RESUMO
Plants grow within a complex web of species that interact with each other and with the plant1-10. These interactions are governed by a wide repertoire of chemical signals, and the resulting chemical landscape of the rhizosphere can strongly affect root health and development7-9,11-18. Here, to understand how interactions between microorganisms influence root growth in Arabidopsis, we established a model system for interactions between plants, microorganisms and the environment. We inoculated seedlings with a 185-member bacterial synthetic community, manipulated the abiotic environment and measured bacterial colonization of the plant. This enabled us to classify the synthetic community into four modules of co-occurring strains. We deconstructed the synthetic community on the basis of these modules, and identified interactions between microorganisms that determine root phenotype. These interactions primarily involve a single bacterial genus (Variovorax), which completely reverses the severe inhibition of root growth that is induced by a wide diversity of bacterial strains as well as by the entire 185-member community. We demonstrate that Variovorax manipulates plant hormone levels to balance the effects of our ecologically realistic synthetic root community on root growth. We identify an auxin-degradation operon that is conserved in all available genomes of Variovorax and is necessary and sufficient for the reversion of root growth inhibition. Therefore, metabolic signal interference shapes bacteria-plant communication networks and is essential for maintaining the stereotypic developmental programme of the root. Optimizing the feedbacks that shape chemical interaction networks in the rhizosphere provides a promising ecological strategy for developing more resilient and productive crops.
Assuntos
Arabidopsis/microbiologia , Comamonadaceae/classificação , Comamonadaceae/fisiologia , Microbiota/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Comamonadaceae/genética , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Microbiota/genética , Óperon/genética , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/genética , Rizosfera , Transdução de SinaisRESUMO
Methodological advances over the past two decades have propelled plant microbiome research, allowing the field to comprehensively test ideas proposed over a century ago and generate many new hypotheses. Studying the distribution of microbial taxa and genes across plant habitats has revealed the importance of various ecological and evolutionary forces shaping plant microbiota. In particular, selection imposed by plant habitats strongly shapes the diversity and composition of microbiota and leads to microbial adaptation associated with navigating the plant immune system and utilizing plant-derived resources. Reductionist approaches have demonstrated that the interaction between plant immunity and the plant microbiome is, in fact, bidirectional and that plants, microbiota, and the environment shape a complex chemical dialogue that collectively orchestrates the plantmicrobiome. The next stage in plant microbiome research will require the integration of ecological and reductionist approaches to establish a general understanding of the assembly and function in both natural and managed environments.
Assuntos
Bactérias/genética , Ecologia , Microbiota , Plantas/imunologia , Plantas/microbiologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Bactérias/classificação , Bactérias/isolamento & purificação , Evolução Molecular , Filogenia , Fenômenos Fisiológicos VegetaisRESUMO
Biomass degrading thermophiles play an indispensable role in building lignocellulose-based supply chains. They operate at high temperatures to improve process efficiencies and minimize mesophilic contamination, can overcome lignocellulose recalcitrance through their native carbohydrate-active enzyme (CAZyme) inventory, and can utilize a wide range of sugar substrates. However, sugar transport in thermophiles is poorly understood and investigated, as compared to enzymatic lignocellulose deconstruction and metabolic conversion of sugars to value-added chemicals. Here, we review the general modes of sugar transport in thermophilic bacteria and archaea, covering the structural, molecular, and biophysical basis of their high-affinity sugar uptake. We also discuss recent genetic studies on sugar transporter function. With this understanding of sugar transport, we discuss strategies for how sugar transport can be engineered in thermophiles, with the potential to enhance the conversion of lignocellulosic biomass into renewable products. ONE-SENTENCE SUMMARY: Sugar transport is the understudied link between extracellular biomass deconstruction and intracellular sugar metabolism in thermophilic lignocellulose bioprocessing.
Assuntos
Archaea , Bactérias , Lignina , Açúcares , Lignina/metabolismo , Archaea/metabolismo , Archaea/genética , Transporte Biológico , Açúcares/metabolismo , Bactérias/metabolismo , Bactérias/genética , Biomassa , Metabolismo dos Carboidratos , Temperatura AltaRESUMO
Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.
