Kye Man Cho

Endophytic Bacterial Communities in Ginseng and their Antifungal Activity Against Pathogens

Plant roots are associated with diverse communities of endophytic bacteria which do not exert adverse effects. The diversity of bacterial endophytes associated with ginseng roots cultivated in three different areas in Korea was investigated. Sixty-three colonies were isolated from the interior of ginseng roots. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to three major phylogenetic groups: the high G+C Gram-positive bacteria (HGCGPB), low G+C Gram-positive bacteria (LGCGPB), and the Proteobacteria. The dominant species at the three different ginseng growing areas were: HGCGPB at Ganghwa (55.0%), LGCGPB at Geumsan (45.5%), and Proteobacteria at Jinan (61.9%). Most cellulase-, xylanase-, and pectinase-producing colonies among the isolates belong to the LGCGPB group, except for Pectobacterium carotovora which belonged to the Proteobacteria. The 13 isolates belonging to LGCGPB and Proteobacteria were assessed for their antifungal activity against phytopathogenic fungi such as Rhizoctonia solani. Among them, Paenibacillus polymyxa GS01, Bacillus sp. GS07, and Pseudomonas poae JA01 show potential activity as biocontrol agents against phytopathogenic fungi. Finally, most of the low G+C Gram-positive bacteria with antifungal activity against phytopathogenic microorganisms showed cellulolytic enzyme activity while some Proteobacteria with the antifungal activity and the high G+C Gram-positive bacteria did not show any cellulolytic activity.

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu91 and Glu222 may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.

Assembling a novel bifunctional cellulase–xylanase from Thermotoga maritima by end-to-end fusion

An artificial, bifunctional, thermostable cellulase–xylanase enzyme from Thermotoga maritima by gene fusion. The fusion protein exhibited both cellulase and xylanase activity when xynA was fused downstream of cel5C but no activities were shown when xynA was fused upstream of cel5C. The enzyme was optimally active at pH 5.0 and 80°C over 30xa0min. E. coli expressed the fusion enzyme, with an apparent molecular mass of approximately 152xa0kDa by carboxymethyl cellulose- and xylan-SDS-PAGE.

Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation

We developed a multiplex PCR assay for the detection of lactic acid bacteria (LAB) species, and used it to examine the LAB species involved in kimchi fermentation. The LAB profile during kimchi fermentation varied with pH and acidity. Leuconostoc mesenteroides was observed during early fermentation (pH 5.64-4.27 and acidity 0.48-0.89%), and Lactobacillus sakei become dominant later in fermentation (pH <or=4.15 and acidity >or=0.98%). The efficiency of the multiplex PCR ranged from 86.5% at day 0 (pH 6.17 and acidity 0.24%) to 100% at day 96 (pH 4.16 and acidity 1.14). This multiplex PCR assay will facilitate study of the microbial ecosystem of kimchi and its impact on kimchi fermentation.

Biodegradation of Chlorpyrifos by Lactic Acid Bacteria during Kimchi Fermentation

We examined the role of microorganisms in the degradation of the organophosphorus (OP) insecticide chlorpyrifos (CP) during kimchi fermentation. During the fermentation of kimchi, 30 mg L(-1) of CP was added and its stability assayed during fermentation. CP was degraded rapidly until day 3 (83.3%) and degraded completely by day 9. Four CP-degrading lactic acid bacteria (LAB) were isolated from kimchi fermentation in the presence of 200 mg L(-1) CP and were identified as Leuconostoc mesenteroides WCP907, Lactobacillus brevis WCP902, Lactobacillus plantarum WCP931, and Lactobacillus sakei WCP904. CP could be utilized by these four strains as the sole source of carbon and phosphorus. Coumaphos (CM), diazinon (DZ), parathion (PT), and methylparathion (MPT) were also degraded by WCP907, WCP902, WCP931, and WCP904 when provided as sole sources of carbon and phosphorus.

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.

Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C-man 26A

Endophytic bacteria are acknowledged as a new source of genes, proteins and other biochemical compounds, which are often used in biochemical processes. In this study, Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). Two cellulase genes, cel 5A and cel 5B, were cloned from GS01, and encode 334 aa and 573 aa proteins, respectively. Cel5A and Cel5B each contain a glycosyl hydrolase family 5 (GH5) catalytic domain. The molecular mass of Cel5A and Cel5B were estimated to be 33 kDa and 61 kDa, respectively, by CMC‐SDS‐PAGE. When purified from Escherichia coli Cel5A and Cel5B both displayed cellulase activity with pH optima of 7.0 and 6.0, respectively and shared a temperature optimum of 50 °C. Neither enzyme had detectable xylanase, lichenase, or mannase activity, in contrast to the multifunctional Cel44C‐Man26A enzyme of P. polymyxa which displays cellulase, xylanase, lichenase and mannanase activities. However, Cel5A and Cel5B exhibited higher specific cellulase activity than Cel44C‐Man26A (120% and 140%, respectively). Cel5A and Cel5B mutants with alanine substitutions at a conserved glutamic acid in the GH5 domain (Glu 179 of Cel5A and Glu184 of Cel5B) lacked cellulase activity, suggesting that this residue is important for GH5 domain function. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

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.

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Ω mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. γ-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.