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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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Effect of organic solvents on cell growth of P. putida JUCT1 (open bar) and its parent strain JUCS (gray bar).The strains were initially grown in nutrient medium at 37°C till OD660 reached 0.2, and then 60% (v/v) organic solvent was added for further incubation of 5 h.
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pone-0055858-g001: Effect of organic solvents on cell growth of P. putida JUCT1 (open bar) and its parent strain JUCS (gray bar).The strains were initially grown in nutrient medium at 37°C till OD660 reached 0.2, and then 60% (v/v) organic solvent was added for further incubation of 5 h.

Mentions: An OST strain P. putida JUCT1 was obtained through gradient adaptation in medium containing cyclohexane over 12 serial transfers. Fig. 1 shows the tolerant level of P. putida JUCT1 towards various organic solvents including decalin (Log Pow = 4.8), methyl cyclohexane (Log Pow = 3.7), cyclohexane (Log Pow = 3.2), and toluene (Log Pow = 2.5). Log Pow is defined as the common logarithm of a partition coefficient (Pow) of the solvent between n-octanol and water, and is regarded as an index of the solvent toxicity for most microorganisms [6], [10]. Compared with cell growth in medium without solvent (1.0 OD660 for both P. putida JUCT1 and JUCS), OST adapted strain P. putida JUCT1 could grow well in the presence of 60% (v/v) of all solvents tested. Cell densities (OD660) of 0.94 (decalin), 0.91 (methyl cyclohexane), 0.86 (cyclohexane) and 0.78 (toluene) were attained after 5-h incubation in various solvents. In contrast, the growth of parent strain P. putida JUCS was almost restrained by high concentration of toluene (OD660 increase <0.1), while around 0.15 to 0.45 OD660 increase was observed with decalin, methyl cyclohexane and cyclohexane. Our results also indicate that the Log Pow of organic solvent is closely related to their inhibition on cell growth, the lower the Log Pow value, the higher the toxicity of the solvent [9], [10].


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

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

Effect of organic solvents on cell growth of P. putida JUCT1 (open bar) and its parent strain JUCS (gray bar).The strains were initially grown in nutrient medium at 37°C till OD660 reached 0.2, and then 60% (v/v) organic solvent was added for further incubation of 5 h.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055858-g001: Effect of organic solvents on cell growth of P. putida JUCT1 (open bar) and its parent strain JUCS (gray bar).The strains were initially grown in nutrient medium at 37°C till OD660 reached 0.2, and then 60% (v/v) organic solvent was added for further incubation of 5 h.
Mentions: An OST strain P. putida JUCT1 was obtained through gradient adaptation in medium containing cyclohexane over 12 serial transfers. Fig. 1 shows the tolerant level of P. putida JUCT1 towards various organic solvents including decalin (Log Pow = 4.8), methyl cyclohexane (Log Pow = 3.7), cyclohexane (Log Pow = 3.2), and toluene (Log Pow = 2.5). Log Pow is defined as the common logarithm of a partition coefficient (Pow) of the solvent between n-octanol and water, and is regarded as an index of the solvent toxicity for most microorganisms [6], [10]. Compared with cell growth in medium without solvent (1.0 OD660 for both P. putida JUCT1 and JUCS), OST adapted strain P. putida JUCT1 could grow well in the presence of 60% (v/v) of all solvents tested. Cell densities (OD660) of 0.94 (decalin), 0.91 (methyl cyclohexane), 0.86 (cyclohexane) and 0.78 (toluene) were attained after 5-h incubation in various solvents. In contrast, the growth of parent strain P. putida JUCS was almost restrained by high concentration of toluene (OD660 increase <0.1), while around 0.15 to 0.45 OD660 increase was observed with decalin, methyl cyclohexane and cyclohexane. Our results also indicate that the Log Pow of organic solvent is closely related to their inhibition on cell growth, the lower the Log Pow value, the higher the toxicity of the solvent [9], [10].

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