Adaptation of Comamonas testosteroni TA441 to utilization of phenol by spontaneous mutation of the gene for a trans-acting factor

Comamonas testosferoni TA441 adapts to utilization of phenol upon incubatio n with phenol as the major carbon source. Strain TA441 has a cluster of gen es (aphKLMNOPQB) encoding the catabolic enzymes phenol hydroxylase and cate chol 2,3-dioxygenase, and a divergently transcribed regulatory gene (aphR), but these genes are silent until adaptation occurs. We found another regul atory gene (aphS) downstream of aphR. AphS belongs to the GntR family of tr anscriptional regulators. All adapted strains were found to have mutations in the aphS gene or in the aphR-aphS intervening region. The adapted strain s expressed phenol hydroxylase and catechol 2,3-dioxygenase activity in the presence of phenol. The transcriptional activity of both the aphK and the aphR promoters was elevated in the adapted strains. A strain whose aphS gen e was artificially disrupted was found to be able to grow using phenol, and the cells showed high levels of the above-mentioned transcriptional and en zymatic activities, indicating that adaptation was caused only by the mutat ion in the aphS gene. Gel retardation analysis revealed that AphS bound to two specific sites in the promoter region between aphK and aphR. These resu lts indicate that the active aphS gene product acts as a trans-acting facto r and represses transcription of the aph genes in strain TA441.