A novel degradation pathway of chloroaniline in Diaphorobacter sp. PCA039 entails initial hydroxylation

Abstract

A novel degradation pathway of chloroaniline in Diaphorobacter sp. PCA039 entails initial hydroxylation. A novel gene cluster (pca), involved in the degradation of p-chloroaniline, was identified from Diaphorobacter sp. PCA039 capable of utilizing p-chloroaniline as sole carbon, nitrogen and energy source for growth. There were 29 ORFs in the pca cluster, in the order of pcaTRKLMNOPR1D1CIU1R2D2C2U2EFGXJHYWZI1I2QS. Based on sequence analysis, they were putatively identified as encoding a multicomponent phenol-hydroxylating oxygenase (pcaKLMNOP), two extradiol ring-cleavage dioxygenases, transcriptional regulatory proteins, enzymes mediating chlorocatechol degradation, and transportation functions. The genes pcaKLMNOP exhibited significant sequence identity (94%) to those of phenol hydroxylases (PH) from other bacteria, inferring that they might encode a multicomponent PH. This PH activity was also functionally characterized with the recombinant strain E. coli TOP10-S201 showing only PH activity, indicating that the degradation of p-chloroaniline by strain PCA039 was initiated by hydroxylation instead of normal dioxygenation. The other nineteen genes, pcaR1D1CIU1R2D2C2U2EFGXJHYWZI1I2, encode for further degradation of p-chloroaniline to intermediates of the TCA-cycle and transposases. RT-PCR revealed that the hydrolytic (pcaF) and dehydrogenetic pathways (pcaE, pcaH), the two degrading branches, are both necessary for complete degradation of aniline, p-chloroaniline, phenol and also 4-aminophenol; and of the two C23O sets, PcaR1D1C1U1 and PcaR2D2C2U2, PcaC1 is produced in the degradation of above four substrates, while PcaC2 is only expressed in p-chloroaniline degradation, suggesting that both C23O sets are needed for complete degradation of p-chloroaniline.