title: Degradation Pathways of 2- and 4-Nitrobenzoates in Cupriavidus sp. Strain ST-14 and Construction of a Recombinant Strain, ST-14::3NBA, Capable of Degrading 3-Nitrobenzoate.
abstract: Strain ST-14, characterized as a member of the genus Cupriavidus, was capable of utilizing 2- and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2- and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed in Escherichia coli identified two gene clusters, onb and pob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2- and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain.
  
  
  
  Mononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate a Cupriavidus sp. strain capable of assimilating both 2- and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.
authors.affiliation: Department of Microbiology, Bose Institute, Kolkata, India.
authors.forename: Soumik
authors.initials: S
authors.lastname: Basu
authors.affiliation: Department of Microbiology, Bose Institute, Kolkata, India.
authors.forename: Piyali
authors.initials: P
authors.lastname: Pal Chowdhury
authors.affiliation: Department of Microbiology, Bose Institute, Kolkata, India.
authors.forename: Satamita
authors.initials: S
authors.lastname: Deb
authors.affiliation: Department of Microbiology, Bose Institute, Kolkata, India tapan@jcbose.ac.in.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Dutta
mesh_terms: D002244:Carbon / Q000378:metabolism; D049921:Cupriavidus / Q000235:genetics* / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D009579:Nitrobenzoates / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D009579:Nitrobenzoates; D002244:Carbon