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Proteomic analysis of Pseudomonas putida reveals an organic solvent tolerance-related gene mmsB.

Ni Y, Song L, Qian X, Sun Z - PLoS ONE (2013)

Bottom Line: Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control.Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli.Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, PR China. yni@jiangnan.edu.cn

ABSTRACT
Organic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD(660), whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.

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2-DE images of protein extracts of P. putida JUCT1 grown under different solvent conditions.a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.
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pone-0055858-g002: 2-DE images of protein extracts of P. putida JUCT1 grown under different solvent conditions.a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.

Mentions: Various chaotropes, surfactants, and reducing agents were added to extract the total cellular protein of P. putida JUCT1, and about 6 mg protein was extracted from 0.1 g wet cells. From the image of 2-D gels, 486 spots were detected (Fig. 2). The quantity of total protein spots is similar to P. putida UW4 in a previous report [28]. In total cellular protein of P. putida JUCT1 grown in the presence of 60% (v/v) cyclohexane (Fig. 2b), the expression level of 22 proteins were detected to be significantly higher than their counterpart without solvent (Fig. 2a), showing over 50% discrepancies in intensity values between two samples. In the 2-DE images, a majority of 22 protein spots are low-abundance proteins, and 5 high-abundance proteins whose expression levels were up-regulated for over 60% were chosen for further study (Fig. 2b, 2c).


Proteomic analysis of Pseudomonas putida reveals an organic solvent tolerance-related gene mmsB.

Ni Y, Song L, Qian X, Sun Z - PLoS ONE (2013)

2-DE images of protein extracts of P. putida JUCT1 grown under different solvent conditions.a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569466&req=5

pone-0055858-g002: 2-DE images of protein extracts of P. putida JUCT1 grown under different solvent conditions.a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.
Mentions: Various chaotropes, surfactants, and reducing agents were added to extract the total cellular protein of P. putida JUCT1, and about 6 mg protein was extracted from 0.1 g wet cells. From the image of 2-D gels, 486 spots were detected (Fig. 2). The quantity of total protein spots is similar to P. putida UW4 in a previous report [28]. In total cellular protein of P. putida JUCT1 grown in the presence of 60% (v/v) cyclohexane (Fig. 2b), the expression level of 22 proteins were detected to be significantly higher than their counterpart without solvent (Fig. 2a), showing over 50% discrepancies in intensity values between two samples. In the 2-DE images, a majority of 22 protein spots are low-abundance proteins, and 5 high-abundance proteins whose expression levels were up-regulated for over 60% were chosen for further study (Fig. 2b, 2c).

Bottom Line: Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control.Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli.Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, PR China. yni@jiangnan.edu.cn

ABSTRACT
Organic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD(660), whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.

Show MeSH
Related in: MedlinePlus