Min Keun Kim

Endophytic bacillus sp. isolated from the interior of balloon flower root.

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.

Analysis of bgl Operon Structure and Characterization of β-Glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various β-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS–PAGE. The purified β-glucosidase exhibited maximal activity at pH 7.0 and 40 °C, and its activity was enhanced in the presence of Mg2+. Two glutamate residues (Glu173 and Glu362) were found to be essential for enzyme activity.

Functional innovations of three chronological mesohexaploid Brassica rapa genomes

BackgroundThe Brassicaceae family is an exemplary model for studying plant polyploidy. The Brassicaceae knowledge-base includes the well-annotated Arabidopsis thaliana reference sequence; well-established evidence for three rounds of whole genome duplication (WGD); and the conservation of genomic structure, with 24 conserved genomic blocks (GBs). The recently released Brassica rapa draft genome provides an ideal opportunity to update our knowledge of the conserved genomic structures in Brassica, and to study evolutionary innovations of the mesohexaploid plant, B. rapa.ResultsThree chronological B. rapa genomes (recent, young, and old) were reconstructed with sequence divergences, revealing a trace of recursive WGD events. A total of 636 fast evolving genes were unevenly distributed throughout the recent and young genomes. The representative Gene Ontology (GO) terms for these genes were ‘stress response’ and ‘development’ both through a change in protein modification or signaling, rather than by enhancing signal recognition. In retention patterns analysis, 98% of B. rapa genes were retained as collinear gene pairs; 77% of those were singly-retained in recent or young genomes resulting from death of the ancestral copies, while others were multi-retained as long retention genes. GO enrichments indicated that single retention genes mainly function in the interpretation of genetic information, whereas, multi-retention genes were biased toward signal response, especially regarding development and defense. In the recent genome, 13,302, 5,790, and 20 gene pairs were multi-retained following Brassica whole genome triplication (WGT) events with 2, 3, and 4 homoeologous copies, respectively. Enriched GO-slim terms from B. rapa homomoelogues imply that a major effect of the B. rapa WGT may have been to acquire environmental adaptability or to change the course of development. These homoeologues seem to more frequently undergo subfunctionalization with spatial expression patterns compared with other possible events including nonfunctionalization and neofunctionalization.ConclusionWe refined Brassicaceae GB information using the latest genomic resources, and distinguished three chronologically ordered B. rapa genomes. B. rapa genes were categorized into fast evolving, single- and multi-retention genes, and long retention genes by their substitution rates and retention patterns. Representative functions of the categorized genes were elucidated, providing better understanding of B. rapa evolution and the Brassica genus.

Cloning and expression of ophB gene encoding organophosphorus hydrolase from endophytic Pseudomonas sp. BF1-3 degrades organophosphorus pesticide chlorpyrifos.

Chlorpyrifos is an organophosphate pesticide that has adverse effect on animals and plants. We isolated endophytic bacterial strain, Pseudomonas sp. BF1-3, from balloon flower root which can hydrolyze chlorpyrifos. A gene (ophB) encoding a protein involved in chlorpyrifos degradation from this strain was cloned into Escherichia coli DH5α for confirming enzyme activity. After sequencing, total 1024bp nucleotide sequences were found in the open reading frame of ophB. The chlorpyrifos degradation patterns by E. coli DH5α (ophB) were observed. During incubation in minimal salt (M9) medium supplemented with chlorpyrifos (100mgL(-1)), the E. coli DH5α harboring ophB degraded about 97% initial chlorpyrifos (100mgL(-1)) and accumulated 86mgL(-1) 3,5,6-trichloro-2-pyridinol (TCP) within 9 days. In addition, optical density (OD) of E. coli DH5α (ophB) culture at 600nm was increased from 0.172 to 1.118 within 2 days of inoculation in the chlorpyrifos supplemented M9 medium. The estimated molecular weight of purified OphB protein was determined to be 31.4kDa by SDS-PAGE. The OphB enzyme was most active at pH 8 and an optimal temperature around 35°C. These results indicate that endophytic bacteria are supposed to be useful for biological control of environments contaminated with pesticides.

Effect of mild alkali pretreatment on structural changes of reed (Phragmites communis Trinius) straw

The effect of dilute sodium hydroxide (NaOH) on reed straw structural change at 105°C temperature was evaluated in this study. Various concentrations of NaOH (1% to 2.5%) were used for pretreatment of reed straw at 105°C for 10 min. Scanning electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy studies showed that 2% and 2.5% NaOH pretreated sample exposed more cellulose fibers compared with other treatments. The cellulose crystalline index was increased by the 1% to 2.0% NaOH treatments and slightly lowered by the 2.5% NaOH treatment due to destructing cellulose fibres. Two per cent NaOH pretreatment caused 69.9% lignin removal, whereas 2.5% NaOH pretreatment removed 72.4% lignin. Besides, reed straw, when pretreated at 2% and 2.5% NaOH, resulted 56.4% and 60.5% hemicellulose removal, respectively. However, the difference in removal of lignin and hemicellulose between 2% and 2.5% NaOH treated reed straw was very marginal. In addition, very negligible increase of cellulose level was estimated, amounting 78.8% and 76.6% in 2.5% and 2% NaOH-treated sample, respectively. Moreover, after 72 h, reducing sugar yield was 81.2% and 83.3% using enzyme loading of 15 FPU (g dry biomass)−1 and 30 IU (g dry biomass)−1 and xylanase 4 FXU (g dry biomass)−1 from 2% and 2.5% NaOH pretreated reed straw, respectively. Reducing sugar yield was increased very marginally when NaOH concentration increased from 2% to 2.5% for reed straw pretreatment. Therefore, 2% NaOH is supposed to be effective for reed straw pretreatment at this mentioned condition.

