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.

The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.Ralstonia pickettii PKO1 has been investigated by our laboratory for several years as a model microorganism representative of those bacteria capable of metabolizing alkylaromatic hydrocarbons in oxygen-limited (hypoxic) aquifer environments (23,34,42,43). The tbu pathway of R. pickettii PKO1, which encodes enzymes for utilization of benzene, toluene, and related alkylaromatic hydrocarbons as well as enabling this strain to transform trichloroethylene (TCE), has been cloned as a 26.5-kbp DNA fragment designated pRO1957 (41). The genes encoding enzymes for this catabolic pathway have been shown previously to be organized into three operons: the tbuA1UBVA2C and tbuT operon encoding the initial toluene-3-monooxygenase and the transcriptional activator TbuT (6), the tbuD operon encoding phenol/cresol hydroxylase (24, 26), and the tbuWEFGKIHJ operon encoding enzymes of the metacleavage pathway for conversion of catechol and methylcatechols to tricarboxylic acid cycle intermediates (25). We have previously shown through physiological analysis as well as through transcriptional fusion analysis of promoter regions that TbuT controls transcription of each of these operons in response to aromatic effector compounds. Moreover, it has been shown that the unique organization of tbuT, which is located immediately dow...

An aerobic, gliding, yellow-pigmented bacterium lacking flagella and showing strong tyrosinase activity, designated strain EM41T , was isolated from seawater on the eastern coast of Jeju Island in Korea. Growth was observed at 15-35 6C (optimum, 25-30 6C) 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

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

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.

A Gram-staining-negative, strictly aerobic and rod-shaped bacterium, designated strain CNURIC013 T , was isolated from seawater collected on the coast of Jeju Island, South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CNURIC013 T belonged to the genus Tenacibaculum, within the family Flavobacteriaceae. Sequence similarities between the novel strain and the type strains of recognized species of the genus Tenacibaculum were 93.6-96.0 %, the highest value being with Tenacibaculum litopenaei B-I T (96 %). The DNA G+C content of the novel strain was 34.5 mol% and the major respiratory quinone was menaquinone-6. The major fatty acids were summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH; 26.0 %), iso-C 15 : 0 (24.4 %), iso-C 15 : 1 G (18.5 %) and iso-C 17 : 0 3-OH (8.1 %). The polar lipids consisted of phosphatidylethanolamine, one unknown aminophospholipid and nine unknown polar lipids. On the basis of the phenotypic, phylogenetic and genotypic data, strain CNURIC013 T represents a novel species within the genus Tenacibaculum, for which the name Tenacibaculum jejuense sp. nov. is proposed. The type strain is CNURIC013 T (5KCTC 22618 T 5JCM 15975 T ).The genus Tenacibaculum was first proposed by Suzuki et al. (2001), who described Tenacibaculum mesophilum and Tenacibaculum amylolyticum and reclassified Flexibacter maritimus (Wakabayashi et al., 1986) and Flexibacter ovolyticus (Hansen et al., 1992) as Tenacibaculum maritimum and Tenacibaculum ovolyticum, respectively. The genus Tenacibaculum belongs to the family Flavobacteriaceae and currently comprises 15 recognized species isolated from different marine habitats. T. maritimum, T. ovolyticum, Tenacibaculum discolor, Tenacibaculum aiptasiae and Tenacibaculum soleae were isolated from diseased marine animals (Wakabayashi et al., 1986;Hansen et al., 1992;Wang et al., 2008; Piñeiro-Vidal et al., 2008a, b). Although T. mesophilum, Tenacibaculum adriaticum and Tenacibaculum crassostreae were also isolated from marine animals (Suzuki et al., 2001;Heindl et al., 2008;Lee et al., 2009), T. amylolyticum was isolated from marine macroalgae (Suzuki et al., 2001), Tenacibaculum lutimaris, Tenacibaculum litoreum and Tenacibaculum aestuarii were isolated from tidal-flat sediments (Yoon et al., 2005;Choi et al., 2006;Jung et al., 2006) and Tenacibaculum skagerrakense, Tenacibaculum litopenaei and Tenacibaculum gallaicum were isolated from seawater (Frette et al., 2004;Sheu et al., 2007; Piñeiro-Vidal et al., 2008a). In this study, a Tenacibaculumlike bacterial strain, CNURIC013 T , was isolated from a seawater sample collected from the coast of Jeju Island, South Korea, before its taxonomy was investigated using a polyphasic approach. Strain CNURIC013 T was isolated, using a standard dilutionplating technique, on marine agar 2216 (MA; Difco), after incubation at 25 u C for 5 days. Subcultivation was routinely performed on MA at 30 u C for 3 days under aerobic conditions and the strain was preserved at 280 u C in marine broth (M...

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl-or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl-and chloroaromatic monooxygenases in strain JS150.Biodegradation of the monoaromatic hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (collectively designated BTEX) has been extensively investigated as a basis for understanding the intrinsic biodegradation potential of these fuel hydrocarbons when they occur as groundwater contaminants (17,21,39,57,61). Toluene has been studied as a model compound representative of this group of aromatic hydrocarbons (14), and its biodegradation under aerobic conditions has been found to proceed by the six pathways shown in Fig. 1. Implicit in much of the literature on biodegradation of toluene is the assumption that each toluene degrader elaborates a single pathway for toluene degradation. This is seen, for example, for Pseudomonas putida mt-2 (PaW1), which carries the TOL plasmid pWW0 (64); for P. putida F1 (13); for Burkholderia cepacia G4 (52); for Ralstonia pickettii PKO1 (25); or for Pseudomonas mendocina KR-1 (63). However, Burkholderia sp. strain JS150 seems to be an exception to this rule. Previous research conducted with this strain by Haigler and coworkers (15) has demonstrated that a broad-substrate-range toluene dioxygenase is at least partly responsible for the extended aromatic substrate range of JS150. Subsequently, the studies of Johnson...

Two genes encoding lipolytic enzymes were isolated from a metagenomic library constructed from oil-polluted mud flats. An esterase gene, est3K, encoded a protein of 299 amino acids (ca. 32,364 Da). Est3K was a family IV esterase with typical motifs, HGGG, and HGF. Although est3K showed high identity to many genes with no information on their enzymatic properties, Est3K showed the highest identity (36 %) to SBLip5.1 from forest soil metagenome when compared to the enzymes with reported properties. A lipase gene, lip3K, encoded a protein of 616 amino acids (ca. 64,408 Da). Lip3K belonged to family I.3 lipase with a C-terminal secretion signal and showed the highest identity (93 %) to the lipase of Pseudomonas sp. MIS38. The presence of several newly identified conserved motifs in Est3K and Lip3K are suggested. Both Est3K and Lip3K exerted their maximal activity at pH 9.0 and 50 °C. The activity of Lip3K was significantly increased by the presence of 30 % methanol. The ability of the enzymes to retain activities in the presence of methanol and the substrates may offer a merit to the biotechnological applications of the enzymes such as transesterification. The activity and the thermostability of Lip3K were increased by Ca(2+). Est3K and Lip3K preferred p-nitrophenyl butyrate (C4) and octanoate (C8), respectively, as the substrate and acted independently on the substrates with no synergistic effect.