RESUMO
Recalcitrance of plant biomass is a major barrier for commercially feasible cellulosic biofuel production. Chemical and enzymatic assays have been developed to measure recalcitrance and carbohydrate composition; however, none of these assays can directly report which polysaccharides a candidate microbe will sense during growth on these substrates. Here, we propose using the transcriptomic response of the plant biomass-deconstructing microbe, Caldicellulosiruptor saccharolyticus, as a direct measure of how suitable a sample of plant biomass may be for fermentation based on the bioavailability of polysaccharides. Key genes were identified using the global gene response of the microbe to model plant polysaccharides and various types of unpretreated, chemically pretreated and genetically modified plant biomass. While the majority of C. saccharolyticus genes responding were similar between plant biomasses; subtle differences were discernable, most importantly between chemically pretreated or genetically modified biomass that both exhibit similar levels of solubilization by the microbe. Furthermore, the results here present a new paradigm for assessing plant-microbe interactions that can be deployed as a biological assay to report on the complexity and recalcitrance of plant biomass.
Assuntos
Proteínas de Bactérias/genética , Firmicutes/genética , Firmicutes/metabolismo , Lignina/metabolismo , Populus/química , Populus/genética , Ácidos/química , Proteínas de Bactérias/metabolismo , Biotransformação , Populus/metabolismo , Populus/microbiologia , TranscriptomaRESUMO
A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.
Assuntos
Flagelos/genética , Genes Bacterianos , Mutação , Pyrococcus furiosus/genética , Proliferação de Células , Flagelos/ultraestrutura , Fenótipo , Pyrococcus furiosus/crescimento & desenvolvimento , Pyrococcus furiosus/metabolismo , Pyrococcus furiosus/fisiologia , TranscriptomaRESUMO
Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of â¼ 80 °C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.
Assuntos
Metabolismo dos Carboidratos/genética , Genoma Bacteriano , Bactérias Gram-Positivas/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Biomassa , Perfilação da Expressão Gênica , Genes Bacterianos , Genômica , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/ultraestrutura , Plantas/metabolismo , ProteômicaRESUMO
Phylogenetic, microbiological, and comparative genomic analyses were used to examine the diversity among members of the genus Caldicellulosiruptor, with an eye toward the capacity of these extremely thermophilic bacteria to degrade the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii, C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA gene phylogeny and cross-species DNA-DNA hybridization to a whole-genome C. saccharolyticus oligonucleotide microarray, revealing that C. saccharolyticus was the most divergent within this group. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid-pretreated switchgrass, only C. saccharolyticus, C. bescii, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman no. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that the cellulolytic species also had diverse secretome fingerprints. The C. saccharolyticus secretome contained a prominent S-layer protein that appears in the cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interactions. Growth physiology also correlated with glycoside hydrolase (GH) and carbohydrate-binding module (CBM) inventories for the seven bacteria, as deduced from draft genome sequence information. These inventories indicated that the absence of a single GH and CBM family was responsible for diminished cellulolytic capacity. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and this argues for continued efforts to isolate new members from high-temperature terrestrial biotopes.
Assuntos
Biomassa , Variação Genética , Glicosídeo Hidrolases/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Plantas/microbiologia , Proteínas de Bactérias/análise , Metabolismo dos Carboidratos , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Eletroforese em Gel Bidimensional , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/metabolismo , Temperatura Alta , Análise em Microsséries , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , Proteoma/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO(2), and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Bactérias Gram-Positivas/genética , Proteínas de Bactérias/genética , Metabolismo dos Carboidratos/genética , Enzimas/genética , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Extremely thermophilic fermentative anaerobes (growth T(opt) > or = 70 degrees C) have the capacity to use a variety of carbohydrates as carbon and energy sources. As such, a wide variety of glycoside hydrolases and transferases have been identified in these microorganisms. The genomes of three model extreme thermophiles-an archaeon Pyrococcus furiosus (T(opt) = 98 degrees C), and two bacteria, Thermotoga maritima (T(opt) = 80 degrees C) and Caldicellulosiruptor saccharolyticus (T(opt) = 70 degrees C)-encode numerous carbohydrate-active enzymes, many of which have been characterized biochemically in their native or recombinant forms. In addition to their voracious appetite for polysaccharide degradation, polysaccharide production has also been noted for extremely thermophilic fermentative anaerobes; T. maritima generates exopolysaccharides that aid in biofilm formation, a process that appears to be driven by intraspecies and interspecies interactions.
Assuntos
Polissacarídeos/metabolismo , Pyrococcus furiosus/metabolismo , Thermotoga maritima/metabolismo , Anaerobiose , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Polissacarídeos/biossíntese , Pyrococcus furiosus/crescimento & desenvolvimento , Thermotoga maritima/crescimento & desenvolvimentoRESUMO
Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h(-1) at 90 degrees C. In the absence of S(0), cellobiose-grown cells generated twice as much protein and had 50%-higher specific H(2) generation rates than maltose-grown cultures. Addition of S(0) to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H(2) to H(2)S. In contrast, the presence of S(0) in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H(2) to H(2)S production, with 2.5 times more H(2) than H(2)S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in alpha- or beta-glucan uptake and processing were up-regulated in an S(0)-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S(0) in cellobiose-grown cells also responded to S(0) in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S(0) was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S(0) metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.
Assuntos
Glicosídeos/metabolismo , Hidrogênio/metabolismo , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/fisiologia , Enxofre/metabolismo , Archaea , Fenômenos Químicos , Química , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Temperatura Alta , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Pyrococcus furiosus/genéticaRESUMO
In the genome of the hyperthermophilic bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.
Assuntos
Genes Bacterianos , Genoma Bacteriano , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Bacteriano/genética , Thermotoga maritima/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Cloranfenicol/farmacologia , Técnicas de Cocultura , DNA Bacteriano/genética , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mathanococcus , RNA Bacteriano/análise , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sintenia , Transcrição GênicaRESUMO
A novel protocol for the generation of sulfur ylides is described. The overall process involves thermal decarboxylation of a carboxymethylsulfonium betaine to give a sulfur ylide that, in the presence of an aldehyde, affords the corresponding terminal oxirane. Yields were found to correlate with the electron deficiency of the aryl aldehyde. In situ generation of betaine in the presence of an aldehyde successfully afforded the desired oxirane in moderate yield, thus demonstrating the feasibility of a catalytic process. [reaction: see text]