21 research outputs found
Genomic and transcriptomic analysis of the anaerobic fungus Orpinomyces strain C1A, a versatile biodegrader of plant biomass
No full textThe anaerobic fungi represent a basal fungal lineage, members of which reside in the rumen and alimentary tract of herbivores. Due to their reported capacity to degrade plant materials, the anaerobic fungi have recently been touted as promising agents for biofuel production. In the first part of this thesis, I present the first reported genomic analysis of a member of the anaerobic gut fungi, Orpinomyces sp. strain C1A. The genome of strain C1A was sequenced using a combination of Illumina and PacBio SMRT technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large non-coding intergenic regions (73.1%), a proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in basal fungal lineages and/or non-fungal Opisthokonts. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments.In the second part of my thesis, I analyzed the transcriptomic profiles of C1A when grown on four different types of lignocellulosic biomass (alfalfa, energy cane, corn stover, and sorghum) versus a soluble sugar monomer (glucose). My overall goal was to understand the mechanistic and regulatory basis of biomass deconstruction in anaerobic fungi. Transcriptomic sequencing yielded a total of 468.2 million reads (70.2 GB) that were assembled into 27,506 distinct transcripts. Transcripts belonging to Carbohydrate Active Enzymes (CAZYmes) included 385, 246, and 44 transcripts belonging to 44, 13, and 8 different glycoside hydrolases (GH), carbohydrate esterases (CE), and polysaccharide lyases (PL) families, respectively. Examination of CAZyme transcriptional patterns indicates that strain C1A constitutively transcribes a high baseline level of CAZyme transcripts on glucose. Although growth on lignocellulosic biomass substrates was associated with a significant increase in transcriptional levels in few GH families, including the highly transcribed GH1 B-glucosidase, GH6 cellobiohydrolase, and GH9 endogluconase, the transcriptional levels of the majority of CAZymes families and transcripts were not significantly altered in glucose grown versus lignocelluosic biomass-grown cultures. Further, strain C1A co-transcribes multiple functionally redundant enzymes for cellulose and hemicellulose saccharification that are mechanistically and structurally distinct. Analysis of fungal dockerin domain (FDD)- containing transcripts strongly suggests that anaerobic fungal cellulosomes represent distinct catalytic units capable of independently attacking and converting intact plant fibers to sugar monomers. Collectively, these results demonstrate that strain C1A achieves fast, effective biomass degradation by the simultaneous employment of a wide array of constitutively-transcribed cellulosomal-bound and free enzymes with considerable functional overlap.The thesis hence represents the first in-depth evaluation of the genome and transcriptome of a member of this poorly studied group of fungi. Collectively, my work has revealed multiple novel insights into the metabolic capabilities, cell biology, and genomic architecture of anaerobic fungi such as the presence of unique pathways and processes not encountered in higher fungi, genomic features shaped by its unique evolutionary trajectory, extensive lignocellulolytic gene repertoire, and regulatory mechanisms employed to achieve fast and efficient biomass degradation within the herbivore gut
Genome Sequence of a Spontaneous Nonhemolytic Mutant of Mannheimia haemolytica 16041065 GH
No full textABSTRACT
We report here the draft genome sequence of a spontaneous nonhemolytic mutant of
Mannheimia haemolytica
16041065 GH. This mutant arose during routine passage and was devoid of hemolytic activity on standard blood agars. This genome sequence had a total size of 2.7 Mb with an
N
50
of 117 kb.
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Genome Sequence of Mannheimia haemolytica Serotype 1 Strain 16041065 BH
No full textABSTRACT
Here, we report the genome sequence of
Mannheimia haemolytica
serotype 1 strain 16041065 BH, which was recently isolated from a Midwestern calf that died due to
Mannheimia haemolytica
–induced pneumonia. This genome comprised a total of 2.7 Mb, with an
N
50
of 122 kb, and maintained hemolytic activity when grown on blood heart infusion agar supplemented with 5% sheep’s blood.
