• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• Biomedical Science Letters
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• v.5 no.1
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• pp.27-40
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• 1999

This study was focused on the evaluation of MarB alongside with MarA for its regulatory effects upon the efflux function of AcrAB pump, which were induced or not, perhaps as a target. Transductions of marR and/or acrAB mutation which were derived from Mar and/or AcrAB mutants of wild type E. coli K-12, respectively, into the multicopy plasmid in wild type E. coli backgrounds or into the chromosome of isogenic parents were done. Minimal inhibitory concentration (MIC) of transduced mutants was compared with their original mutants. This study reports the indirect evidences that suggests a model in which MarB along with MarA have a putative negative regulatory effect upon the efflux function of AcrAB/TolC pump while MarA alone have a positive regulatory effect to the expression of acrRAB operon at transcription level. The target of MarB with MarA for its putative negative regulator might be the AcrAB efflux pump. Another efflux system (s) might be negatively regulated by MarB with MarA, and be involved in the efflux of antibiotics which were otherwise extruded preferentially by AcrAB efflux pump.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1605-1612
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• 2016

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.100-108
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• 1989

Thirty-six aromatic compound biodegraders; 10 strains for benzoate, 10 for salicylate, 6 for m-toluate, and 10 for DL-camphor were isolated and taxonomically characterized. A mutant Pseudomonas strain, Ben 6-2, derived from Ben 6 revealed remarkably improved ability to metabolize benzoate. Thus enhancement of the average substrate removal rate from 5.2 to 11.0mg/$\ell$/ hr was attained by the mutant. Both of strains Sal 7 and Tol 2, degraders of salicylate and m-toluate respectively, were classified as Pseudomonas sup. Both strains were found to be extremely effective in metabolizing each aromatic substrates. The average substrate degradation rates in minimal salt media containing 2,200mg/$\ell$ of the substrate were calculated to be 40.1 mg/$\ell$/ hr for strain Sal 7 and 33.0mg/$\ell$/ hr for Tol 2. Cam 10, a camphor degrading strain was demonstrated to be capable of mineralizing benzoate, phenol, toluene, octane, cyclohexane and xylene as well as camphor. Strain 1040 isolated from Cam 10 after repented adaptation to 1,000 mg/$\ell$ m-toluate gained the ability to utilize toluate as a sole carbon source. The mutant Brew actively at the expense of a mixture of car-bon sources; camphor, m-toluate, benzoate and phenol (each: 200 mg/$\ell$) and utilized the substances in the preferential order of camphor, phenol, benzoate, and m-toluate. Among the biodegraders examined Cam 1040 and Tol 2 were detected to harbor plasmid. The plasmid from Cam 1001 was determined to be about 98kb, and evidenced to encode the enzyme(s) for the degradation of camphor. For the further diversification of the metabolic potentials of Cam 1040, the NAH 2 plasmid of Pseudomonas putida NCIB 9816 was transferred to Cam 1040 by conjugation. The exconjugant obtained, Cam 1043, proved to gain an additional ability to metabolize salicylate and naphthalene.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.185-190
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• 2014

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from raw bulk milk and to characterize the resistance determinants. In this study, the gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced from quinolone resistant E. coli isolates. Also, the presence of plasmid-mediated quinolone resistance (PMQR) and the expression of efflux pump genes based on quantitative real-time PCR (qRT-PCR) were investigated. Of the 487 coliform bacteria, 9 strains showed nalidixic acid resistance, and 6 of the 9 nalidixic acid resistant isolates were also ciprofloxacin resistant. These 9 strains had a single mutation at codon 83 (S83L) in gyrA, 2 of them had double mutations at codon 83 and 87 (S83L and D87N) in gyrA and 3 of the 9 isolates had single mutations at codon 80 (S80I) in parC. None of the 9 isolates harbored PMQR determinants. Compared with wild-type E. coli ATCC 25922, an over-expression of the acrB gene (2.15-5.74 fold), encoding the pump component of the AcrAB-TolC efflux pump was observed in 4 of 6 ciprofloxacin resistant isolates. This study identified the quinolone resistance mechanism of E. coli isolated from raw milk samples in Gyeonggi-do.

• Journal of Korean Society of Environmental Engineers
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• v.27 no.5
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• pp.507-512
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• 2005

This research focuses on the development and application of a method for the detection of m-toluate in soils using a genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 produces light by direct (m-toluate and benzoate) and indirect (toluene analogs) inducers. For detection of m-toluate in soil system, 9.9 mL strain was amended with 0.1 mL soil ethanol extractant. A high correlation ($r^2>0.97$) was observed between bioluminescence and m-toluate concentration. The unknown concentrations of m-toluate in soil samples were pre-determined using a method developed based on bioluminescence activity of strain with extracted inducers. Values between by LC analysis and bioluminescence activity show moderate statistical results. These results demonstrate the feasibility of recombinant bioluminescent microorganism, engineered to generate a quantifiable bioluminescence signal in response to specific pollutants, may serve as combined sensing and reporting tools in environmental monitoring.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.700-705
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• 2003

Pseudomonas sp. S-47 is capable of degrading benzoate and 4-chlorobenzoate as well as catechol and 4-chlorocatechol via the meta-cleavage pathway. The three enzymes of 2-oxopenta-4-enoate hydratase (OEH), acetaldehyde dehydrogenase (acylating) (ADA), and 2-oxo-4-hydroxypentonate aldolase (HOA) encoded by xylJQK genes are responsible for the three steps after the meta-cleavage of catechol. The nucleotide sequence of the xylJQK genes located in the chromosomal DNA was cloned and analyzed. GC content of xylJ, xylQ, and xylK was 65% and consisted of 786, 924, and 1,041 nucleotides, respectively. The deduced amino acid sequences of xylJ, xylQ, and xylK genes from Pseudomonas sp. S-47 showed 93%, 99%, and 99% identity, compared with those of nahT, nahH, and nahI in Pseudomonas stutzeri An10. However, there were only about 53% to 85% identity with xylJQK of Pseudomonas putida mt-2, dmpEFG of P. putida CF600, aphEFG of Comamonas testosteroni TA441, and ipbEGF of P. putida RE204. On the other hand, the xylLTEGF genes located upstream of xylJQK in the strain S-47 showed high homology with those of TOL plasmid from Pseudomonas putida mt-2. These findings suggested that the xylLTEGFIJQK of Pseudomonas sp. S-47 responsible for complete degradation of benzoate and then catechol via the meta-pathway were phylogenetically recombinated from the genes of Pseudomonas putida mt-2 and Pseudomonas stutzeri An10.

• BMB Reports
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• v.35 no.4
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• pp.432-436
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• 2002

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.