Alkali Pretreatment of Wheat Straw (Triticum aestivum) at Boiling Temperature for Producing a Bioethanol Precursor

We evaluated the effect of dilute sodium hydroxide (NaOH) on wheat straw at boiling temperature for removing lignin and increasing the yield of reducing sugar. Various concentrations of NaOH (0.5% to 2%) were used for pretreating wheat straw at 105 °C for 10 min. Scanning electron microscopy, atomic force microscopy, and Fourier transform infrared spectroscopy studies revealed that the 2% NaOH-pretreated sample exposed more cellulose fiber. The maximum respective removal of lignin and hemicellulose was 70.3% and 68.2% from the 2% NaOH-pretreated liquor. The reducing sugar yield was 84.6% using an enzyme dose containing 20 FPU of cellulase, 40 IU of β-glucosidase and 4 FXU of xylanase/g of substrate. However, 2% NaOH-treated wheat straw had the lowest crystalline index of 52.5%, due to destructured cellulose fibers. The results indicate the effectiveness of producing the bioethanol precursor from wheat straw by using 2% NaOH at boiling temperature.

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons of Pectobacterium carotovorum subsp. carotovorum LY34

A third bgl operon containing bglE, bglF, bglI, and bglA was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). The sequences of BglE, BglF, and Bgll were similar to those of the phosphotransferase system (PTS) components IIB, IIC, and IIA respectively. BglF contains important residues for the phosphotransferase system. The amino acid sequence of BglA showed high similarity to various 6-phospho-β-glucosidases and to a member of glycosyl hydrolase family 1. Sequence and structural analysis also revealed that these four genes were organized in a putative operon that differed from two operons previously isolated from Pcc LY34, bglTPB (accession no. AY542524) and ascGFB (accession no. AY622309). The transcription regulator for this operon was not found, and the EII complexes for PTS were encoded separately by three genes (bglE, bglF, and bglI). The BglA enzyme had a molecular weight estimated to be 57,350 Da by SDS–PAGE. The purified β-glucosidase hydrolyzed salicin, arbutin, ρNPG, ρNPβG6P, and MUG, exhibited maximal activity at pH 7.0 and 40 °C, and displayed enhanced activity in the presence of Mg2+ and Ca2+. Two glutamate residues (Glu178 and Glu378) were found to be essential for enzyme activity.

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

Fungicide Sensitivity and Characterization of Cobweb Disease on a Pleurotus eryngii Mushroom Crop Caused by Cladobotryum mycophilum.

In 2009–2010, unusual symptoms were observed on Pleurotus eryngii grown in mushroom farms in Gyeongnam Province, Republic of Korea. One of the main symptoms was a cobweb-like growth of fungal mycelia over the surface of the mushroom. The colonies on the surface rapidly overwhelmed the mushrooms and developed several spores within 3–4 days. The colonized surface turned pale brown or yellow. The fruit body eventually turned dark brown and became rancid. Koch’s postulates were completed by spraying and spotting using isolated strains. The phylogenetic tree obtained from the internal transcribed spacer sequence analysis showed that the isolated fungal pathogen corresponded to Cladobotryum mycophilum (99.5%). In the fungicide sensitivity tests, the ED50 values for the isolate with respect to benomyl and carbendazim were from 0.29 to 0.31 ppm. Benzimidazole fungicides were most effective against C. mycophilum, a causal agent of cobweb disease in P. eryngii.

Impacts of Soil Texture on Microbial Community of Orchard Soils in Gyeongnam Province

Soil management for orchard depends on the effects of soil microbial activities. The present study evaluated the soil microbial community of 25 orchard (5 sites for sandy loam, 7 sites for silt loam, and 13 sites for loam) in Gyeongnam Province by fatty acid methyl ester (FAME) method. The average values for 25 orchard soil samples were 270 nmol g -1 of total FAMEs, 72 nmol g -1 of total bacteria, 34 nmol g -1 of Gram-negative bacteria, 34 nmol g -1 of Gram-positive bacteria, 6 nmol g -1 of actinomycetes, 49 nmol g -1 of fungi, and 7 nmol g -1 of arbuscular mycorrhizal fungi. In addition, silt loam soils had significantly low ratio of cy17:0 to 16:1ω7c and cy19:0 to 18:1ω7c compared with those of loam soils (p < 0.05), indicating that microbial activity increased. The average soil microbial communities in the orchard soils were 26.7% of bacteria, 17.9% of fungi, 12.6% of Gram-negative bacteria, 12.5% of Gram-positive bacteria, 2.5% of arbuscular mycorrhizal fungi, and 2.2% of actinomycetes. The soil microbial community of Gram-negative bacteria in silt loam soils was significantly higher than those of sandy loam and loam soils (p < 0.05).