• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.6
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• pp.449-455
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• 2000

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of the meta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, and meta-cleavage of protocatechuate. The pcbC gene responsible for the meta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that of Pseudomonas sp. CBS3, yet only a 50% homology with that of Arthrobacter spp. However, the fcb genes for the hydrolytic dechlorination of 4CBA in Pseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBA completely via meta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.259-270
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• 2020

Listeria monocytogenes is a gram-positive, facultative anaerobe food pathogen responsible for the listeriosis that mostly occurs during the low-temperature storage of a cold cut or dairy products. To understand the systemic response to a wide range of growth temperatures, L. monocytogenes were cultivated at a different temperature from 10℃ to 42℃, then whole cell proteomic analysis has been performed both exponential and stationary cells. The specific growth rate increased proportionally with the increase in growth temperature. The maximum growth rate was observed at 37℃ and was maintained at 42℃. Global protein expression profiles mainly depended on the growth temperatures showing similar clusters between exponential and stationary phases. Expressed proteins were categorized by their belonging metabolic systems and then, evaluated the change of expression level in regard to the growth temperature and stages. DnaK, GroEL, GroES, GrpE, and CspB, which were the heat&cold shock response proteins, increased their expression with increasing the growth temperatures. In particular, GroES and CspB were expressed more than 100-fold than at low temperatures during the exponential phase. Meanwhile, CspL, another cold shock protein, overexpressed at a low temperature then exponentially decreased its expression to 65-folds. Chemotaxis protein CheV and flagella proteins were highly expressed at low temperatures and stationary phases. Housekeeping proteins maintained their expression levels constant regardless of growth temperature or growth phases. Most of the growth related proteins, which include central carbon catabolic enzymes, were highly expressed at 30℃ then decreased sharply at high growth temperatures.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.934-944
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• 2007

Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the strain was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1,000 spots in the cell and 1,100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E68l cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG, spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-alanine ligase A, isopentenyl-diphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.673-679
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• 2003

Glutamine and urea, abundant in body fluids or plasma, yield net ammonium ions upon hydrolysis by ${\gamma}-glutamyl$ transpeptidase (${\gamma}-GTP$) and urease, respectively, and these two enzymes are largely produced from Helicobacter pylori. To investigate bacterial potential of ammonium production, we first quantified those in whole-cell systems and found that the relative ratio of their amounts varied greatly, especially with pH values and the cell's aging. During the H. pylori cultivation, the ratio appeared to be inversely proportional to each other, showing a progressive increase of the ${\gamma}-GTP$ with decreasing of the urease. Under the urease-defective conditions due to low pH or coccoids, the bacterial cells still possessed a considerable amount of ${\gamma}-GTP$, which was found exclusively in the external compartment, therefore, the cell's ammonium production was found to be solely dependent upon glutamine, and the external ammonium concentration was constant without any contribution of urea concentration. Such ammonium constancy would definitely have an adverse effect on the host, because of its absolute requirement for vacuolar degeneration by H. pylori VacA, maximized at approximately 10 mM $NH_4Cl$. It was also found that, by using the metal-saturated membrane vesicles, ammonium ions were likely to be involved in the pH-dependent cation-flux across the H. pylori membrane, where the role of ${\gamma}-GTP$ in ammonium homeostasis around cells was suggested, especially under the hostile conditions against H. pylori.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.155-160
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• 2005

Abstract A direct approach was used to retrieve active lipases from Paenibacillus polymyxa genome databases. Twelve putative lipase genes were tested using a typical lipase sequence rule built on the basis of a consensus sequence of a catalytic triad and oxyanion hole. Among them, six genes satisfied the sequence rule and had similarity (about 25%) with known bacterial lipases. To obtain the six lipase proteins, lipase genes were expressed in E. coli cells and lipolytic activities were measured by using tributyrin plate and pnitrophenyl caproate. One of them, contig 160-26, was expressed as a soluble and active form in E. coli cell. After purifying on Ni-NTA column, its detailed biochemical properties were characterized. It had a maximum hydrolytic activity at $30^{\circ}C$ and pH 7- 8, and was stable up to $40^{\circ}C$ and in the range of pH 5- 8. It most rapidly hydrolyzed pNPC$_6$ among various PNPesters. The other contigs were expressed more or less as soluble forms, although no lipolytic activities were detected. As they have many conserved regions with lipase 160-26 as well as other bacterial lipases throughout their equence, they are suggested as true lipase genes.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• Archives of Pharmacal Research
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• v.28 no.4
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• pp.433-437
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• 2005

