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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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Colony formation of E. coli JM109 strains over-expressing mmsB, tsf, and PSEEN0851 on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.
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pone-0055858-g005: Colony formation of E. coli JM109 strains over-expressing mmsB, tsf, and PSEEN0851 on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.

Mentions: Colony-formation efficiency, a commonly used experiment to evaluate the OST of microbial cells [25], was also adopted in this study to examine the effect of three genes. Approximately 107, 106, 105, 104, and 103 cells were dropped in the spots on agar plate which was then overlaid with decalin (Fig. 5). Similar as liquid cultivation in 4% cyclohexane, mmsB transformants exhibited the highest OST among three genes. The colony-formation efficiency of E. coli JM109 was markedly increased by over-expression of PSEEN0851. The expression of tsf also slightly increased the solvent tolerance of JM109. Therefore, three genes (mmsB, tsf and PSEEN0851) identified in 2-DE analysis of P. putida JUCT1 could be associate with the OST performance of other Gram-negative strains such as E. coli.


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

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

Colony formation of E. coli JM109 strains over-expressing mmsB, tsf, and PSEEN0851 on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055858-g005: Colony formation of E. coli JM109 strains over-expressing mmsB, tsf, and PSEEN0851 on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.
Mentions: Colony-formation efficiency, a commonly used experiment to evaluate the OST of microbial cells [25], was also adopted in this study to examine the effect of three genes. Approximately 107, 106, 105, 104, and 103 cells were dropped in the spots on agar plate which was then overlaid with decalin (Fig. 5). Similar as liquid cultivation in 4% cyclohexane, mmsB transformants exhibited the highest OST among three genes. The colony-formation efficiency of E. coli JM109 was markedly increased by over-expression of PSEEN0851. The expression of tsf also slightly increased the solvent tolerance of JM109. Therefore, three genes (mmsB, tsf and PSEEN0851) identified in 2-DE analysis of P. putida JUCT1 could be associate with the OST performance of other Gram-negative strains such as E. coli.

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