Hyung-Yeel Kahng

Cellular Responses and Proteomic Analysis of Escherichia coli Exposed to Green Tea Polyphenols

The purpose of this study was to characterize the cellular response and proteomic analysis of Escherichia coli exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L). TPP showed a dose-dependent bactericidal effect on E. coli. Analysis of cell-membrane fatty acids of E. coli cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, whereas scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from E. coli cultures exposed to TPP showed 17 protein spots increased or decreased by TPP. Nine upregulated proteins were identified (including GroEL and proteins involved in cellular defense, such as GyrA, RpoS, SodC, and EmrK), whereas the expression of eight proteins was downregulated by exposure to TPP (including proteins involved in carbon and energy metabolism, such as Eno, SdhA, and UgpQ, as well as those involved in amino-acid biosynthesis, such as GltK and TyrB). These results provide clues for understanding the mechanism of TPP-induced stress and cytotoxicity on E. coli.

Analysis of TNT (2,4,6-trinitrotoluene)-inducible cellular responses and stress shock proteome in Stenotrophomonas sp. OK-5.

In this study, the cellular responses of Stenotrophomonas sp. OK-5 to explosive 2,4,6-trinitrotoluene (TNT) have been extensively analyzed. The stress shock proteins, which might contribute to enhancing cellular resistance to TNT-mediated toxicity, were induced at different concentrations of TNT used as a substrate for cell culture of Stenotrophomonas sp. OK-5 capable of utilizing TNT. Proteomic analysis for 2-DE of soluble protein fractions from the culture of OK-5 exposed to TNT demonstrated approximately 300 spots on the silver-stained gel ranging from pH 3 to pH 10. Among them, 10 spots significantly induced and expressed in response to TNT were selected and analyzed. As the result of internal amino acid sequencing with ESI-Q TOF mass spectrometry, TNT-mediated stress shock proteins such as DnaK, OmpW, and OsmC were identified and characterized. Survival of strain OK-5 was periodically monitored in the presence of different concentrations of TNT along with the production of the stress shock proteins. Cells of strain OK-5 pre-exposed to TNT had in improved survival tolerance. Analysis of total cellular fatty acids in strain OK-5 suggested that several saturated or unsaturated fatty acids might increase or decrease under TNT-mediated stress condition. Scanning electron microscopy of cells treated with 0.8 mM TNT for 12 h revealed irregular rod shapes with wrinkled surfaces.

Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation.

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.

Cellulophaga tyrosinoxydans sp. nov., a tyrosinase- producing bacterium isolated from seawater

An aerobic, gliding, yellow-pigmented bacterium lacking flagella and showing strong tyrosinase activity, designated strain EM41(T), was isolated from seawater on the eastern coast of Jeju Island in Korea. Growth was observed at 15-35 degrees C (optimum, 25-30 degrees C) and at pH 6.5-9.0 (optimum, pH 7.0-8.5). Cells were Gram-negative, negative for flexirubin pigments and catalase- and oxidase-positive. The G+C content of the genomic DNA was 33.5 mol% and the major respiratory quinone was menaquinone-6 (MK-6). Comparative 16S rRNA gene sequence analysis showed that strain EM41(T) formed a distinct phyletic line within the genus Cellulophaga with a 100 % bootstrap value and was most closely related to Cellulophaga pacifica KMM 3664(T) (97.0 % sequence similarity). The level of DNA-DNA relatedness between strain EM41(T) and C. pacifica KMM 3664(T) was about 17.8 %. On the basis of phenotypic and genotypic data, strain EM41(T) is considered to represent a novel species of the genus Cellulophaga, for which the name Cellulophaga tyrosinoxydans sp. nov. is proposed. The type strain is EM41(T) (=KCTC 22297(T)=DSM 21164(T)).

Sphingobacterium cladoniae sp. nov., isolated from lichen, Cladonia sp., and emended description of Sphingobacterium siyangense

A strictly aerobic, Gram-stain-negative bacterium, designated strain No.6(T), was isolated from a lichen (Cladonia sp.) collected in Geogeum Island, Korea, and its taxonomic status was established by a polyphasic study. Cells of strain No.6(T) were non-motile, catalase- and oxidase-positive, non-spore-forming rods. Growth was observed at 15-35 °C (optimum, 25-30 °C), at pH 5.0-10.0 (optimum, pH 6.0-8.0) and with 0-3 % NaCl (optimum, 0-2 %). The predominant cellular fatty acids were summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c, 41.5 %), iso-C(15 : 0) (26.7 %) and C(16 : 0) (9.6 %), and menaquinone MK-7 was the only respiratory quinone. The G+C content of the genomic DNA of strain No.6(T) was 36.8 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain No.6(T) fell within the evolutionary group encompassed by the genus Sphingobacterium. Levels of 16S rRNA gene sequence similarity between the novel strain and the type strains of recognized Sphingobacterium species ranged from 92.1 to 99.1 %, the highest values being with Sphingobacterium siyangense SY1(T) (99.1 %) and Sphingobacterium multivorum IAM 14316(T) (98.5 %). DNA-DNA relatedness between strain No.6(T) and these two type strains were 32.0 and 5.7 %, respectively. The polar lipids found in strain No.6(T) were phosphatidylethanolamine, two unidentified phospholipids, three unidentified aminophospholipids, one glycolipid and four unidentified lipids. One unidentified sphingolipid was also found. On the basis of phenotypic and genotypic data, strain No.6(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium cladoniae sp. nov. is proposed. The type strain is No.6(T) ( = KCTC 22613(T) = JCM 16113(T)). An emended description of Sphingobacterium siyangense is also proposed.

