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We report the complete genome sequences of five Variovorax strains isolated from the roots of Populus species in greenhouse and field settings. Genome sizes range from 7.44 to 7.71 Mbp and encode 6,077–6,879 predicted protein-coding genes. These genome assemblies expand the known genomic catalog of Variovorax in plant-associated environments.
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There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in genetically obese, diet-induced obese, and lean mice. Samples of feces, urine, colon tissue, and plasma were analyzed using targeted LC-MS/MS quantification of metabolites, immunoassays of inflammatory cytokine levels, genome-resolved metagenomics, and metaproteomics. Genetic obesity, diet-induced obesity, and morphine treatment in lean mice each showed increases in distinct inflammatory cytokines. Metagenomic assembly and binning uncovered over 400 novel gut bacterial genomes and species. Morphine administration impacted the microbiome’s composition and function, with the strongest effect observed in lean mice. This microbiome effect was less pronounced than either diet or genetically driven obesity. Based on inferred microbial physiology from the metaproteome datasets, a high-fat diet transitioned constituent microbes away from harvesting diet-derived nutrients and towards nutrients present in the host mucosal layer. Considered together, these results identified novel host-dependent phenotypes, differentiated the effects of genetic obesity versus diet induced obesity on gut microbiome composition and function, and showed that chronic morphine administration altered the gut microbiome.
ABSTRACT Horizontal genetic transfer (HGT) is a significant driver of genomic novelty in all domains of life. HGT has been investigated in many studies however, the focus has been on conspicuous protein‐coding DNA transfers that often prove to be adaptive in recipient organisms and are therefore fixed longer‐term in lineages. These results comprise a subclass of HGTs and do not represent exhaustive (coding and non‐coding) DNA transfer and its impact on ecology. Uncovering exhaustive HGT can provide key insights into the connectivity of genomes in communities and how these transfers may occur. In this study, we use the term frequency‐inverse document frequency (TF‐IDF) technique, that has been used successfully to mine DNA transfers within real and simulated high‐quality prokaryote genomes, to search for exhaustive HGTs within an extremophilic microbial community. We establish a pipeline for validating transfers identified using this approach. We find that most DNA transfers are within‐domain and involve non‐coding DNA. A relatively high proportion of the predicted protein‐coding HGTs appear to encode transposase activity, restriction‐modification system components, and biofilm formation functions. Our study demonstrates the utility of the TF‐IDF approach for HGT detection and provides insights into the mechanisms of recent DNA transfer.
As humanity moves from earth to the moon and beyond, there are many challenges, both known and unknown, which will complicate our path. One unknown variable is how microorganisms will be affected by the space environment, and how this may impact mission success. There is a serious need for greater research to be conducted on how space environments alter microbial organisms genetically and physiologically, and to understand the threats that potential microbial changes may present to future missions.
Experimental Data for Comparison of microbial strains as candidate hosts and genetic reservoirs for the valorization of lignin streams
Comparison of microbial strains for tolerance to and catabolism of lignin stream constituents toward evaluating microbial hosts for lignin bioconversion.
This report describes the results of work conducted at NASA Ames Research Center, in collaboration with investigators at Lawrence Livermore National Laboratory, during the time period 12/15/2014 to 12/15/2017. The work was an extension of a collaboration with LLNL that began in 2008. Work this reporting period focused primarily on tasks related to microbial mats from both the Elkhorn Slough and Baja California field sites, including the analysis and publication of data produced prior to 12/15/2014. During this reporting period, in addition to the microbial mat work, we also conducted a series of experiments with a particular algal-bacterial association (C. sorokiniana and A. brasilense). A number of aspects of this well-characterized algal-bacterial association enabled us not only to further our understanding of interactions between these particular organisms, but to apply that knowledge and techniques to our collection of microorganisms isolated from microbial mats. Microbial mat experiments focused both on intact mat communities and on cultures of organisms isolated from the mats (the Microbial Mat Living Library). In a series of co-culture experiments, we documented both positive and negative interactions amongst heterotrophic and autotrophic isolates of microbial mat microorganisms. One particularly important and novel mat cyanobacterium (ESFC-1) was studied in detail. We developed techniques to pursue an investigation of functional redundancy (the idea that closely related organisms perform identical roles in a community) in cyanobacterial autotrophs in the Elkhorn Slough microbial mat. In those experiments, we documented numerous instances of increases in growth rate between co-cultures of two organisms (relative to their growth rate in isolation) performing what would seem to be identical functional roles. Simplified microbial mat communities consisting of organisms from the Microbial Mat Living Library were constructed and studied in laboratory experiments. The “constructed mats” were grown in standard laboratory glassware and plasticware, but cultures were also combined with artificial materials in an effort to re-create the three-dimensional structure of naturally occurring microbial mats. These constructed mats re-created a number of functions attributable to natural microbial mats (N-fixation, H2-production) and simplified their study using molecular ecological techniques due to the smaller number of microbial “species” present. Cyanobacteria which migrated in response to vertical variations in light intensity (irradiance) obtained characteristic positions in constructed mats that were similar to their positions in natural mats. A detailed investigation of nitrogen cycling in both the Baja California and Elkhorn Slough microbial mats was initiated during this reporting period. The nitrogen cycle in the microbial mats has not been studied in depth, despite the fact that nitrogen usually limits growth in marine environments. The organisms conducting nitrogen cycling in both microbial mats were studied using the genomes available from earlier work on these mats, as well as with a targeted approach using molecular ecological methods developed here. Quantitative measurements of rates of nitrogen transformations were made using stable isotopically labeled nitrogen. The processes quantified and examined using molecular ecological methods included: nitrogen fixation, ammonification, ammonium and nitrate assimilation, denitrification, anaerobic oxidation of ammonium, and dissimilatory nitrate reduction to ammonium. In a series of experiments designed to examine a potential application for microbial mats, we demonstrated that both Elkhorn Slough and Baja California microbial mats were very effective in removing large quantities of both nitrate and ammonium from waste streams containing these compounds at levels where they would be considered to be pollutants. Considerable effort was expended during this reporting period to analyze, and prepare for publication, metagenomes and metatranscriptomes collected from two experimental manipulations of microbial mats. The first, a diel study, was conducted in November 2011, while the second, an investigation of the effects of sulfate on community composition, was conducted during this reporting period. For the earlier study, reference-based and reference-free methods were used to assess microbial ecology and genetic partitioning in these complex microbial systems, and that work is available as a pre-print. Four metagenomes from the same experiment were published. In the second, longer (nearly 200 day) manipulation, rRNA iTag libraries for cDNA and DNA were analyzed to determine the effects of sulfate concentration on microbial mat community composition over the course of the experiment. Work on the algal-bacterial association (Chlorella sorokiniana and Azospirillum brasilense) focused initially on demonstrating the transfer of carbon and nitrogen between the partners, using stable isotopic labeling and the LLNL NanoSIMS. Subsequently, the potential role of diazotrophy (nitrogen fixation) by the bacterial partner in supporting the growth of the association was assessed in a series of experiments. It appears to be the case that, although the bacterial partner demonstrably increases the fitness of the algae under nitrogen limitation, and cells of C. sorokiniana receive nitrogen originally fixed from atmospheric nitrogen by A. brasilense, the amount of nitrogen obtained by diazotrophy is too small to explain the increase in fitness.
