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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.

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Related in: MedlinePlus

Cultural broth analysis by GC-MSD. Cells of R. opacus R7 were exposed to n-dodecane and at different times, the cultural broth were analysed in GC-MSD. The intermediate metabolites identified are reported in the graph.
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Figure 2: Cultural broth analysis by GC-MSD. Cells of R. opacus R7 were exposed to n-dodecane and at different times, the cultural broth were analysed in GC-MSD. The intermediate metabolites identified are reported in the graph.

Mentions: Metabolic intermediates of n-alkanes in R. opacus R7 were determined from cultural broths during the growth on n-dodecane as reference substrate, chosen on the basis of the kinetic parameters determined for biodegradation runs. R7 growth on C12 was followed during the exposition to the hydrocarbon as a function of time. At time 0 h, 3 h, 5 h, 8 h and 24 h, culture samples were acidified, extracted, and analyzed by GC-MSD as reported in Material and Methods. Cultural broth analysis showed the presence of 1-dodecanol and the corresponding carboxylic acid (Figure 2) which were identified by comparison with the relative standards. We identified the 1-dodecanol only and not the mixture of 1-dodecanol and 2-dodecanol, as reported in literature for many cases of Rhodococcus strains (Whyte et al., [1998]; Whyte et al., [2002]). To confirm these data, both 1-dodecanol and 2-dodecanol were supplied separately as carbon and energy source and growth was observed only in presence of 1-dodecanol and no growth was observed in presence of 2-dodecanol. Moreover, we observed growth when the dodecanoic acid was supplied as the only carbon and energy source.


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)

Cultural broth analysis by GC-MSD. Cells of R. opacus R7 were exposed to n-dodecane and at different times, the cultural broth were analysed in GC-MSD. The intermediate metabolites identified are reported in the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Cultural broth analysis by GC-MSD. Cells of R. opacus R7 were exposed to n-dodecane and at different times, the cultural broth were analysed in GC-MSD. The intermediate metabolites identified are reported in the graph.
Mentions: Metabolic intermediates of n-alkanes in R. opacus R7 were determined from cultural broths during the growth on n-dodecane as reference substrate, chosen on the basis of the kinetic parameters determined for biodegradation runs. R7 growth on C12 was followed during the exposition to the hydrocarbon as a function of time. At time 0 h, 3 h, 5 h, 8 h and 24 h, culture samples were acidified, extracted, and analyzed by GC-MSD as reported in Material and Methods. Cultural broth analysis showed the presence of 1-dodecanol and the corresponding carboxylic acid (Figure 2) which were identified by comparison with the relative standards. We identified the 1-dodecanol only and not the mixture of 1-dodecanol and 2-dodecanol, as reported in literature for many cases of Rhodococcus strains (Whyte et al., [1998]; Whyte et al., [2002]). To confirm these data, both 1-dodecanol and 2-dodecanol were supplied separately as carbon and energy source and growth was observed only in presence of 1-dodecanol and no growth was observed in presence of 2-dodecanol. Moreover, we observed growth when the dodecanoic acid was supplied as the only carbon and energy source.

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