Sung Uk Kim

Novel GH10 Xylanase, with a Fibronectin Type 3 Domain, from Cellulosimicrobium sp. Strain HY-13, a Bacterium in the Gut of Eisenia fetida

ABSTRACT The gene encoding a novel modular xylanase from Cellulosimicrobium sp. strain HY-13 was identified and expressed in Escherichia coli, and its truncated gene product was characterized. The enzyme consisted of three distinct functional domains, an N-terminal catalytic GH10 domain, a fibronectin type 3 domain, and C-terminal carbohydrate-binding module 2.

Synthesis and antifungal activity of a novel series of 13-(4-isopropylbenzyl)berberine derivatives.

By replacing the methyl group of 13-(4-isopropylbenzyl)berberine 2 with various acyl, alkyl, and benzyl groups via the demethylated intermediate, 13-(4-isopropylbenzyl)berberrubine 4, a novel series of 9-O-alkyl-13-(4-isopropylbenzyl)berberine derivatives was synthesized and examined for antifungal activities against various human pathogenic fungi. The introduction of various alkyl groups led to enhanced antifungal activity but that of acyl groups resulted in decrease of the activity. Among them, 9-O-butyl-13-(4-isopropylbenzyl)berberine 6d exhibited the most potent antifungal activities against Cryptococcus neoformans, Candida species (MIC=0.25-1 μg/ml), and Aspergillus species (MIC=2-4 μg/ml). The compound was found to be relatively safe up to 900 mg/kg in oral administration to mice.

Antifungal Activity of CHE-23C, a Dimeric Sesquiterpene from Chloranthus henryi

An antifungal compound was isolated from methanol extracts of stems and roots of Chloranthus henryi Hemsl. using ethyl acetate extraction and various chromatographic techniques. On the basis of spectroscopic analyses including mass and various NMR, the structure of the compound was identified as a dimeric sesquiterpene, CHE-23C. The compound showed potent antifungal activities (MICs = 1-32 microg/mL) in vitro against various phytopathogenic fungi such as Alternaria kikuchiana , Botrytis cinerea , Colletotrichum lagenarium , Magnaporthe grisea , Pythium ultimum , and Phytophthora infestans . In particular, it exhibited 91 and 100% disease-control activity in vivo against tomato late blight (P. infestans) and wheat leaf rust ( Puccinia recondita ) at concentrations of 33 and 100 microg/mL, respectively. The disease-control activity of this compound was stronger than that of the commercially available fungicide chlorothalonil, but weaker than that of dimethomorph. Therefore, the compound might serve as an interesting lead to develop effective antifungal agents.

Pedobacter luteus sp. nov., isolated from soil

Two strains of Gram-staining-negative, rod-shaped bacteria that were motile by gliding, N7d-4(T) and B4a-b5, were isolated during a study of culturable bacteria in soil cultivated with potatoes. These isolates grew at 15-37 °C and at pH 6.5-7.0. The major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. The major polar lipids were phosphatidyl-N-methylethanolamine and phosphatidylethanolamine. The strains contained d-18 : 0 and d-19 : 0 sphingosines. The DNA G+C contents of strains N7d-4(T) and B4a-b5 were 48.5 and 46.9 mol% (HPLC), respectively. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains N7d-4(T) and B4a-b5 were affiliated with Pedobacter species in the family Sphingobacteriaceae. Strains N7d-4(T) and B4a-b5 shared 99.9 % sequence similarity, and the most closely related Pedobacter type strains were Pedobacter composti TR6-06(T) (96.5 and 96.7 % sequence similarity, respectively), P. oryzae N7(T) (95.4 and 95.6 %) and P. caeni LMG 22862(T) (94.0 and 94.4 %). Phenotypic data and phylogenetic inference clearly distinguished the two isolates from other Pedobacter species. Based on these data, the isolates are considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter luteus sp. nov. is proposed. The type strain is N7d-4(T) ( = KCTC 22699(T)  = DSM 22385(T)).

A Phenylpropanoid Glycoside as a Calcineurin Inhibitor Isolated from Magnolia obovata Thunb.