Small molecules encoded by biosynthetic pathways mediate cross-species interactions and harbor untapped potential, which has provided valuable compounds for medicine and biotechnology. Since studying biosynthetic gene clusters in their native context is often difficult, alternative efforts rely on heterologous expression, which is limited by host-specific metabolic capacity and regulation. Here, in this work, we describe a computational-experimental technology to redesign genes and their regulatory regions with hybrid elements for cross-species expression in Gram-negative and -positive bacteria and eukaryotes, decoupling biosynthetic capacity from host-range constraints to activate silenced pathways. These synthetic genetic elements enabled the discovery of a class of microbiome-derived nucleotide metabolites—tyrocitabines—from Lactobacillus iners. Tyrocitabines feature a remarkable orthoester-phosphate, inhibit translational activity, and invoke unexpected biosynthetic machinery, including a class of “Amadori synthases” and “abortive” tRNA synthetases. Our approach establishes a general strategy for the redesign, expression, mobilization, and characterization of genetic elements in diverse organisms and communities.
Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. So, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.
Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature. Furthermore, our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.
Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.
Microorganisms are essential to all life on Earth through critical roles in key biological processes and diverse interactions with other organisms that shape ecosystems, drive biogeochemical cycles and influence both human health and environmental health. High-throughput sequencing from environmental samples has revolutionized the understanding of microbial diversity and functions. With vast amounts of genomes now available across Earth’s biomes, these data provide a blueprint of microbial life that can be harnessed for a more holistic understanding of microbiome structure and function across the various ecosystems on Earth. Here we review the application of genome-centric approaches, including recent advances in single-cell sequencing and functional profiling, to survey microbial and viral diversity. Furthermore, we highlight some of the most impactful evolutionary and functional discoveries, explore the spatial diversity and temporal dynamics of microorganisms across diverse environments, and discuss genome-enabled insights into host-associated microorganisms.
Magnetotactic bacteria (MTB) are prokaryotes that possess genes for the synthesis of membrane-bounded crystals of magnetite or greigite, called magnetosomes. Despite over half a century of studying MTB, only about 60 genomes have been sequenced. Most belong to Proteobacteria, with a minority affiliated with the Nitrospirae, Omnitrophica, Planctomycetes, and Latescibacteria. Due to the scanty information available regarding MTB phylogenetic diversity, little is known about their ecology, evolution and about the magnetosome biomineralization process. This study presents a large-scale search of magnetosome biomineralization genes and reveals 38 new MTB genomes. Several of these genomes were detected in the phyla Elusimicrobia, Candidatus Hydrogenedentes, and Nitrospinae, where magnetotactic representatives have not previously been reported. Analysis of the obtained putative magnetosome biomineralization genes revealed a monophyletic origin capable of putative greigite magnetosome synthesis. The ecological distributions of the reconstructed MTB genomes were also analyzed and several patterns were identified. These data suggest that open databases are an excellent source for obtaining new information of interest.
Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans , which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2 , respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1 . In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.
Plant-microbe symbioses span a continuum from pathogenic to mutualistic with functional consequences for both organisms in the symbiosis. In order to increase sustainable food and fuel production in the future, it is imperative that we harness these symbioses. The tree genus Populus is an excellent model system for studies examining plant-microbe interactions due to the wealth of genomic information available and the molecular tools that have been developed to manipulate Populus-microbe symbioses. In this review, we highlight how Populus can serve as a model system to explore plant-microbe interactions. Specifically, highlighting research linking Populus-microbe interactions from the gene to the ecosystem level. We explore why Populus is an excellent model for perennial plant systems, the molecular underpinnings of Populus-microbe interactions, how host genetics influence microbial community composition, and how microbial communities vary at fine spatial scales and between Populus species. Further, we explore how the patterns of the microbiome may affect ecosystem level functions in managed and natural ecosystems. Understanding and manipulating these interactions in Populus has the potential to improve plant health and impact ecosystem sustainability and processes as Populus trees function as foundational species in many natural ecosystems and are also deployed in managed ecosystems for various agroforestry applications.