Transcription of nitrification genes by the methane-oxidizing bacterium, Methylococcus capsulatus strain Bath

Methylococcus capsulatus strain Bath, a methane-oxidizing bacterium, and ammonia-oxidizing bacteria (AOB) carry out the first step of nitrification, the oxidation of ammonia to nitrite, through the intermediate hydroxylamine. AOB use hydroxylamine oxidoreductase (HAO) to produce nitrite. M. capsulatus Bath was thought to oxidize hydroxylamine with cytochrome P460 (cytL), until the recent discovery of an hao gene in its genome. We used quantitative PCR analyses of cDNA from M. capsulatus Bath incubated with CH4 or CH4 plus 5mM (NH4)(2)SO4 to determine whether cytL and hao transcript levels change in response to ammonia. While mRNA levels for cytL were not affected by ammonia, hao mRNA levels increased by 14.5- and 31-fold in duplicate samples when a promoter proximal region of the transcript was analyzed, and by sixfold when a region at the distal end of the transcript was analyzed. A conserved open reading frame, orf2, located 3' of hao in all known AOB genomes and in M. capsulatus Bath, was cotranscribed with hao and showed increased mRNA levels in the presence of ammonia. These data led to designating this gene pair as haoAB, with the role of haoB still undefined. We also determined mRNA levels for additional genes that encode proteins involved in N-oxide detoxification: cytochrome c'-beta (CytS) and nitric oxide ( NO) reductase (NorCB). Whereas cytS mRNA levels increased in duplicate samples by 28.5- and 40-fold in response to ammonia, the cotranscribed norC-norB mRNA did not increase. Our results strongly suggest that M. capsulatus Bath possesses a functional, ammonia-responsive HAO involved in nitrification.