Winogradskyella lutea sp. nov., isolated from seawater, and emended description of the genus Winogradskyella

A novel gram-negative, orange-pigmented, rod-shaped, strictly aerobic, gliding, oxidase- and catalase-positive bacterial strain, A73(T), was isolated from seawater collected off Jeju, South Korea. 16S rRNA gene sequence similarity between A73(T) and type strains of Winogradskyella species with validly published names ranged from 94.1 to 96.2 %. The dominant fatty acids of strain A73(T) were iso-C(15 : 1) G (19.1 %), iso-C(15 : 0) (13.3 %), iso-C(17 : 0) 3-OH (10.0 %) and iso-C(15 : 0) 3-OH (7.2 %). The DNA G+C content of strain A73(T) was 36.0 mol% and its major respiratory quinone was MK-6. On the basis of combined data from phenotypic and phylogenetic analyses, strain A73(T) represents a novel species of the genus Winogradskyella, for which the name Winogradskyella lutea sp. nov. is proposed. The type strain is A73(T) ( = KCTC 23237(T)  = DSM 22624(T)). An emended description of the genus Winogradskyella is also provided.

Polaribacter gangjinensis sp. nov., isolated from seawater

A strictly aerobic, orange-pigmented and Gram-staining-negative bacterium, designated K17-16(T), was isolated from seawater of Gangjin Bay, Korea. Comparative 16S rRNA gene sequence analysis revealed that strain K17-16(T) was a member of the genus Polaribacter in the family Flavobacteriaceae and showed 94.0-95.6 % sequence similarity with the type strains of recognized species of the genus Polaribacter. The G+C content of the genomic DNA was 34.6 mol% and the major respiratory lipoquinone was MK-6. The major polar lipids detected were phosphatidylethanolamine, three unidentified amino-group-containing lipids and an unidentified aminophospholipid. The predominant cellular fatty acids were iso-C(15 : 0) (15.4 %), C(15 : 0) (12.4 %), summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c; 10.6 %), C(15 : 1)ω6c (9.8 %) and iso-C(15 : 0) 3-OH (8.6 %). On the basis of phenotypic and genotypic data, strain K17-16(T) represents a novel species in the genus Polaribacter, for which the name Polaribacter gangjinensis sp. nov. is proposed. The type strain is K17-16(T) ( = KCTC 22729(T) = JCM 16152(T)).

Tenacibaculum crassostreae sp. nov., isolated from the Pacific oyster, Crassostrea gigas

A Gram-stain-positive, endospore-forming, rod-shaped, aerobic bacterium, designated LPB0068T, was isolated from a Pacific oyster (Crassostrea gigas) in Korea. This isolate was found to share the highest 16S rRNA gene sequence similarity with Paenibacillus macquariensis subsp. macquariensis DSM 2T (98.1 %) and Paenibacillus macquariensis subsp. defensor JCM 14954T (98.0 %). To establish the genomic relatedness of this isolate to its phylogenetic neighbours, its genome sequence and those of Paenibacillus antarcticus CECT 5836T, P. macquariensis subsp. macquariensis DSM 2T, P. macquariensis subsp. defensor JCM 14954T, and Paenibacillus glacialis DSM 22343T were determined. The low average nucleotide identity and digital DNA-DNA hybridization values exhibited by LPB0068T in relation to the other strains in this analysis revealed that it is distinct from other Paenibacillus species. The genome of strain LPB0068T consists of one chromosome and three circular plasmids, and had a DNA G+C content of 40.0 mol%. The major respiratory quinone was menaquinone-7 and the diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, one unidentified glycolipid, and two unidentified polar lipids. The major cellular fatty acids were anteiso-C15 : 0, C14 : 0, and C16 : 0. Based on genomic, phylogenetic, and phenotypic characteristics, this strain was clearly distinguished from other Paenibacillus species with validly published names and should therefore be classified as a novel species of the genus. The name Paenibacillus crassostreae sp. nov. is proposed, the type strain of which is LPB0068T (=KACC 18694T=JCM 31183T).

Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1.

Erythrobacter gangjinensis sp. nov., a marine bacterium isolated from seawater

A novel Gram-negative, aerobic, orange-pigmented bacterial strain, designated K7-2(T), was isolated from seawater of Gangjin Bay, Korea, and subjected to a polyphasic taxonomic study. Strain K7-2(T) contained ubiquinone-10 (Q-10) as the predominant respiratory lipoquinone and did not produce bacteriochlorophyll a. Major fatty acids were C(18 : 1)omega7c (51.4 %), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (15.0 %) and C(17 : 1)omega6c (8.8 %). Major polar lipids were phosphatidylethanolamine and phosphatidylcholine. The DNA G+C content was 61.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain K7-2(T) formed a distinct phylogenetic lineage within the cluster comprising Erythrobacter strains. Similarities between the 16S rRNA gene sequences of strain K7-2(T) and the type strains of Erythrobacter species ranged from 95.0 % (Erythrobacter litoralis DSM 8509(T)) to 96.8 % (Erythrobacter citreus RE35F/1(T)). On the basis of polyphasic taxonomic data, strain K7-2(T) (=KCTC 22330(T)=JCM 15420(T)) is classified in a novel species within the genus Erythrobacter, for which the name Erythrobacter gangjinensis sp. nov. is proposed.