Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.
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.
Hommes
N G
NG
Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis 97331-2902, USA.
Sayavedra-Soto
L A
LA
Arp
D J
DJ
D000114:Acetylene / Q000378:metabolism; D017931:DNA Primers; D005798:Genes, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009606:Nitrosomonas / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D009876:Operon; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C045582:ammonia monooxygenase; D001616:beta-Galactosidase; D000114:Acetylene