MUTAGENESIS AND EXPRESSION OF AMO, WHICH CODES FOR AMMONIA MONOOXYGENASE IN NITROSOMONAS-EUROPAEA

Nitrosomonas europaea has two copies of the operon encoding ammonia mo nooxygenase (AR IO). The nucleotide sequences of the two copies of amo A were obtained, and they were found to differ by one nucleotide. To d etermine if both copies of amoA were functional, insertional mutagenes is was performed to inactivate either copy of amoA alone. A DNA casset te containing the lacZ and kan genes inserted into amoA was constructe d. Mutagenesis was done by using transformation and homologous recombi nation to mobilize the cassette into the chromosomal copies of amoA, M utations were obtained in both copies of amoA. Either copy of amoA was sufficient to support growth when the other copy was disrupted. Howev er, inactivation of one copy of amoA, but not the other, resulted in s lower growth, Measurements of ammonia dependent O-2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower ac tivity while the faster-growing mutant had near wild-type levels of ac tivity. Similarly, as measured by [C-14]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than i n the faster-growing mutant or in the wild type. Northern blot analysi s of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.