(A) Proposed initial reactions of ONP catabolism catalyzed by the onpABC gene products in vivo. (B) Organization of the ONP gene cluster involved in the initial reactions of ONP degradation in Alcaligenes sp. strain NyZ215. The arrows indicate the sizes and directions of transcription of each ORF or gene. The arrow above onpC indicates the region where the genome walking started in both directions. Plasmid pZWXW1 was constructed by cloning the 5-kb fragment from the first cycle of genome walking into T-vector. E. coli containing this plasmid exhibited ONP 2-monooxygenase activity. The locations of primer sets RTA, RTB, RTC, RTAB, and RTBC and the amplified DNA fragments for RT-PCR are indicated. (C) Analysis of onpABC transcription by RT-PCR. Total RNAs of ONP-grown strain NyZ215 were prepared for RT-PCR, and reactions performed without RT were used as negative controls. Lanes M, molecular size markers; lanes 2, 4, 6, 8, and 10, products amplified using the RTA, RTB, RTC, RTAB and RTBC primer sets, respectively, with products of RT; lanes 1, 3, 5, 7, and 9, corresponding negative controls.