Woo Jin Lim

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.

Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides.

Lactobacillus brevis WCP902 that is capable of biodegrading chlorpyrifos was isolated from kimchi. The opdB gene cloned from this strain revealed 825 bp, encoding 274 aa, and an enzyme molecular weight of about 27 kDa. OpdB contains the same Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic esterase, lipase, and serine hydrolases, yet it is a novel member of the GDSVG family of esterolytic enzymes. Its conserved serine residue, Ser82, is significantly involved with enzyme activity that may have application for removing some pesticides. Optimum organophosphorus hydrolase (OpdB) activity appeared at pH 6.0 and 35 degrees C and during degradation of chlorpyrifos, coumaphos, diazinon, methylparathion, and parathion.

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.

Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 107 cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.

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 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.

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.

Cloning and characterization of cel8A gene from Rhizobium leguminosarum bv. trifolii 1536

Aims:  To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536.