Poret-Peterson Amisha T, Graham James E, Gulledge Jay, Klotz Martin G
Evolutionary and Genomic Microbiology Laboratory, Department of Biology, University of Louisville, Louisville, KY 40292, USA.
ISME J. 2008 Dec;2(12):1213-20. doi: 10.1038/ismej.2008.71. Epub 2008 Jul 24.
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 CH(4) or CH(4) plus 5 mM (NH(4))(2)SO(4) 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.
荚膜甲基球菌巴斯菌株是一种甲烷氧化细菌，与氨氧化细菌（AOB）一样，通过中间产物羟胺进行硝化作用的第一步，即将氨氧化为亚硝酸盐。AOB利用羟胺氧化还原酶（HAO）生成亚硝酸盐。直到最近在荚膜甲基球菌巴斯菌株的基因组中发现hao基因之前，人们一直认为该菌株是利用细胞色素P460（cytL）氧化羟胺。我们对与CH₄或CH₄加5 mM硫酸铵（(NH₄)₂SO₄）一起培养的荚膜甲基球菌巴斯菌株的cDNA进行了定量PCR分析，以确定cytL和hao转录水平是否会因氨而发生变化。虽然cytL的mRNA水平不受氨的影响，但在分析转录本的启动子近端区域时，重复样本中hao的mRNA水平分别增加了14.5倍和31倍，而在分析转录本远端区域时增加了6倍。在所有已知的AOB基因组以及荚膜甲基球菌巴斯菌株中，位于hao 3'端的一个保守开放阅读框orf2与hao共转录，并且在有氨存在时mRNA水平升高。这些数据导致将该基因对命名为haoAB，其中haoB的作用仍未明确。我们还确定了编码参与N-氧化物解毒的蛋白质的其他基因的mRNA水平：细胞色素c'-β（CytS）和一氧化氮（NO）还原酶（NorCB）。虽然重复样本中cytS的mRNA水平因氨而分别增加了28.5倍和40倍，但共转录的norC-norB的mRNA水平并未增加。我们的结果有力地表明，荚膜甲基球菌巴斯菌株拥有一种参与硝化作用的功能性、对氨有反应的HAO。
