• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.295-301
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• 1986

The bacteria, which were capable of producing ${\beta}-1$, 3-glucanase inducibly by utilizing cell wall of Aspergillus fumigatus as a sole carbon source, were isolated from soil in the campus of Kyungpook National University. Among them, the strain which produced the enzyme excellently was selected and identified to be Pseudomonas stutzeri KF 13 by morphological, cultural and physiological examination. The optimal conditions for the enzyme production from Pseudomonas stutzeri KF 13 were investigated. the enzyme production was reached maximum state shen the broth cultured for 72hr at $30^{\circ}C$. And the enzyme showed the highest activity in the medium containing 3.5% cell wall as an inducer, 15% yeast autolysate as a nitrogen source and 0.05% $MnSO_4$ at pH 7.5.

• Journal of the Korea Organic Resources Recycling Association
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• v.19 no.4
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• pp.84-89
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• 2011

Cellulose-degrading bacteria were isolated and identified from cocopeat which has a good quality as a bulking agent in composting. Various bacteria from different sourecs of cocopeat were detected on CMC agar media, and these were found to be Burkholderi2a sp., Bacillu subtilis, Sphingomonas sp., Rhodotorula sp. & Pseudomonas sp. etc. Among these, four bacteria were further selected and analyzed for their biochemical characteristics and CMCase activities. CMCase activities of four bacteria, P. aeruginosa, P. stutzeri, B. subtilis, and P. luteola were found to be 83%, 40%, 8%, 6%, respectively, compared with that of the standard strain Cellulomonas sp.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1339-1348
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• 2015

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

• Korean journal of applied entomology
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• v.26 no.2 s.71
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• pp.89-97
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• 1987

Antagonistic microorganisms from rhizosphere soil were isolated, identified, and applied successfully as the biocontrol agents of damping-off caused by Rhizoctonia spp. Rhizosphere antagonists isolated from rhizosphere soil were identified as Trichoderma viride, T. harzianum, T. hamatum, T. polysporum, Gliocladium sp., Pseudomonas fluorescence, P. stutzeri, P. cepacia, Enterobacter sp., Serratia sp. and Erwinia herbicola. Of these, the most promising ones in vitro were T. virdie, T. harzianum, Gliocladium sp., Serratia sp., P. stutzeri, and P. cepacia. These above six antagonists were efficient in reducing disease incidence to $40{\sim}70%$ when the reselected rhizosphere antagonists preparations were applied to the soil at $10^6$ propagules per gram. Among six antagonists, T. viride was the most promising biocontrol agents against R. solani isolates in soil. The suppressive effect was more evident in steam-sterilized soil than in non-sterilized field soil.

• Korean Journal of Environmental Agriculture
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• v.26 no.4
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• pp.332-336
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• 2007

Pseudomonas stutzeri strain KCCM 34719 was used in this experiment to determine the effects of increasing Pb(II) concentrations on its growth rate. To obtain optimum growth conditions, strain KCCM 34719 was cultivated in nutrient broth under various conditions, such as temperature, pH, and NaCl concentration. Optimal conditions for cell growth were $30^{\circ}C$ of temperature, 8.0 of pH, and 3% of NaCl concentration, respectively. Growth response of bacterial cell to Pb(II) showed tolerance to concentrations ranging from 10 to 100 mg ${\ell}^{-1}$ in liquid culture, following a growth pattern similar to the control. Growth rate was greatly inhibited at 200 mg ${\ell}^{-1}$ of Pb(II).

• Korean Journal of Environmental Agriculture
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• v.16 no.1
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• pp.55-60
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• 1997

