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Applied and Environmental Microbiology, March 2006, p. 1825-1832, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1825-1832.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6

Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan

Received 26 October 2005/ Accepted 16 December 2005

We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphR_IC_IA2_IA3_IB_IA1_I and tphR_IIC_IIA2_IIA3_IIB_IIA1_II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2_I or tphA2_II singly; however, the tphA2_I tphA2_II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphR_IIC_IIA2_IIA3_IIB_IIA1_II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphR_II or tphC_II on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1_II, tphA2_II, tphA3_II, and tphB_II were expressed in Escherichia coli, and the resultant cell extracts containing TphA1_II, TphA2_II, and TphA3_II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphB_II. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.

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