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Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB.

Khara P, Roy M, Chakraborty J, Ghosal D, Dutta TK - FEBS Open Bio (2014)

Bottom Line: Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins.The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads.The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.

ABSTRACT
Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

No MeSH data available.


Related in: MedlinePlus

Real-time PCR analysis of genes in Sphingobium sp. PNB involved in the metabolism of aromatic hydrocarbons. Heat map representing expression levels of different genes, induced with phenanthrene and biphenyl. The fold change is shown in shades of red, yellow and green which indicate decreased, unchanged and increased levels of expression, respectively. Fold change denotes change in expression level of a gene in induced cells compared to the uninduced (succinate grown) cells. Double slash (//) represents gap between distantly located genes or genes present in different loci. Orientation of catA, identified in different loci, with respect to sequenced gene clusters is not known. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0025: Real-time PCR analysis of genes in Sphingobium sp. PNB involved in the metabolism of aromatic hydrocarbons. Heat map representing expression levels of different genes, induced with phenanthrene and biphenyl. The fold change is shown in shades of red, yellow and green which indicate decreased, unchanged and increased levels of expression, respectively. Fold change denotes change in expression level of a gene in induced cells compared to the uninduced (succinate grown) cells. Double slash (//) represents gap between distantly located genes or genes present in different loci. Orientation of catA, identified in different loci, with respect to sequenced gene clusters is not known. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: Results obtained from real-time PCR analyses with cDNA synthesized from the respective RNAs isolated from cells grown in presence of either phenanthrene or biphenyl are shown in Fig. 5. With the exception of ahdA1b and ahdA4, most of the genes were overexpressed in phenanthrene-grown cells as compared with succinate-grown cells. However, ahdA1b, ahdA4, nahD, ahdC or xylE were not overexpressed in biphenyl-grown cells. On the other hand, catA (GenBank: KC683533), encoding catechol 1,2-dioxygenase, was found to be upregulated in biphenyl grown cells but marginally downregulated in phenanthrene grown culture.


Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB.

Khara P, Roy M, Chakraborty J, Ghosal D, Dutta TK - FEBS Open Bio (2014)

Real-time PCR analysis of genes in Sphingobium sp. PNB involved in the metabolism of aromatic hydrocarbons. Heat map representing expression levels of different genes, induced with phenanthrene and biphenyl. The fold change is shown in shades of red, yellow and green which indicate decreased, unchanged and increased levels of expression, respectively. Fold change denotes change in expression level of a gene in induced cells compared to the uninduced (succinate grown) cells. Double slash (//) represents gap between distantly located genes or genes present in different loci. Orientation of catA, identified in different loci, with respect to sequenced gene clusters is not known. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048848&req=5

f0025: Real-time PCR analysis of genes in Sphingobium sp. PNB involved in the metabolism of aromatic hydrocarbons. Heat map representing expression levels of different genes, induced with phenanthrene and biphenyl. The fold change is shown in shades of red, yellow and green which indicate decreased, unchanged and increased levels of expression, respectively. Fold change denotes change in expression level of a gene in induced cells compared to the uninduced (succinate grown) cells. Double slash (//) represents gap between distantly located genes or genes present in different loci. Orientation of catA, identified in different loci, with respect to sequenced gene clusters is not known. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: Results obtained from real-time PCR analyses with cDNA synthesized from the respective RNAs isolated from cells grown in presence of either phenanthrene or biphenyl are shown in Fig. 5. With the exception of ahdA1b and ahdA4, most of the genes were overexpressed in phenanthrene-grown cells as compared with succinate-grown cells. However, ahdA1b, ahdA4, nahD, ahdC or xylE were not overexpressed in biphenyl-grown cells. On the other hand, catA (GenBank: KC683533), encoding catechol 1,2-dioxygenase, was found to be upregulated in biphenyl grown cells but marginally downregulated in phenanthrene grown culture.

Bottom Line: Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins.The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads.The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.

ABSTRACT
Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

No MeSH data available.


Related in: MedlinePlus