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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

Mapping of aromatic hydrocarbon catabolic genes obtained from Sphingobium sp. PNB in comparison to the related catabolic genes in other sphingomonads. Numerical value below each gene indicates its sequence identity with the homologous gene in strain PNB. Shaded regions between the maps of a pair of organisms represent the locus of homologous gene segments. Dotted lines indicate presence of genes, which are not related to aromatic hydrocarbon catabolism.
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f0005: Mapping of aromatic hydrocarbon catabolic genes obtained from Sphingobium sp. PNB in comparison to the related catabolic genes in other sphingomonads. Numerical value below each gene indicates its sequence identity with the homologous gene in strain PNB. Shaded regions between the maps of a pair of organisms represent the locus of homologous gene segments. Dotted lines indicate presence of genes, which are not related to aromatic hydrocarbon catabolism.

Mentions: Out of 1000 fosmid clones, eight supported desired PCR amplification with one and/or the other set of primer(s) corresponding to different RHO α-subunit genes (Table S1). Among them, fosmid clone FC-31 was found to harbor α-subunit gene specific for ahdA1f, clone FC-183 for ahdA1c and ahdA1d while clone FC-781 for ahdA1b, ahdA1e and xylX. Subcloning, screening based on the presence of RHO α-subunit gene(s) and sequencing, followed by sequence alignment and blast searches revealed the identification of putative ORFs. The subclones which did not serve as template for the amplification of RHO α-subunit gene(s), were also sequenced using M13 forward and reverse primers and analyzed. Those which showed the presence of putative genes involved in the metabolism of aromatic compounds were further sequenced by primer walking and analyzed as described above to identify additional putative genes and proteins of the degradative gene clusters. Further, gaps between genes expected to be in close proximity were bridged by a conventional primer walking method, using primers designed from the sequences at the proximal ends of the genes. Examination of sequence revealed 37 complete, 5 partial and 2 disrupted ORFs. Putative genes and proteins, identified from the above analyses are listed in Table S2. Based on protein sequence homology and conserved domain analyses, a number of genes are likely to be involved in PAHs or other aromatic degradation pathways. Sequenced aromatic catabolic gene clusters of strain PNB were mapped and compared with the homologous gene clusters reported in various sphingomonads (Fig. 1).


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)

Mapping of aromatic hydrocarbon catabolic genes obtained from Sphingobium sp. PNB in comparison to the related catabolic genes in other sphingomonads. Numerical value below each gene indicates its sequence identity with the homologous gene in strain PNB. Shaded regions between the maps of a pair of organisms represent the locus of homologous gene segments. Dotted lines indicate presence of genes, which are not related to aromatic hydrocarbon catabolism.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0005: Mapping of aromatic hydrocarbon catabolic genes obtained from Sphingobium sp. PNB in comparison to the related catabolic genes in other sphingomonads. Numerical value below each gene indicates its sequence identity with the homologous gene in strain PNB. Shaded regions between the maps of a pair of organisms represent the locus of homologous gene segments. Dotted lines indicate presence of genes, which are not related to aromatic hydrocarbon catabolism.
Mentions: Out of 1000 fosmid clones, eight supported desired PCR amplification with one and/or the other set of primer(s) corresponding to different RHO α-subunit genes (Table S1). Among them, fosmid clone FC-31 was found to harbor α-subunit gene specific for ahdA1f, clone FC-183 for ahdA1c and ahdA1d while clone FC-781 for ahdA1b, ahdA1e and xylX. Subcloning, screening based on the presence of RHO α-subunit gene(s) and sequencing, followed by sequence alignment and blast searches revealed the identification of putative ORFs. The subclones which did not serve as template for the amplification of RHO α-subunit gene(s), were also sequenced using M13 forward and reverse primers and analyzed. Those which showed the presence of putative genes involved in the metabolism of aromatic compounds were further sequenced by primer walking and analyzed as described above to identify additional putative genes and proteins of the degradative gene clusters. Further, gaps between genes expected to be in close proximity were bridged by a conventional primer walking method, using primers designed from the sequences at the proximal ends of the genes. Examination of sequence revealed 37 complete, 5 partial and 2 disrupted ORFs. Putative genes and proteins, identified from the above analyses are listed in Table S2. Based on protein sequence homology and conserved domain analyses, a number of genes are likely to be involved in PAHs or other aromatic degradation pathways. Sequenced aromatic catabolic gene clusters of strain PNB were mapped and compared with the homologous gene clusters reported in various sphingomonads (Fig. 1).

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