title: Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB.
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.
authors.affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.
authors.forename: Pratick
authors.initials: P
authors.lastname: Khara
authors.affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.
authors.forename: Madhumita
authors.initials: M
authors.lastname: Roy
authors.affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.
authors.forename: Joydeep
authors.initials: J
authors.lastname: Chakraborty
authors.affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.
authors.forename: Debajyoti
authors.initials: D
authors.lastname: Ghosal
authors.affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Dutta
keywords: Catabolic genes; ET, electron transport; GR, glutathione reductase; IPTG, isopropyl-β-thiogalactopyranoside; ISP, iron-sulfur protein; NBB, n-butylboronate; NR, non-redundant; PAHs, polycyclic aromatic hydrocarbons; PDB, Protein Data Bank; Polycyclic aromatic hydrocarbons; RHO, ring-hydroxylating oxygenase; Sphingobium sp.; Sphingomonads; Substrate specificities