ABSTRACT
Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.
Subject(s)
Fungi/metabolism , Lignin/metabolism , Metabolic Networks and Pathways , Biopolymers/metabolism , Biotransformation , Ecosystem , Organic Chemicals/metabolism , Soil MicrobiologyABSTRACT
Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.
Subject(s)
Cellulosomes , Cellulosomes/genetics , Cellulosomes/chemistry , Cellulosomes/metabolism , Escherichia coli/metabolism , Anaerobiosis , Bacterial Proteins/chemistry , Spores/metabolism , Adenosine Triphosphate/metabolism , FungiABSTRACT
Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.
Subject(s)
RNA, Fungal/genetics , RNA, Guide, Kinetoplastida/genetics , Saccharomyces cerevisiae/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Expression Regulation, Fungal , Gene Library , Plasmids/genetics , Plasmids/metabolism , Promoter Regions, Genetic , RNA, Fungal/metabolism , RNA, Guide, Kinetoplastida/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.
Subject(s)
Cell Membrane/metabolism , Cellulosomes/metabolism , Kluyveromyces/genetics , Kluyveromyces/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cellulase/genetics , Cellulase/metabolism , Cellulose/metabolism , Cellulosomes/enzymology , Cellulosomes/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/genetics , Clostridium thermocellum/genetics , Ethanol/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Kluyveromyces/enzymology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , beta-Glucosidase/genetics , beta-Glucosidase/metabolism , CohesinsABSTRACT
The oxygen transport function of hemoglobin (HB) is thought to have arisen â¼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.
Subject(s)
Erythrocytes/metabolism , Evolution, Molecular , Fish Proteins , Gene Expression Regulation/physiology , Hemoglobins , Lampreys , Animals , Fish Proteins/biosynthesis , Fish Proteins/genetics , Hemoglobins/biosynthesis , Hemoglobins/genetics , Lampreys/genetics , Lampreys/metabolism , Multigene FamilyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Evolution, Molecular , Galactose/metabolism , Phosphoglucomutase/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/genetics , Transcription Factors/metabolism , Phosphoglucomutase/metabolism , Saccharomycetales/metabolismABSTRACT
Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Gene Library , Genes, Fungal , Yarrowia/geneticsABSTRACT
BACKGROUND: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. RESULTS: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. CONCLUSIONS: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.
Subject(s)
Metabolic Engineering , Promoter Regions, Genetic , Rhodotorula/genetics , Base Sequence , Rhodotorula/metabolism , Transformation, GeneticABSTRACT
Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.
Subject(s)
Archaea/classification , Archaea/genetics , Bacteria/classification , Bacteria/genetics , Metagenomics , Phylogeny , Archaea/isolation & purification , Archaea/metabolism , Bacteria/isolation & purification , Bacteria/metabolism , Ecosystem , Genome, Archaeal/genetics , Genome, Bacterial/genetics , Metagenome/genetics , Molecular Sequence Data , Sequence Analysis, DNA , Single-Cell AnalysisABSTRACT
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.
Subject(s)
Adaptation, Physiological/genetics , Genes, Plant , Genetic Variation , Panicum/anatomy & histology , Panicum/genetics , Base Sequence , Biomass , Chromosome Mapping , Gene Flow , Genetics, Population , Mutation/genetics , Panicum/physiology , Phylogeography , Polymorphism, Single Nucleotide/genetics , Principal Component Analysis , United StatesABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Multigene Family , Phenazines/metabolism , Biosynthetic Pathways/genetics , Escherichia coli/genetics , Escherichia coli/metabolismABSTRACT
BACKGROUND: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composition and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for large initial DNA template quantities during library preparation. RESULTS: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. CONCLUSIONS: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.
Subject(s)
Metagenome , Metagenomics , Microbiota , Archaea/genetics , Bacteria/genetics , Base Composition , Biomass , Contig Mapping , Gene Library , Metagenomics/methods , Sequence Analysis, DNAABSTRACT
Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic 'phylogenomic' efforts to compile a phylogeny-driven 'Genomic Encyclopedia of Bacteria and Archaea' in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.
Subject(s)
Archaea/classification , Archaea/genetics , Bacteria/classification , Bacteria/genetics , Genome, Archaeal/genetics , Genome, Bacterial/genetics , Phylogeny , Actins/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Biodiversity , Databases, Genetic , Genes, rRNA/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/ß and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.
Subject(s)
Fishes/genetics , Fishes/immunology , Immune System , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Animals , Exons , Genes, Immunoglobulin/genetics , Genes, Immunoglobulin/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/immunology , Phylogeny , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , SyntenyABSTRACT
Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize gamma-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, approximately 1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation-combined loop assays showed that Mecp2 mediated the silent chromatin-derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin-associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.
Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Genomic Imprinting , Homeodomain Proteins/genetics , Repressor Proteins/genetics , Rett Syndrome/genetics , Animals , Chromatin/genetics , CpG Islands/physiology , DNA Methylation , Gene Expression Profiling , Homeodomain Proteins/metabolism , Humans , Methyl-CpG-Binding Protein 2 , Mice , Multigene Family , Neurons/metabolism , Precipitin Tests , Transcription Factors , gamma-Aminobutyric Acid/metabolismABSTRACT
The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a ß-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as ß-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing ß-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.
ABSTRACT
Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.
Subject(s)
Hemeproteins , Synechocystis , Heme , Zinc , Histidine , Hemeproteins/genetics , Synechocystis/genetics , Carbon , IronABSTRACT
High-throughput genetic screening is frequently employed to rapidly associate gene with phenotype and establish sequence-function relationships. With the advent of CRISPR technology, and the ability to functionally interrogate previously genetically recalcitrant organisms, non-model organisms can be investigated using pooled guide RNA (gRNA) libraries and sequencing-based assays to quantitatively assess fitness of every targeted locus in parallel. To aid the construction of pooled gRNA assemblies, we have developed an in silico design workflow for gRNA selection using the gRNA Sequence Region Extraction Tool (gRNA-SeqRET). Built upon the previously developed CCTop, gRNA-SeqRET enables automated, scalable design of gRNA libraries that target user-specified regions or whole genomes of any prokaryote or eukaryote. Additionally, gRNA-SeqRET automates the bulk extraction of any regions of sequence relative to genes or other features, aiding in the design of homology arms for insertion or deletion constructs. We also assess in silico the application of a designed gRNA library to other closely related genomes and demonstrate that for very closely related organisms Average Nucleotide Identity (ANI) > 95% a large fraction of the library may be of relevance. The gRNA-SeqRET web application pipeline can be accessed at https://grna.jgi.doe.gov. The source code is comprised of freely available software tools and customized Python scripts, and is available at https://bitbucket.org/berkeleylab/grnadesigner/src/master/ under a modified BSD open-source license (https://bitbucket.org/berkeleylab/grnadesigner).
ABSTRACT
CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.
Subject(s)
Archaea , Symbiosis , Archaea/genetics , Archaea/metabolism , Symbiosis/genetics , Genomics , Plasmids , DNA/metabolismABSTRACT
BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.