Bon-Sung Koo

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.

Characterization of a gene encoding cellulase from uncultured soil bacteria.

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.

Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

ABSTRACT It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.

Massilia niabensis sp. nov. and Massilia niastensis sp. nov., isolated from air samples

Two bacterial isolates, designated strains 5420S-26(T) and 5516S-1(T), were recovered from air samples collected in Suwon, Korea. Cells of both strains were aerobic, Gram-negative, motile rods. Phylogenetically, these strains were positioned within the radius of the genus Massilia. 16S rRNA gene sequence analysis showed that the strains shared 97.3 % sequence similarity and had sequence similarities of 94.9-98.1 % with respect to type strains of species belonging to the genus Massilia. In DNA-DNA hybridization tests, the two strains showed <39 % relatedness with respect to strains of closely related species of the genus Massilia and 27 % relatedness to each other. Both strains contained Q-8 as the predominant isoprenoid quinone and possessed summed feature 3 (comprising C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) as the major fatty acid. Strain 5516S-1(T) was found to contain the fatty acid C(20 : 0) (in small amounts), a feature that served to distinguish it from both 5420S-26(T) and recognized members of the genus Massilia. The DNA G+C contents of 5420S-26(T) and 5516S-1(T) were 67.8 and 66.6 mol%, respectively. Phylogenetic, phenotypic and chemotaxonomic data accumulated in this study revealed that 5420S-26(T) and 5516S-1(T) represent novel species of the genus Massilia, for which the names Massilia niabensis sp. nov. (type strain 5420S-26(T) =KACC 12632(T) =DSM 21312(T)) and Massilia niastensis sp. nov. (type strain 5516S-1(T) =KACC 12599(T) =DSM 21313(T)) are proposed, respectively.

Chitinophaga niabensis sp. nov. and Chitinophaga niastensis sp. nov., isolated from soil.

Two yellow-coloured bacterial strains, designated JS13-10(T) and JS16-4(T), were isolated from soil from Jeju Island, Republic of Korea. On the basis of 16S rRNA gene sequence analysis, the strains were found to be affiliated with members of the genus Chitinophaga. Phenotypically, the novel strains were identified as being different from each other and from recognized species of the genus Chitinophaga. DNA-DNA hybridization tests between the two novel strains and closely related Chitinophaga reference strains produced DNA relatedness values that were significantly lower (<36 %) than those generally accepted as the highest threshold for the phylogenetic definition of a species. On the basis of their distinct taxonomic characteristics, these strains represent two novel species of the genus Chitinophaga, for which the names Chitinophaga niabensis sp. nov. (type strain JS13-10(T)=KACC 12952(T)=JCM 15440(T)) and Chitinophaga niastensis sp. nov. (type strain JS16-4(T)=KACC 12954(T)=JCM 15441(T)) are proposed.

Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library Ki Hyun Nam,1y Min-Young Kim,2y Soo-Jin Kim, Amit Priyadarshi, Suk-Tae Kwon, Bon-Sung Koo, Sang-Hong Yoon, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 2National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea 4Department of Genetic Engineering, Sungkyunkwan University, Jangan-gu, Suwon 440-746, Korea 5 Fermentation and Food Processing Division, Department of Korean Food Research for Globalization, National Academy of Agricultural Science,

Production and proteolytic assay of lethal factor from Bacillus anthracis.

Bacillus anthracis is the causative agent of anthrax. The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively. These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells. In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli. rLF was rapidly purified through a single affinity purification step to near homogeneity. Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide. The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.

Solitalea koreensis gen. nov., sp. nov. and the reclassification of [Flexibacter] canadensis as Solitalea canadensis comb. nov.

A Gram-negative, rod-shaped, non-spore-forming bacterium, designated strain R2A36-4T, was isolated from greenhouse soil and subjected to a polyphasic taxonomic analysis. 16S rRNA gene sequence analysis revealed that the strain represented a novel member of the family Sphingobacteriaceae. Its nearest phylogenetic neighbour was the type strain of [Flexibacter] canadensis CIP 104802T (93.2% 16S rRNA gene sequence similarity). Strain R2A36-4T and [F.] canadensis CIP 104802T fell in a distinct cluster within the family Sphingobacteriaceae. Strain R2A36-4T contained MK-7 as the predominant quinone. Strain R2A36-4T and [F.] canadensis CIP 104802T had iso-C15:0, iso-C17:0 3-OH, summed feature 3 and C15:1omega6c as the major fatty acids. Strain R2A36-4T could be distinguished from [F.] canadensis on the basis of several physiological properties and fatty acid compositions. Based on phenotypic characterization and 16S rRNA gene sequence analysis, strain R2A36-4T represents a novel species within a new genus, for which the name Solitalea koreensis gen. nov., sp. nov. is proposed. The type strain of Solitalea koreensis is strain R2A36-4T (=KACC 12953T=DSM 21342T). It is also proposed that [F.] canadensis be transferred to this genus as Solitalea canadensis comb. nov. (type strain UASM 9DT=ATCC 29591T=CIP 104802T=DSM 3403T=NBRC 15130T=JCM 21819T=KACC 13276T=LMG 8368T).

Niabella soli sp. nov., isolated from soil from Jeju Island, Korea

A dark yellow-coloured bacterium, JS13-8(T), was isolated from a soil sample from Jeju Island, Republic of Korea. The cells were aerobic, Gram-negative, non-motile, short rods (0.5-0.7 x 0.8-1.4 microm). Growth occurred at 15-35 degrees C (optimally at 30 degrees C), at pH 5.0-8.0 (optimally at pH 6.0-7.0) and at 0-1 % NaCl (w/v). Flexirubin pigment was produced. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JS13-8(T) was closely related to Niabella aurantiaca KACC 11698(T) (95.0 % sequence similarity). The major respiratory quinone system was MK-7 and the predominant cellular fatty acids were iso-C(15 : 0), iso-C(15 : 1) G, iso-C(17 : 0) 3-OH and summed feature 3. The DNA G+C content was 45 mol%. On the basis of the phylogenetic, physiological and chemotaxonomic data, strain JS13-8(T) represents a novel species of the genus Niabella, for which the name Niabella soli sp. nov. is proposed. The type strain is strain JS13-8(T) (=KACC 12604(T)=DSM 19437(T)).