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Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

Zampolli J, Collina E, Lasagni M, Di Gennaro P - AMB Express (2014)

Bottom Line: This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed.By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation.Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, Milano, 20126, Italy ; Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milano, Italy.

ABSTRACT
Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

No MeSH data available.


Related in: MedlinePlus

Kinetic analyses ofn-dodecane degradation inR. erytropolisAP (pTipQT1-alkR7) recombinant strain (square in the graph). Cells of the recombinant strain were grown on rich medium and then, after induction with thiostrepton, were collected and exposed to n-dodecane. For comparison, kinetics of n-dodecane degradation in R. erytropolis AP (triangle in the graph) without the cloned fragment expressing the alkB gene, is also showed.
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Figure 4: Kinetic analyses ofn-dodecane degradation inR. erytropolisAP (pTipQT1-alkR7) recombinant strain (square in the graph). Cells of the recombinant strain were grown on rich medium and then, after induction with thiostrepton, were collected and exposed to n-dodecane. For comparison, kinetics of n-dodecane degradation in R. erytropolis AP (triangle in the graph) without the cloned fragment expressing the alkB gene, is also showed.

Mentions: The alkB gene cluster was isolated from R7 genomic DNA as PCR product and cloned into the pDrive vector giving the plasmid pDrive-alkR7. The region was isolated as NcoI/NdeI fragment and cloned into the shuttle-vector E. coli-Rhodococcus pTipQT1. The recombinant plasmid pTipQT1-alkR7 was isolated from E. coli DH5a and transferred into Rhodococcus erythropolis AP, because the Ptip/regulator system of the shuttle-vector is more efficient in Rhodococcus erythropolis species (Nakashima and Tamura [2004b]). In order to verify the expression of the alkB gene cluster under the control of the Ptip/regulator system, experiments with resting cells of Rhodococcus erytropolis AP(pTipQT1-alkR7) exposed to C12 were performed. The expression of the alk region was determined comparing the biodegradation kinetics of the recombinant strain R. erythropolis AP (pTipQT1-alkR7) with the wild type strain R. erythropolis AP. Results are reported in Figure 4. The percentage of biodegradation in 6 h was near 80% in the recombinant strain and 37% in the wild type strain. From kinetics analysis we can observe that the initial degradation rate was higher in the recombinant strain with respect to the wild type strain indicating a difference in the activity levels of the alkB system. This difference was confirmed by a statistical test for the comparison of the slopes of the regression lines at 95% significance level.


Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

Zampolli J, Collina E, Lasagni M, Di Gennaro P - AMB Express (2014)

Kinetic analyses ofn-dodecane degradation inR. erytropolisAP (pTipQT1-alkR7) recombinant strain (square in the graph). Cells of the recombinant strain were grown on rich medium and then, after induction with thiostrepton, were collected and exposed to n-dodecane. For comparison, kinetics of n-dodecane degradation in R. erytropolis AP (triangle in the graph) without the cloned fragment expressing the alkB gene, is also showed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230829&req=5

Figure 4: Kinetic analyses ofn-dodecane degradation inR. erytropolisAP (pTipQT1-alkR7) recombinant strain (square in the graph). Cells of the recombinant strain were grown on rich medium and then, after induction with thiostrepton, were collected and exposed to n-dodecane. For comparison, kinetics of n-dodecane degradation in R. erytropolis AP (triangle in the graph) without the cloned fragment expressing the alkB gene, is also showed.
Mentions: The alkB gene cluster was isolated from R7 genomic DNA as PCR product and cloned into the pDrive vector giving the plasmid pDrive-alkR7. The region was isolated as NcoI/NdeI fragment and cloned into the shuttle-vector E. coli-Rhodococcus pTipQT1. The recombinant plasmid pTipQT1-alkR7 was isolated from E. coli DH5a and transferred into Rhodococcus erythropolis AP, because the Ptip/regulator system of the shuttle-vector is more efficient in Rhodococcus erythropolis species (Nakashima and Tamura [2004b]). In order to verify the expression of the alkB gene cluster under the control of the Ptip/regulator system, experiments with resting cells of Rhodococcus erytropolis AP(pTipQT1-alkR7) exposed to C12 were performed. The expression of the alk region was determined comparing the biodegradation kinetics of the recombinant strain R. erythropolis AP (pTipQT1-alkR7) with the wild type strain R. erythropolis AP. Results are reported in Figure 4. The percentage of biodegradation in 6 h was near 80% in the recombinant strain and 37% in the wild type strain. From kinetics analysis we can observe that the initial degradation rate was higher in the recombinant strain with respect to the wild type strain indicating a difference in the activity levels of the alkB system. This difference was confirmed by a statistical test for the comparison of the slopes of the regression lines at 95% significance level.

Bottom Line: This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed.By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation.Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, Milano, 20126, Italy ; Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milano, Italy.

ABSTRACT
Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

No MeSH data available.


Related in: MedlinePlus