Heavy metal-tolerant microorganisms, such as Pseudomonas putida, P. aeruginosa, P. chlororaphis and P. stutzeri which possessed the ability to accumulate cadmium, lead, zinc and copper, respectively, were isolated from industrial wastewaters and mine wastewaters polluted with various heavy metals. The distribution of heavy metal in the cell components, and amino acid compositions, was investigated. The distribution of heavy metal in the cell fractions of each heavy metal-tolerant microorganism grown for 20 hours in the basal medium containing 100mg/l of each heavy metal was investigated. In the case of cadmium-tolerant P. putida, lead-tolerant P. aeruginosa and copper-tolerant P. stutzeri, approximately $50{\sim}60%,\;30{\sim}40%$ and $10{\sim}17%$ of each heavy metal absorbed were distributed to cell wall, cell membrane and cytoplasm fractions, respectively. In the case of zinc-tolerant P. chlororaphis, approximately 32%, 55% and 13% of zinc were distributed to cell wall, cell membrane and cytoplasm fractions, respectively. These results indicated that the cell wall was a major adsorbing fraction of cadmium, lead and copper, and the cell membrane was that of zinc. Total amino acid content per gram of the cell grown in the culture media with heavy metal was higher than that of the cell grown in the culture media without heavy metal, and the content of acidic amino acids, such as aspartic acid(Asp.+Asn.) and glutamic acid(Glu.+Gln.) was higher than that of basic amino acids, such as histidine, lysine and arginine.

• Korean Journal of Microbiology
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• v.29 no.3
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• pp.195-198
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• 1991

Isolation and identification of mesophilic and psychrotrophic bacteria distributed in Korean refrigerated beef were attempted. Total isolated colonies were 192, and identified as 5 genera and 10 species. Among them, mesophilic bacteria were Enterobacter aerogenes, E. agglomerans, Serratia liquefaciens, Proteus mirabilis, and "psychrotrophic" bacteria were Pseudomons fluorescens, P. putida, P. pickettii, P. mendocina, P. stutzeri, Alcaligenes faecalis. Dominant species was Serratia liquefaciens as mesophiles, and Pseudomonas putida as psychrotroph.chrotroph.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.483-489
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• 2004

Pseudomonas sp. S-47 is a bacterium capable of degrading benzoate as well as 4-chlorobenzoate (4CBA). Benzoate and 4CBA are known to be degraded via a meta-cleavage pathway characterized by a series of enzymes encoded by xyl genes. The meta-cleavage pathway operon in Pseudomonas sp. S-47 encodes a set of enzymes which transform benzoate and 4CBA into TCA cycle intermediates via the meta-cleavage of (4-chloro )catechol to produce pyruvate and acetyl-CoA. In the current study, the meta-pathway gene cluster was cloned from the chromosomal DNA of S-47 strain to obtain pCS1, which included the degradation activities for 4CBA and catechol. The genetic organization of the operon was then examined by cloning the meta-pathway genes into a pBluescript SKII(+) vector. As such, the meta-pathway operon from Pseudomonas sp. S-47 was found to contain 13 genes in the order of xylXYZLTEGFlQKIH. The two regulatory genes, xylS and xylR, that control the expression of the meta-pathway operon, were located adjacently downstream of the meta-pathway operon. The xyl genes from strain S-47 exhibited a high nucleoside sequence homology to those from Pseudomonas putida mt-2, except for the xylJQK genes, which were more homologous to the corresponding three genes from P. stutzeri AN10. One open reading frame was found between the xylH and xylS genes, which may playa role of a transposase. Accordingly, the current results suggest that the xyl gene cluster in Pseudomonas sp. S-47 responsible for the complete degradation of benzoate was recombined with the corresponding genes from P. putida mt-2 and P. stutzeri AN10.

• Korean Journal of Environmental Agriculture
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• v.16 no.1
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• pp.48-54
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• 1997

This study was performed to develop the biological treatment technology of wastewater polluted with heavy metals. The copper-tolerant bacteria, Pseudomonas stutzeri which possessed the ability to accumulate copper, was isolated from mine wastewaters polluted with various heavy metals. The characteristics of copper accumulation in the cells and the recovery of the copper from the cells accumulating zinc, were investigated. Removal rate of copper from the solution containing 100mg/l of copper by copper-tolerant bacteria was more than 78% at 2 days after inoculation with the cells. A large number of the electron-dense granules were found mainly on the cell wall and cell membrane fractions, when determined by transmission electron microscopy. Energy dispersive X-ray spectroscopy revealed that the electron-dense granules were copper complex with the substances binding copper. The copper accumulated into the cells was not desorbed by deistilled water, but more than 80% of the copper accumulated was desorbed by 0.1M-EDTA solution. The residues of the cells after combustion at $550^{\circ}C$ amounted to about 23.2% of the dry weight of the cells. EDS analysis showed that residues were relatively pure copper compound containing more than 78.4% of copper.