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.