</jats:p
Draft genome sequence and detailed analysis of Pantoea eucrina strain Russ and implication for opportunistic pathogenesis
No full textAbstractThe genus Pantoea is a predominant member of host-associated microbiome. We here report on the genomic analysis of Pantoea eucrina strain Russ that was isolated from a trashcan at Oklahoma State University, Stillwater, OK. The draft genome of Pantoea eucrina strain Russ consists of 3,939,877bp of DNA with 3704 protein-coding genes and 134 RNA genes. This is the first report of a genome sequence of a member of Pantoea eucrina. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of all amino acids as well as glucose, fructose, mannose, xylose, arabinose and galactose, suggesting the organism is a versatile heterotroph. The genome also encodes an extensive secretory machinery including types I, II, III, IV, and Vb secretion systems, and several genes for pili production including the new usher/chaperone system (pfam 05,229). The implications of these systems for opportunistic pathogenesis are discussed
Molecular dating of the emergence of anaerobic rumen fungi and the impact of laterally acquired genes
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Molecular dating of the emergence of anaerobic rumen fungi and the impact of laterally acquired genes
No full textAbstract
The anaerobic gut fungi (AGF) or Neocallimastigomycota inhabit the rumen and alimentary tract of herbivorous mammals, where they play an important role in the degradation of plant fiber. Comparative genomic and phylogenomic analysis of the AGF has long been hampered by their fastidious growth pattern as well as their large and AT-biased genomes. We sequenced 21 AGF transcriptomes and combined them with 5 available genome sequences of AGF taxa to explore their evolutionary relationships, time their divergence, and characterize patterns of gene gain/loss associated with their evolution. We estimate that the most recent common ancestor of the AGF diverged 66 (±10) million years ago, a timeframe that coincides with the evolution of grasses (Poaceae), as well as the mammalian transition from insectivory to herbivory. The concordance of these independently estimated ages of AGF evolution, grasses evolution, and mammalian transition to herbivory suggest that AGF have been important in shaping the success of mammalian herbivory transition by improving the efficiency of energy acquisition from recalcitrant plant materials. Comparative genomics identified multiple lineage-specific genes and protein domains in the AGF, two of which were acquired from an animal host (galectin) and rumen gut bacteria (carbohydrate-binding domain) via horizontal gene transfer (HGT). Four of the bacterial derived “Cthe_2159” genes in AGF genomes also encode eukaryotic Pfam domains (“Atrophin-1”, “eIF-3_zeta”, “Nop14”, and “TPH”) indicating possible gene fusion events after the acquisition of “Cthe_2159” domain. A third AGF domain, plant-like polysaccharide lyase N-terminal domain (“Rhamnogal_lyase”), represents the first report from fungi that potentially aids AGF to degrade pectin. Analysis of genomic and transcriptomic sequences confirmed the presence and expression of these lineage-specific genes in nearly all AGF clades supporting the hypothesis that these laterally acquired and novel genes in fungi are likely functional. These genetic elements may contribute to the exceptional abilities of AGF to degrade plant biomass and enable metabolism of the rumen microbes and animal hosts
Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A
No full textMembers of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute, Neurospora crassa QDE-3 homolog DNA helicase, Argonaute-interacting protein, and Neurospora crassa QIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lower ldhD transcriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade
Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus<i>Pecoramyces ruminantium</i>strain C1A
No full textMembers of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungusPecoramyces ruminantiumstrain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute,Neurospora crassaQDE-3 homolog DNA helicase, Argonaute-interacting protein, andNeurospora crassaQIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lowerldhDtranscriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.</jats:p
Genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader
No full textAnaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.Peer reviewedMicrobiology and Molecular GeneticsBiosystems and Agricultural Engineerin
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Molecular Dating of the Emergence of Anaerobic Rumen Fungi and the Impact of Laterally Acquired Genes
No full textThe anaerobic gut fungi (AGF), or Neocallimastigomycota, inhabit the rumen and alimentary tract of herbivorous mammals, where they play important roles in the degradation of plant fiber. Comparative genomic and phylogenomic analyses of the AGF have long been hampered by their fastidious growth condition, as well as their large (up to 200 Mb) and AT-biased (78 to 84%) genomes. We sequenced 21 AGF transcriptomes and combined them with 5 available AGF genome sequences to explore their evolutionary relationships, time their divergence, and characterize gene gain/loss patterns associated with their evolution. We estimate that the most recent common ancestor of the AGF diverged 66 (±10) million years ago, a time frame that coincides with the evolution of grasses (Poaceae), as well as the mammalian transition from insectivory to herbivory. The concordance of independent estimations suggests that AGF have been important in shaping the success of mammalian herbivory transition by improving the efficiency of energy acquisition from recalcitrant plant materials. Comparative genomics identified multiple lineage-specific genes in the AGF, two of which were acquired from rumen gut bacteria and animal hosts via horizontal gene transfer (HGT). A third AGF domain, plant-like polysaccharide lyase, represents a novel gene in fungi that potentially aids AGF to degrade pectin. Analysis of genomic and transcriptomic sequences confirmed both the presence and expression of these lineage-specific genes in nearly all AGF clades. These genetic elements may contribute to the exceptional abilities of AGF to degrade plant biomass and enable metabolism of the rumen microbes and animal hosts.IMPORTANCE Anaerobic fungi living in the rumen of herbivorous mammals possess an extraordinary ability to degrade plant biomass. We examined the origin and genomic composition of these poorly characterized anaerobic gut fungi using both transcriptome and genomic data. Phylogenomics and molecular dating analyses found remarkable concurrence of the divergence times of the rumen fungi, the forage grasses, and the dietary shift of ancestral mammals from primarily insectivory to herbivory. Comparative genomics identified unique machinery in these fungi to utilize plant polysaccharides. The rumen fungi were also identified with the ability to code for three protein domains with putative functions in plant pectin degradation and microbial defense, which were absent from all other fungal organisms (examined over 1,000 fungal genomes). Two of these domains were likely acquired from rumen gut bacteria and animal hosts separately via horizontal gene transfer. The third one is a plant-like polysaccharide lyase, representing a unique fungal enzyme with potential pectin breakdown abilities