Human cytochromes P450 (GYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various GYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under $30^{\circ}C$). Expression levels of GYPs 2A6 and 2E1 at $37^{\circ}C$ were compared to those at $28^{\circ}C$, which is a usual temperature used in most bacterial expression systems for human GYP expression. Within 18 h the expression levels of GYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at $37^{\circ}C$, respectively, which are compatible with those of 36 h culture at $28^{\circ}C$. The activities of GYPs expressed at $37^{\circ}C$ were also comparable to those expressed at $28^{\circ}C$. The present over-expression system can be useful for rapid production of large amounts of active human GYPs 2A6 and 2E1 in E. coli.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.152-154
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• 2018

Klebsiella pneumoniae is a Gram-negative, rod-shape bacterium causing disease in human and animal lungs. K. pneumoniae has been often found to gain antimicrobial resistance, thus it has been difficult to treat K. pneumoniae infection with antibiotics. For such infection, bacteriophage can provide an alternative approach for pathogenic bacterial infection with antimicrobial resistance, because of its sensitivity and specificity to the host bacteria. Bacteriophage KP1 was isolated in sewage and showed specific infectivity to K. pneumoniae. Here, we report the draft genome sequence of Klebsiella pneumoniae phage KP1. The draft genome of KP1 is 167,989 bp long, and the G + C content is 39.6%. The genome has 295 predicted ORFs and 14 tRNA genes. In addition, it encodes various enzymes which involve in lysis of the host cell such as lysozyme and holin.

• Restorative Dentistry and Endodontics
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• v.20 no.2
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• pp.549-576
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• 1995

Bacteria are one of the most important causative agents of the pulpal and periapical diseases. Streptococci are one of the most frequently isolated facultative anarerobic bacteria in the infected root canals. Bacterial cell wall components have a direct effect in the pathogenesis of the pulpal and periapical infections. Hyaluronidase produced by bacteria has been implicated in dissemination of the diseases. The purpose of this study was to evaluate the effect of cell wall extract of streptococci on the L929 cells using inverted microscope and the transmission electron microscopy (TEM). Hyaluronidase production of streptococcal strains were investigated to determine the correlation between the severity of cell damage and the activity of enzymes. Bacterial cell wall extracts of S. sanguis, S. mitis and S. uberis isolated from infected root canals and ATCC type strains of S. mutans (ATCC 10449) and E. faecalis (ATCC 19433) were prepared by sonication and confirmed with SDS-PAGE. Silver stain of SDS-PAGE of sonic extract was efficient at $100{\mu}g$/ml concentration of cell wall protein, while Coomasie blue stain was efficient at $100{\mu}g$/ml concentration. Inverted microscope showed that sonic extract-treated L929 cells were round and detached from the substratum while others lost their fibroblastic shapes. Transmission electron microscopic examination revealed that streptococcal extracts induced death of L929 cells. Sonic extracts of streptococci had variable effect on microstructure of L929 cells. significant chromatin condensation was observed in the nucleus of the cells. Disappearance of cell surface microvilli and nuclear fragments with dense chromatin were observed. The cell nucleus had an irregular shape and numerous large vacuoles were seen in the cytoplasm and some breaks of the cell membrane could be seen. Cell organelles were in various stages of destruction and cristae of mitochondria were disoriented or disappeared. Eighteen strains of streptococci did not produce hyaluronidase.

• The Plant Pathology Journal
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• v.37 no.2
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• pp.173-181
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• 2021

The genus Streptomyces demonstrates enormous promise in promoting plant growth and protecting plants against various pathogens. Single and consortium treatments of two selected Streptomyces strains (Streptomyces shenzhenensis TKSC3 and Streptomyces sp. SS8) were evaluated for their growth-promoting potential on rice, and biocontrol efficiency through induced systemic resistance (ISR) mediation against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS) disease. Seed bacterization by Streptomyces strains improved seed germination and vigor, relative to the untreated seed. Under greenhouse conditions, seed bacterization with consortium treatment TKSC3 + SS8 increased seed germination, root length, and dry weight by 20%, 23%, and 33%, respectively. Single and consortium Streptomyces treatments also successfully suppressed Xoc infection. The result was consistent with defense-related enzyme quantification wherein single and consortium Streptomyces treatments increased peroxidase (POX), polyphenol oxidase, phenylalanine ammonia-lyase, and β,1-3 glucanase (GLU) accumulation compared to untreated plant. Within all Streptomyces treatments, consortium treatment TKSC3 + SS8 showed the highest disease suppression efficiency (81.02%) and the lowest area under the disease progress curve value (95.79), making it the best to control BLS disease. Consortium treatment TKSC3 + SS8 induced the highest POX and GLU enzyme activities at 114.32 µmol/min/mg protein and 260.32 abs/min/mg protein, respectively, with both enzymes responsible for plant cell wall reinforcement and resistant interaction. Our results revealed that in addition to promoting plant growth, these Streptomyces strains also mediated ISR in rice plants, thereby, ensuring protection from BLS disease.