Assuntos
Microbiota/fisiologia , Imunidade Vegetal/imunologia , Raízes de Plantas/microbiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Bactérias/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Imunidade , Microbiota/imunologia , Doenças das Plantas/microbiologia , Raízes de Plantas/imunologia , Plantas/microbiologia , Microbiologia do Solo , Simbiose/imunologia , VirulênciaRESUMO
Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter, Fervidobacterium, Caloramator, and Clostridium). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator, had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic.
Assuntos
Firmicutes/genética , Firmicutes/metabolismo , Genoma Bacteriano , Lignina/metabolismo , Metagenoma , Celulose/metabolismo , Firmicutes/classificação , Genômica , MetagenômicaRESUMO
Six multidomain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation.
Assuntos
Celulose , Firmicutes , Glicosídeo Hidrolases , Celulose/química , Celulose/metabolismo , Firmicutes/enzimologia , Firmicutes/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , HidróliseRESUMO
Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.
Assuntos
Firmicutes/genética , Microbiologia Industrial/métodos , Regiões Promotoras Genéticas , Xilose/metabolismo , Biotransformação , Celulose/metabolismo , Firmicutes/efeitos dos fármacos , Firmicutes/crescimento & desenvolvimento , Firmicutes/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Termotolerância , Xilose/farmacologiaRESUMO
The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensisencodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptorspecies genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique toC. kronotskyensisand contains SLH domains. Calkro_0111 was produced recombinantly inEscherichia colias two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo- and exoglucanase activity on the ß-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs inC. kronotskyensis, when produced inE. coli, was active on a variety of xylans and ß-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptorand among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii As such, the gene encoding Calkro_0402 was inserted into the C. besciigenome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-typeC. bescii The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptorspecies.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Genoma Bacteriano , Glicosídeo Hidrolases/metabolismo , Madeira/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Clostridiales/química , Clostridiales/classificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucanos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Cinética , Mutação , Filogenia , Polissacarídeos/metabolismo , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilanos/metabolismoRESUMO
Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(-) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (TâMâTâM) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the TâMâTâM treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.
Assuntos
Bactérias Gram-Positivas/metabolismo , Lignina/metabolismo , Panicum/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Polissacarídeos/metabolismo , Biomassa , Fermentação , Bactérias Gram-Positivas/genética , Temperatura Alta , Lignina/química , Panicum/química , Panicum/genética , Panicum/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/químicaRESUMO
Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii Furthermore, a facile approach for assessing genomic stability in C. bescii has been established.IMPORTANCECaldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies.
Assuntos
Genoma Bacteriano , Instabilidade Genômica , Bactérias Gram-Positivas/genética , Lignina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Genética , Bactérias Gram-Positivas/metabolismo , Temperatura AltaRESUMO
The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile.
Assuntos
Proteínas de Bactérias/metabolismo , Celulose/química , Firmicutes/genética , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Panicum/química , Populus/química , Firmicutes/metabolismoRESUMO
A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins ("tapirins," origin from Maori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tapirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tapirins are specific to these extreme thermophiles. Tapirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tapirins for cellulose. Crystallization of a cellulose-binding truncation from one tapirin indicated that these proteins form a long ß-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tapirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Adsorção , Bactérias/genética , Bactérias/ultraestrutura , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Sítios de Ligação , Fímbrias Bacterianas/metabolismo , Genes Bacterianos , Modelos Moleculares , Filogenia , Plantas/microbiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
UNLABELLED: Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii These strains showed resistance to kanamycin at concentrations >50 µg · ml(-1), whereas wild-type C. bescii was sensitive to kanamycin at 10 µg · ml(-1) In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCE: Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.
