Cloning and characterization of the regulatory genes phlR1 and phlR2 involved in phenol metabolism from Alcaligenes eutrophus JMP134.

One mutant (AEK201) of Alcaligenes eutrophus JMP134 deficient in phenol metabolism was isolated by transposon mutagenesis using pSUP2021, a suicide plasmid. The 14.5 kb EcoRI fragment containing Tn5 and flanking DNA was cloned from AEK201 and used to probe a gene bank of wild type by colony hybridization. All five positive cosmids isolated rendered AEK201 to grow on phenol. The data from subcloning revealed that a trans-acting factor encoded on the 2.3 kb SalI-HindIII fragment, which is common to all cosmids, allowed the mutant to restore three enzyme activities tested (phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase). This fragment seems to act as a positive regulator on the entire phenol pathway. Another regulatory segment was subcloned from the 16.8 kb HindIII fragment on which phenol hydroxylase and catechol 2,3-dioxygenase activities were carried [Kim, Y., Ayoubi, P., and Harker, A. R. (1996) Appl. Environ. Microbiol. 62, 3227-3233]. The expression of phenol hydroxylase activity was entirely repressed in the presence of this segment in Pseudomonas aeruginosa PAO1c, but the enzyme activity was increased in A. eutrophus AEK301, suggesting that this trans-acting factor is both an activator and a repressor for phenol hydroxylase. Possible regulatory mechanisms for the phenol pathway in A. eutrophus JMP134 are discussed.