Sasoh, M; Masai, E; Ishibashi, S; Hara, H; Kamimura, N; Miyauchi, K; Fukuda, M
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(I)C(I)A2(I)43(I)B(I)
Al(I), and tphRu(II)C(II)A2(II)A3(III)B(II)A1(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 IcIR-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(II)C(II)A2(II)A3(II)B(II)A1(II) conferred the TPA
utilization phenotype on Comamonas testosteroni LAM 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 LAM 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.