Assuntos
Firmicutes/genética , Firmicutes/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Temperatura Alta , Resistência a Canamicina , Biologia Molecular/métodos , Seleção Genética , Genética Microbiana/métodosRESUMO
Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2, and H2. Caldicellulosiruptor bescii, C. kronotskyensis, and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (Panicum virgatum) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.
Assuntos
Biomassa , Bactérias/metabolismo , Celulase/metabolismo , Panicum/enzimologia , Panicum/metabolismo , Plantas/enzimologiaRESUMO
Microbes orchestrate nearly all major biogeochemical processes. The ability to program their influence on plant growth and development is attractive for sustainable agriculture. However, the complexity of microbial ecosystems and our limited understanding of the mechanisms by which plants and microbes interact with each other and the environment make it challenging to use microbiomes to influence plant growth. Novel technologies at the intersection of microbial ecology, systems biology, and bioengineering provide new tools to probe the role of plant microbiomes across environments. Here, we summarize recent studies on plant and microbe responses to abiotic stresses, showcasing key molecules and micro-organisms that are important for plant health. We highlight opportunities to use synthetic microbial communities to understand the complexity of plant-microbial interactions and discuss future avenues of programming ecology to improve plant and ecosystem health.
Assuntos
Mudança Climática , Microbiota , Plantas , Microbiota/fisiologia , Plantas/microbiologia , Desenvolvimento Vegetal , Ecossistema , Biologia Sintética , Ecologia , Raízes de Plantas/microbiologiaRESUMO
Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 µm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.
Assuntos
Arabidopsis , Microbiota , Raízes de Plantas , Arabidopsis/microbiologia , Arabidopsis/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip , Microscopia Confocal/métodos , Plântula/microbiologia , Plântula/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , MorfogêneseRESUMO
Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon's MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac-like and iad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad-like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Microbiota , Reguladores de Crescimento de Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Plantas/metabolismoRESUMO
Immune systems restrict microbial pathogens by identifying "non-self" molecules called microbe-associated molecular patterns (MAMPs). It is unclear how immune responses are tuned to or by MAMP diversity present in commensal microbiota. We systematically studied the variability of commensal peptide derivatives of flagellin (flg22), a MAMP detected by plants. We define substantial functional diversity. Most flg22 peptides evade recognition, while others contribute to evasion by manipulating immunity through antagonism and signal modulation. We establish a paradigm of signal integration, wherein the sequential signaling outputs of the flagellin receptor are separable and allow for reprogramming by commensal-derived flg22 epitope variants. Plant-associated communities are enriched for immune evading flg22 epitopes, but upon physiological stress that represses the immune system, immune-activating flg22 epitopes become enriched. The existence of immune-manipulating epitopes suggests that they evolved to either communicate or utilize the immune system for host colonization and thus can influence commensal microbiota community composition.
Assuntos
Epitopos/imunologia , Flagelina/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Vegetal , Bactérias/genética , Imunidade , Microbiota , Peptídeos , Ralstonia , SimbioseRESUMO
Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.
Assuntos
Engenharia Genética/métodos , Plantas Geneticamente Modificadas/metabolismo , Pseudomonas/metabolismo , Brachypodium/metabolismo , Brachypodium/microbiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinases/genética , Recombinação Genética , RizosferaRESUMO
Microbial fermentation of lignocellulosic biomass to produce industrial chemicals is exacerbated by the recalcitrant network of lignin, cellulose and hemicelluloses comprising the plant secondary cell wall. In this study, we show that transgenic poplar (Populus trichocarpa) lines can be solubilized without any pretreatment by the extreme thermophile Caldicellulosiruptor bescii that has been metabolically engineered to shift its fermentation products away from inhibitory organic acids to ethanol. Carbohydrate solubilization and conversion of unpretreated milled biomass is nearly 90% for two transgenic lines, compared to only 25% for wild-type poplar. Unexpectedly, unpretreated intact poplar stems achieved nearly 70% of the fermentation production observed with milled poplar as the substrate. The nearly quantitative microbial conversion of the carbohydrate content of unpretreated transgenic lignocellulosic biomass bodes well for full utilization of renewable biomass feedstocks.