Yong-Lark Choi

Novel roles of Bacillus thuringiensis to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.

Hydrolysis of isoflavone glycoside by immobilization of β-glucosidase on a chitosan-carbon in two-phase system

We explored a method to examine the hydrolysis of isoflavone glycoside by immobilizing β-glucosidase on chitosan-carbon beads in an aqueous-organic two-phase system. The chitosan-carbon beads were cross-linked with glutaraldehyde to immobilize β-glucosidase from Exiguobacterium sp. DAU5. The optimal pH and temperature were 9.0 and 55 °C, respectively. Under the optimized conditions, crude and purified enzymes immobilized onto chitosan-carbon beads gave yields of 16.7% and 60%, respectively. The specific activities of immobilized crude and purified enzymes were 4.3 U/g and 6 U/g, respectively. The immobilized enzyme retained more than 80% of its maximum activity at pH 7.0-11.0, while temperature was more influential (80% activity after 40 °C for 1.5 h, but only 40% activity after 55 °C for 0.5 h, respectively. The immobilized enzyme was able to hydrolyze isoflavone glycoside in an aqueous-organic two-phase system, and the hydrolyzed products were enriched in the organic phase, making it easy to recover the products, i.e., genistein and daidein from the reaction system. These results suggest that immobilized β-glucosidase may be applicable for the industrial-scale hydrolysis of isoflavone glycoside.

Discovery of three novel lipase (lipA1, lipA2, and lipA3) and lipase-specific chaperone (lipB) genes present in Acinetobacter sp. DYL129

A microbe isolated from a soil sample obtained on Deog-yu Mountain in Korea, was found to produce an extracellular lipase. This microbe, which was designated as DYL129, was identified as an Acinetobacter sp. based on phylogenetic analysis of its 16S rDNA. A genomic library was constructed by using DYL129 fragment digested with HindIII and a recombinant plasmid, pLip-1, was selected for further analysis by colony polymerase chain reaction (PCR). Sequencing of a 3.8-kb insert in the pLip-1 clone revealed the presence of one incomplete and three complete open reading frames (ORFs). The ORFs were predicted to encode a partial lipase, two putative lipases and a 50S ribosomal protein. Genome-walking PCR also identified transcripts encoding a complete lipase chaperone and three lipaseA proteins. The lipase structural gene present in Acinetobacter sp. DYL129 was similar to the lipase structural gene found in Acinetobacter calcoaceticus BD413 (lipBA). However, the three lipase genes were located downstream of the chaperone gene in Acinetobacter sp. DYL129 (lipBA1A2A3), which differs from the location of these genes in A. calcoaceticus BD413. Although the amino acid sequences of these lipases (LipA1, LipA2, and LipA3) differed, both strains had a common “GHSHG” consensus motif, which is the conserved active-site pentapeptide of lipaseA. Moreover, all three lipases were found to share a conserved domain, the so-called α/β hydrolase fold.

Independently expressed N‐terminal pro‐domain of aqualysin I precursor complements the folding of its mature domain to active form in Escherichia coli

Aqualysin I is a subtilisin‐type serine protease secreted into the culture medium by Thermus aquaticus YT‐1. It is first produced as a large precursor that consists of a signal peptide, an N‐terminal pro‐domain, the mature protease domain and a C‐terminal pro‐domain. To investigate whether the N‐terminal pro‐domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N‐terminal pro‐domain in E. coli has been expressed independent of the mature domain with or without the C‐terminal pro‐domain using an expression system with separate promoters and signal peptides. Protease assay and SDS‐PAGE clearly showed that the N‐terminal pro‐domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N‐terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N‐terminal pro‐domain‐assisted folding of the mature domain.

Complete genome sequence and analysis of three kinds of β-agarase of Cellulophaga lytica DAU203 isolated from marine sediment

Cellulophaga lytica DAU203 (KACC 19187), isolated from the marine sediment in Korea, has a strong ability to degrade agar. The genome of C. lytica DAU203 contains a single chromosome that is 3,952,957bp in length, with 32.02% G+C contents. The genomic information predicted that the DAU203 has the potential to be utilized in various enzymatic industries.

Production and Characterization of Alkaline Protease of Micrococcus sp. PS-1 Isolated from Seawater

The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria- Bertani (LB) medium. Its optimal temperature and pH for growth were 30°C and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and 37°C, and its activity was stable at pH 8.0?11.0 and 25?37°C. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.

Complete genome sequence of cold-adapted enzyme producing Microbulbifer thermotolerans DAU221

Microbulbifer thermotolerans DAU221 was preliminary isolated from the marine sediment samples in the Republic of Korea. Here, we present the complete genome sequence of M. thermotolerans DAU221, which consisted of 3,938,396 base pairs with a GC content of 56.57%. This genomic information should help us find the industrially useful enzymes.

Biological Control of Plant Pathogens by Bacillus sp. AB02.

In the greenhouse fields for fruits and vegetables during the winter in Korea, there are serious damages by the sclerotium diseases due to the low temperature and humidity. This study was carried out to select an antagonic agent for the biological control of the sclerotium diseases. The 55 antagonic agents were selected from the rhizosphere in soil where the fruits and vegetables were cultivated in the green house fields, and strain AB02 among the tested isolates was estimated to be the strongest antagonist against the sclerotium disease. Using strain AB02, the antifungal spectrum was tested against 5 different plant pathogens. According to the results of the test, strain AB02 showed the high antagonistic effect against Botrytis cinerea and Sclerotinia sclerotiorum. For the experiment of biological control against the sclerotium disease, it was estimated the suppression effect and the control effect by the strain in the pot experiment using the green perilla. According to the result of the pot experiments, the suppression effect was 40% and the control effect was 62%, respectively. For the stimulation effect of the tested plant growth by strain AB02 compared to the control, it was improved as 120% for the total length, 141% for the liveweight, 121% for the total number of leaves, 185% for the leaf area, and 327% for the liveweight of the root, respectively. Strain AB02 showing the antagonistic effect against the sclerotium disease and the stimulation effect for the plant growth was identified as Bacillus sp.

Isolation and Characterization of a Novel Bacterium, Bacillus subtilis HR-1019, with Insoluble Phosphates Solubilizing Activity

The objective of this study was to develop a mineral phosphate-solubilizing bacterium as a biofertilizer. A mineral phosphate-solubilizing bacterium HR-1019 was isolated from cultivated soils. It was identified as Bacillus subtilis by 16S rDNA analysis. The phosphate-solubilizing activities of the HR-1019 strain against three types of insoluble phosphate, hydroxyapatite, tri-calcium phosphate, and aluminum phosphate were quantitatively determined. When 5% of glucose concentration was used as a carbon source, the strain showed marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to pH drop in the culture solution of the strain. The pathogenic activity and antifungal effects of the HR-1019 strain were measured inclear zones formed in PDA media.

Growth Promotion of Lettuce by Biofertilizer, BIOACTIVE, Prepared from Bacillus subtilus HR-1019 and N-acetyl-thioproline

A biofertilizer, BIOACTIVE, was manufactured by N-acetyl-thioproline (ATCA) and mineral phosphate solubilizing bacteria. The growth promoting effect of the biofertilizer on lettuce was evaluated under three different pot conditions, and its stability was assessed in the field. According to the results of the pot experiments, plant growth was improved compared with that of control: 128%, 122%, and 153% for the leaf number, leaf length, and leaf mass, respectively. Applying the manufactured biofertilizer increased the concentration of phosphate: 118% and 132% in the cultivation soil and plant cells, respectively. These show that BIOACTIVE may have potential as an effective biofertilizer in agriculture.