates; however, the global spatio-temporal sampling by spaceborne radar is limited. In the present study, the passive/active microwave algorithm is used to construct a more physically-consistent and environment-specific set of candidate solution profiles for the passive microwave algorithm and to help evaluate errors in the passive algorithm s heating estimates. Although satellite estimates of latent heating are based upon instantaneous, footprint- scale data, suppression of random errors requires averaging to at least half-degree resolution. Analysis of mesoscale and larger space-time scale phenomena based upon passive and passive/active microwave heating estimates from TRMM, SSMI, and AMSR data will be presented at the conference.
The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%–50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.
Dissolved organic matter (DOM) is an important component of Earth's carbon cycle and one of the planet's most chemically diverse pools, yet the molecular structures of its constituents remain largely unresolved. This limitation has hindered our ability to link DOM composition to microbial processes and ecosystem function. Here we present ENVnet, a global molecular repository built from tandem mass spectrometry data collected across 13 terrestrial and aquatic environment types, including 419 newly generated samples that expand publicly available DOM metabolomics data and cover previously underrepresented environments. By computationally deconvolving chimeric mass spectra, a longstanding challenge in environmental metabolomics, we recover high-quality fragmentation data for >22,000 distinct molecular features (defined by a specific precursor mass and fragmentation pattern). Using ENVnet, we uncover conserved and environment-specific molecular patterns in DOM composition and underlying biogeochemical processes. We also use molecular features encoded in ENVnet to train predictive models of DOM persistence, allowing molecular-level assessment of microbial turnover in independent systems.
Uptake of uranium (U) by secondary minerals, such as carbonates and iron (Fe)-sulfides, that occur ubiquitously on Earth, may be substantial in deep anoxic environments compared to surficial settings due to different environment-specific conditions. Yet, knowledge of U reductive removal pathways and related fractionation between 238 U and 235 U isotopes in deep anoxic groundwater systems remain elusive. Here we show bacteria-driven degradation of organic constituents that influences formation of sulfidic species facilitating reduction of geochemically mobile U(VI) with subsequent trapping of U(IV) by calcite and Fe-sulfides. The isotopic signatures recorded for U and Ca in fracture water and calcite samples provide additional insights on U(VI) reduction behaviour and calcite growth rate. The removal efficiency of U from groundwater reaching 75% in borehole sections in fractured granite, and selective U accumulation in secondary minerals in exceedingly U-deficient groundwater shows the potential of these widespread mineralogical sinks for U in deep anoxic environments.
Engineered microbes are being programmed using synthetic DNA for applications in soil to overcome global challenges related to climate change, energy, food security, and pollution. However, we cannot yet predict gene transfer processes in soil to assess the frequency of unintentional transfer of engineered DNA to environmental microbes when applying synthetic biology technologies at scale. This challenge exists because of the complex and heterogeneous characteristics of soils, which contribute to the fitness and transport of cells and the exchange of genetic material within communities. Here, we describe knowledge gaps about gene transfer across soil microbiomes. Here, we propose strategies to improve our understanding of gene transfer across soil communities, highlight the need to benchmark the performance of biocontainment measures in situ, and discuss responsibly engaging community stakeholders. We highlight opportunities to address knowledge gaps, such as creating a set of soil standards for studying gene transfer across diverse soil types and measuring gene transfer host range across microbiomes using emerging technologies. By comparing gene transfer rates, host range, and persistence of engineered microbes across different soils, we posit that community-scale, environment-specific models can be built that anticipate biotechnology risks. Such studies will enable the design of safer biotechnologies that allow us to realize the benefits of synthetic biology and mitigate risks associated with the release of such technologies.
Genes belonging to the same functional group may include numerous and variable gene sequences, making characterizing and quantifying difficult. Therefore, high-throughput design tools are needed to simultaneously create primers for improved quantification of target genes. We developed MetaFunPrimer, a bioinformatic pipeline, to design primers for numerous genes of interest. This tool also enables gene target prioritization based on ranking the presence of genes in user-defined references, such as environment-specific metagenomes. Given inputs of protein and nucleotide sequences for gene targets of interest and an accompanying set of reference metagenomes or genomes, MetaFunPrimer generates primers for ranked genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 publicly available soil metagenomes. We demonstrate computationally that these amoA-AOB primers can cover 94% of the amoA-AOB genes observed in the 1,550 soil metagenomes compared with a 49% estimated coverage by previously published primers. Finally, we verified the utility of these primer sets in incubation experiments that used long-term nitrogen fertilized or unfertilized soils. High-throughput quantitative PCR (qPCR) results and statistical analyses showed significant differences in relative quantification patterns between the two soils, and subsequent absolute quantifications also confirmed that target genes enumerated by six selected primer pairs were significantly more abundant in the nitrogen-fertilized soils. This new tool gives microbial ecologists a new approach to assess functional gene abundance and related microbial community dynamics quickly and affordably.