To identify plant-derived cell signaling inhibitors with antifungal properties, a twocomponent screening system using both wild-type Cryptococcus neoformans and a calcineurin mutant was employed owing to their counter-regulatory actions on the Hog1 mitogenactivated protein kinase and calcineurin pathways. Of the 2,000 plant extracts evaluated, a single bioactive compound from M. obovata Thunb. was found to act specifically on the calcineurin pathway of C. neoformans. This compound was identified as magnoloside A, and had potent antifungal activities against various Cryptococcus strains with minimum inhibitory concentration values ranging from 1.0 to 4.0 μg/ml.

9-O-butyl-13-(4-isopropylbenzyl)berberine, KR-72, Is a Potent Antifungal Agent That Inhibits the Growth of Cryptococcus neoformans by Regulating Gene Expression

In this study we explored the mode of action of KR-72, a 9-O-butyl-13-(4-isopropylbenzyl)berberine derivative previously shown to exhibit potent antifungal activity against a variety of human fungal pathogens. The DNA microarray data revealed that KR-72 treatment significantly changed the transcription profiles of C. neoformans, affecting the expression of more than 2,000 genes. Genes involved in translation and transcription were mostly upregulated, whereas those involved in the cytoskeleton, intracellular trafficking, and lipid metabolism were downregulated. KR-72 also exhibited a strong synergistic effect with the antifungal agent FK506. KR-72 treatment regulated the expression of several essential genes, including ECM16, NOP14, HSP10 and MGE1, which are required for C. neoformans growth. The KR-72-mediated induction of MGE1 also likely reduced the viability of C. neoformans by impairing cell cycle or the DNA repair system. In conclusion, KR-72 showed antifungal activity by modulating diverse biological processes through a mode of action distinct from those of clinically available antifungal drugs such as polyene and azole drugs.

Phycicoccus ochangensis sp. nov., isolated from soil of a potato cultivation field

Two novel, Gram-positive, motile, coccal bacteria, strains L1b-b9T and B5a-b5, were isolated from a potato cultivation field in Ochang, Korea. These isolates grew at 10–45°C, pH 5.0–10.0, and in the presence of 8% (w/v) NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major menaquinone was MK-8(H4) and the main cellular fatty acids were iso-C14:0, iso-C15:0, and anteiso-C15:0. Polar lipids in strain L1b-b9T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and an unknown glyco-amino lipid. The G+C content of genomic DNA was 73.6 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains L1b-b9T and B5a-b5 shared 99.36% similarity and formed a robust clade with the type species of the genus Phycicoccus. Strain L1b-b9T is related most closely to Phycicoccus cremeus V2M29T (97.52% 16S rRNA gene sequence similarity). On the basis of phylogenetic characteristics, the name Phycicoccus ochangensis sp. nov. is proposed for strain LIb-b9T (=KCTC 19694T =JCM 17595T).

Qualitative and quantitative detection of agricultural microorganisms expressing iturin and mop cyclase in soils.

The environmental release of genetically engineered microorganisms (GEMs) to improve agriculture or remediate environmental hazards has raised concern over the fate of the organisms and their engineered genes. To detect the microorganisms released into the environment at the molecular level, Bacillus subtilis KB producing iturin and Pseudomonas fluorescens MX1 carrying the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens were employed as model microorganisms. Using specific fusion primers and the TaqMan probes, qualitative and quantitative detections of the model organisms by PCR and real-time PCR were conducted employing a small-scale soil-core device and pots during the six month period. The data indicate that the model bacteria can be easily detected by qualitative and quantitative methods in the test systems employed, and they do not give significant impacts on the other bacteria in soils on the Southern blotting analysis, although long-term observation may be needed.

Inhibitory effect of obovatol from Magnolia obovata on the Salmonella type III secretion system

In many pathogenic Gram-negative bacteria, such as Salmonella, Escherichia coli, Yersinia and Chlamydia spp., which cause diseases in humans, the type III secretion system (TTSS) is an important virulence factor that translocates effector proteins into the cytosol of host cells. Thus, the TTSS is a good target for antibacterial agents. Here we used a hemolysis assay to search for TTSS inhibitors and found that a compound from Magnolia obovata called obovatol blocks the TTSS of Salmonella. Obovatol showed potent inhibitory activity (IC50=19.8 μM) against the TTSS-related hemolysis of Salmonella, which was not due to a reduction of bacterial growth. Instead, the compound inhibited bacterial motility, TTSS-related mRNA expression and effector protein secretion. These data demonstrate the inhibitory effect of obovatol on the Salmonella TTSS and suggest that it could be useful for the prevention and supplementary treatment of bacterial infections.

Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds

Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A–F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.