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Vol. 180, Issue 13, 3353-3359, July 1, 1998
Mutagenesis and Expression of amo, Which Codes for
Ammonia Monooxygenase in Nitrosomonas europaea
Norman G.
Hommes,
Luis A.
Sayavedra-Soto, and
Daniel J.
Arp
Laboratory for Nitrogen Fixation Research,
Oregon State University, Corvallis, Oregon 97331-2902
Nitrosomonas europaea has two copies of the operon
encoding ammonia monooxygenase (AMO). The nucleotide sequences of the
two copies of amoA were obtained, and they were found to
differ by one nucleotide. To determine if both copies of
amoA were functional, insertional mutagenesis was performed
to inactivate either copy of amoA alone. A DNA cassette
containing the lacZ and kan genes inserted into
amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA. Mutations were
obtained in both copies of amoA. Either copy of
amoA was sufficient to support growth when the other copy
was disrupted. However, inactivation of one copy of amoA,
but not the other, resulted in slower growth. Measurements of
ammonia-dependent O2 consumption, which depends on
AMO, confirmed that the slower-growing mutant had lower activity while
the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [14C]acetylene label
incorporation, there was less active AMO present in the slower-growing
mutant than in the faster-growing mutant or in the wild type. Northern
blot analysis of transcription likewise showed that the slower-growing
mutant had less full-sized AMO mRNA.
Copyright © 1998 by American Society for Microbiology
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