With adaptive problem solving, we take a biologically-inspired approach to both stochastic optimization and search in order to enable a spacecraft to adapt its environment-specific bevahior in-situ.
The terrestrial gravitational field serves as an important orientation reference for human perception and movement, being continually monitored by sensory receptors in the skin, muscles, joints, and vestibular otolith organs. Cues from these graviceptors are used by the brain to estimate spatial orientation and to control balance and movement. Changes in these cues associated with the tonic changes in gravity (gravito-inertial force),during the launch and entry phases of space flight missions result in altered perceptions, degraded motor control performance, and in some cases, "motion" sickness during, and for a period of time after, the g-transitions. In response to these transitions, however, physiological and behavioral response mechanisms are triggered to compensate for altered graviceptor cues and/or to adapt to the new sensory environment. Basic research in the neurophysiology discipline is focused on understanding the characteristic features of and the underlying mechanisms for the normal human response to tonic changes in the gravito-inertial force environment. These studies address fundamental questions regarding the role of graviceptors in orientation and movement in the terrestrial environment, as well as the capacity, specificity, and modes for neural plasticity in the sensory-motor and perceptual systems of the brain. At the 2001 workshop basic research studies were presented addressing: neuroanatomical responses to altered gravity environments, the neural mechanisms for resolving the ambiguity between tilting and translational stimuli in otolith organ sensory input, interactions between the vestibular system and the autonomic nervous system , the roles of haptic and visual cues in spatial orientation, mechanisms for training environment-appropriate sensorimotor responses triggered by environment-specific context cues, and studies of sensori-motor control of posture and locomotion in the terrestrial environment with and without recent exposure to space flight. Building on these basic research studies are more applied studies focused on the development of countermeasures to the untoward neurophysiological responses to space flight. At the 2001 workshop, applied research studies were presented addressing issues related to the use of rotational artificial gravity (centripetal acceleration) as a multisystem (bone, muscle, cardiovascular, and, perhaps, neurovestibular) countermeasure. Also presented was a clinical study reporting on a new rating system for clinical evaluation of postflight functional neurological status.
Many conceptual studies for long duration missions beyond Earth orbit have assumed unique habitat designs for each destination and for transit habitation. This may not be the most effective approach. A variable gravity habitat, one designed for use in microgravity, lunar, Martian, and terrestrial environments may provide savings that offset the loss of environment-specific optimization. However, a brief analysis of selected flown spacecraft and Constellation-era conceptual habitat designs suggests that one cannot simply lift a habitat from one environment and place it in another that it was not designed for without incurring significant human performance compromises. By comparison, a conceptual habitat based on the Skylab II framework but designed specifically to accommodate variable gravity environments can be shown to yield significant advantages while incurring only minimal human performance compromises.
The primary goal of the Consortium for the Advancement of Shape Memory Alloy Research and Technology (CASMART) is to enable the design of revolutionary applications based on shape memory alloy (SMA) technology. In order to help realize this goal and reduce the development time and required experience for the fabrication of SMA actuation systems, several modeling tools have been developed for common actuator types and are discussed herein along with case studies, which highlight the capabilities and limitations of these tools. Due to their ability to sustain high stresses and recover large deformations, SMAs have many potential applications as reliable, lightweight, solid-state actuators. Their advantage over classical actuators can also be further improved when the actuator geometry is modified to fit the specific application. In this paper, three common actuator designs are studied: wires, which are lightweight, low-profile, and easily implemented; springs, which offer actuation strokes upwards of 200 at reduced mechanical loads; and torque tubes, which can provide large actuation forces in small volumes and develop a repeatable zero-load actuation response (known as the two-way shape memory effect). The modeling frameworks, which have been implemented in the design tools, are developed for each of these frequently used SMA actuator types. In order to demonstrate the versatility and flexibility of the presented design tools, as well as validate their modeling framework, several design challenges were completed. These case studies include the design and development of an active hinge for the deployment of a solar array or foldable space structure, an adaptive solar array deployment and positioning system, a passive air temperature controller for regulation flow temperatures inside of a jet engine, and a redesign of the Corvette active hatch, which allows for pressure equalization of the car interior. For each of the presented case studies, a prototype or proof-of-concept was fabricated and the experimental results and lessons learned are discussed. This analysis presents a collection of CASMART collaborative best practices in order to allow readers to utilize the available design tools and understand their modeling principles. These design tools, which are based on engineering models, can provide first-order optimal designs and are a basic and efficient method for either demonstrating design feasibility or refining design parameters. Although the design and integration of an SMA-based actuation system always requires application- and environment-specific engineering considerations, common modeling tools can significantly reduce the investment required for actuation system development and provide valuable engineering insight.