• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1125-1131
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• 2006

A kan'::luxCDABE fusion strain that was both highly bioluminescent and responsive to benzoic acid was constructed by transforming E. coli strain W3110 with the plasmid pUCDK, which was constructed by digesting and removing the 7-kb KpnI fragment from the promoterless luxCDABE plasmid pUCD615. Experiments using buffered media showed that this induction was dependent on the pH of the media, which influences the degree of benzoic acid protonation, and the expression levels seen are likely due to acidification of the cytoplasm by uncoupling of benzoic acid. Consequently, the sensitivity of this strain for benzoic acid was increased by nearly 20-fold when the pH was shifted from 8.0 to 6.5. Benzoic acid derivatives and several phenolics also resulted in significantly increased bioluminescent signals. Although these compounds are known to damage membranes and induce the heat-shock response within E. coli, bacterial strains harboring mutations in the fadR and rpoH genes, which are responsible for fatty acid biosynthesis during membrane stress and induction of the heat-shock response, respectively, showed that these mutations had no effect on the responses observed.

• Korean Journal of Plant Resources
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• v.11 no.3
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• pp.245-251
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• 1998

Test plants with 10 days old primary leaves were indouclated by shaking infected seedlings with sporulating colonies over them in an inoculation room under the conditions of 20$\pm$1 $^{\circ}C$ with constant illumination of 2.500 lux and 100% realtive humidity. A seeding reaction of 4 days after inoculation appreared in the trisomic types as opposed to Tri-5B line had been symtoms of a fungus 3 days after inoculation. The infection types of 8 days after inoculation were recognized with higher susceptibility to each trisomics in A genomie than B-genome. Tri-2A line showed less condium and there appeared symptoms of a conditions of mottle and formed papilla, and haustorium was not formed. However, Tri-5B line had much condium one overall leaves and showed a symtom like necrosis compared with normal plant. Moreover, Tri-5B line showed high sensitivity and high germination number of condium. These results inferred that resistant gene located on 2A chromosome and susceptibility gene is located on the chromosome 5B.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.277-285
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• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.

• Journal of Life Science
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• v.25 no.2
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• pp.136-150
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• 2015

Pseudomonas syringae pathovar tabaci is a plant pathogenic bacterium that causes wildfire disease in tobacco plants. In P. syringae pv. tabaci, PsyI, a LuxI-type protein, acts as an AHL synthase, while primary and secondary sequence analysis of PsyR has revealed that it is a homolog of the LuxR-type transcriptional regulator that responds to AHL molecules. In this study, using phenotypic and genetic analyses in P. syringae pv. tabaci, we show the effect of PsyR protein as a quorum-sensing (QS) transcriptional regulator. Regulatory effects of PsyR on swarming motility and production of siderophores, tabtoxin, and N-acyl homoserine lactones were examined via phenotypic assays, and confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Further qRT-PCR showed that PsyR regulates expression of these virulence genes in response to environmental signals. However, an upstream region of the gene was not bound with purified MBP-PsyR protein; rather, PsyR was only able to shift the upstream region of psyI. These results suggested that PsyR may be indirectly controlled via intermediate-regulatory systems and that auto-regulation by PsyR does not occur.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.828-836
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• 2008

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain MI8. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of N-acylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.325-334
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• 2007

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the smcR mutant was approximately $10^2$ times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.3
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• pp.202-206
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• 2000

The genetically-engineered Escherichia coli strain, DPD2540, which contains a fabA:::luxCDAbefusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more pactical application of this strain in the filed as biosensor, freezedrying was adopted. A 12% surcrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be most most effective composition for lyophilization solution among various compositions testitons tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at-7$0^{\circ}C$ and -2$0^{\circ}C$. The biosensing activities of the cells showed a greater sensitivity when the cells from the expontial phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biodencor field was determined.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1644-1653
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• 2014

Wuyiencin is produced by Streptomyces ahygroscopicus var. wuyiensis CK-15 and is widely used as an antifungal agent in agriculture. Analysis of wuyiencin biosynthetic gene clusters reveals wysR, a member of the LAL-family of transcriptional regulatory genes. WysR consists of an N-terminal PAS domain and a LuxR family C-terminal helix-turn-helix motif. However, the roles of wysR in wuyiencin biosynthesis are largely unknown. In this study, we showed that inactivation of wysR resulted in the complete loss of wuyiencin production, which could be restored by complementation with a single copy of wysR. Furthermore, we successfully increased wuyiencin production to a significantly higher level by overexpression of wysR in S. wuyiensis CK-15. Quantitative real-time RT-PCR analysis showed that WysR regulates wuyiencin biosynthesis by modulating other putative regulatory genes. Thus, WysR was identified as an activator of wuyiencin biosynthesis, and overexpression of wysR gene proved to be an effective strategy for improving wuyiencin production.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2104-2111
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• 2017

A new series comprising phenylacetyl-homoserine lactones (HSLs), caffeoyl-HSL and feruloyl-HSL, was biologically synthesized using an artificial de novo biosynthetic pathway. We developed an Escherichia coli system containing artificial biosynthetic pathways that yield phenylacetyl-HSLs from simple carbon sources. These artificial biosynthetic pathways contained the LuxI-type synthase gene (rpaI) in addition to caffeoyl-CoA and feruloyl-CoA biosynthetic genes, respectively. Finally, the yields for caffeoyl-HSL and feruloyl-HSL were $97.1{\pm}10.3$ and $65.2{\pm}5.7mg/l$, respectively, by tyrosine-overproducing E. coli with a $\text\tiny{L}$-methionine feeding strategy. In a quorum sensing (QS) competition assay, feruloyl-HSL and p-coumaroyl-HSL antagonized the QS receptor TraR in Agrobacterium tumefaciens NT1, whereas caffeoyl-HSL did not.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.94-99
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• 2000

Bioluminescence is being used as a prevailing reporter of gene expression in microorganisms and mammalian cells. Bacterial bioluminescence draws special attention from environmental biotechnologists since it has many advantageous characteristics, such as no requirement of extra substractes, highly sensitive, and on-line measurability. Using bacterial bioluminescence as a reporter of toxicity has replaced the classical toxicity monitoring technology of using fish or daphnia with a cutting-edge technology. Fusion of bacterial stress promoters, which control the transcription of stress genes corresponding to heat-shock, DNA-, or oxidative-damaging stress, to the bacterial lux operon has resulted in the development of novel toxicity biosensors with a short measurement time, enhanced sensitivity, and ease and convenient usage. Therefore, these recombinant bioluminescent bacteria are expected to induce bacterial bioluminescence when the cells are exposed to stressful conditions, including toxic chemicals. We have used these recombinant bioluminescent bacteria in order to develop toxicity biosensors in a continuous, portable, or in-situ measurement from for air, water, and soil environments. All the data obtained from these toxicity biosensors for these environments were found to be repeatable and reproducible, and the minimum detection level of toxicity was found to be ppb (part per billion) levels for specific chemicals.