Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

<p>Abstract</p> <p>Background</p> <p>To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms.</p> <p>Results</p> <p>From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in <it>Escherichia coli</it>. Further characterization showed that two enzymes showed significant activity on <it>p</it>-nitrophenyl-α-<smcaps>L</smcaps>-arabinofuranoside, one enzyme had significant activity against <it>p</it>-nitrophenyl-β-<smcaps>D</smcaps>-glucopyranoside, and one enzyme showed significant activity against <it>p</it>-nitrophenyl-β-<smcaps>D</smcaps>-xylopyranoside. Enzymes were also tested in the presence of ionic liquids.</p> <p>Conclusions</p> <p>Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.</p

Genome sequence of the plant growth promoting endophytic bacterium Enterobacter sp. 638.

Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpaxdeltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT-PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar as an energy feedstock on marginal, non-agricultural soils using endophytic bacteria as growth promoting agents

Crystal Structure of ChrR—A Quinone Reductase with the Capacity to Reduce Chromate

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 A ˚ resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction

Bio-inspired Nanowire Arrays as Artificial Photoreceptors for Retinal Prosthesis

PURPOSE: The restoration of light response with complex spatiotemporal features in retinal degenerative diseases towards retinal prosthesis has proven to be a considerable challenge over the past decades. The state-of-art retinal prosthesis utilizes photodiode arrays fabricated on solid substrates, which does not have a complete tiling of the retinal tissue. Polymer based optoelectronic interface to retinal tissue, despite being compatible with flexible substrates, has limited spatial resolution for vision. Herein, inspired by the structure and function of photoreceptors in retinas, we developed bio-inspired artificial photoreceptors, i.e. gold nanoparticle-decorated titania (Au-TiO2) nanowire arrays, for restoration of multi-color visual responses in the retina of blind mice with degenerated photoreceptors. METHODS: single-cell intracellular measurements by patch-clamps, functional calcium imaging, in vivo electrophysiological recording, Pupillary light reflex. RESULTS: When Au-TiO2 nanowire arrays were in direct contact with blind mice retina, single-cell intracellular measurements by patch-clamps in the retinal ganglion cells (RGCs) revealed that green, blue and UV light responses were restored with a spatial resolution approaching or exceeding 100 &micro;m. Light responses in RGCs can be blocked by glutamatergic antagonists, indicating that nanowire array-interfaced retinas were capable of processing visual information through innate retinal circuits. Moreover, cellular-level population responses using functional calcium imaging in the Au-TiO2 nanowire array-interfaced blind retinas indicated that the receptive field is within the range of 200 &micro;m. Neurons in the primary visual cortex responded to light in vivo after subretinal implant of the nanowire arrays into the blind mice. The blind mice had lost a part of ability of the pupillary light reflex. But after subretinal implant of NW array, the blind mice regained the ability of pupillary light reflex. CONCLUSIONS: This study is among the first to show bio-inspired artificial photoreceptors and will shed light on the development of a new generation of optoelectronic toolkits for photo-coded subretinal implants and prosthetic devices.</p

Transcriptional Responses to Sucrose Mimic the Plant-Associated Life Style of the Plant Growth Promoting Endophyte <i>Enterobacter</i> sp. 638

<div><p>Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium <i>Enterobacter</i> sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involved in motility (e.g. flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA <i>csrABCD</i> regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of <i>Enterobacter</i> sp. 638, our results also pointed to the involvement of <i>rcsAB</i> in controlling responses by <i>Enterobacter</i> sp. 638 to a plant-associated life style. Targeted mutagenesis was used to confirm this role and showed that compared to wild-type <i>Enterobacter</i> sp. 638 a Δ<i>rcsB</i> mutant was affected in its plant growth promoting ability.</p></div

Expression levels of <i>Enterobacter sp.</i> 638 genes directly involved in sucrose and lactate metabolisms.

<p>Log₂ of normalized gene expression values (RPKM) are plotted for each condition. L6, L12, S6 and S12 represent the condition lactate and sucrose after 6 and 12 hours growth, respectively. Ent638_2165 is a hypothetical gene that showed homology to <i>ycjM</i>, a putative sucrose phosphorylase.</p

The effects of various mutations on the growth of wheat plants after inoculation with wild-type strain <i>Enterobacter</i> sp. 638 and its derivatives.

<p>Derivatives containing knock-out mutations for the following genes were examined for their effects on wheat growth: <i>budA</i> and <i>budB</i>, required for acetoin and 2,3-butanediol synthesis, respectively; <i>hipA</i>, the toxin of the <i>hipAB</i> toxin-antitoxin system; the <i>rcsB</i> regulator; and <i>osmC</i>, which is important in biofilm formation and under positive control of the <i>rcsAB</i> regulatory network in <i>E. coli</i>. The error bars show the standard deviation (SD). Different letters on top of each column represent the significance at the 0.05 level.</p

Expression levels of various toxin/anti-toxin systems found in <i>Enterobacter sp.</i> 638 in function of carbon source.

<p>Log₂ of normalized gene expression values (RPKM) are plotted for each condition. L6, L12, S6 and S12 represent the condition lactate and sucrose after 6 and 12 hours growth, respectively.</p

The effects of various mutations on the early development of root hairs by tomato plants after inoculation with wild-type strain <i>Enterobacter</i> sp. 638 and its derivatives.

<p>The section of the root, 2–3 mm from the root tip, was visualized using light microscopy 48 hours after inoculation. Root hair formation was determined by the average number of root hairs as counted in triplicate within the visual field image of the microscope:-, 0~5 root hairs; +, 6~20 root hairs; ++, 21~40 root hairs; +++, 41~60 root hairs.</p