Tchawa Yimga Merlin, Dunfield Peter F, Ricke Peter, Heyer Jürgen, Liesack Werner
Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany.
Appl Environ Microbiol. 2003 Sep;69(9):5593-602. doi: 10.1128/AEM.69.9.5593-5602.2003.
Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme.
开展实验以确定在甲烷氧化细菌甲基孢囊菌属菌株SC2（P.F.邓菲尔德、M.查瓦·伊姆加、S.D.德迪什、U.伯杰、W.利萨克和J.海耶，《FEMS微生物生态学》41:17 - 26，2002年）中最近发现的一个新的pmoA样基因（pmoA2）是否存在于其他甲烷氧化细菌（MOB）中，以及它是否表达。一种新开发的引物组合（pmoA206f - pmoA703b）能够对pmoA1和pmoA2进行差异检测。通过使用该引物组合，我们在甲基弯曲菌 - 甲基孢囊菌属群的多种II型MOB中鉴定出了pmoA2。然而，使用新开发的pmoA2特异性寡核苷酸探针进行PCR筛选和Southern杂交也表明，16S rRNA基因序列同一性高于97%的密切相关的II型MOB可能含有也可能不含有pmoA2。在测试的5种I型MOB中未检测到pmoA2：荚膜甲基球菌菌株巴斯、嗜热甲基杆菌菌株E10A、黄色甲基杆菌、白色甲基微菌和甲基单胞菌菌株D1a。在比较序列分析中，所有pmoA2样序列形成一个连贯的簇，明显不同于I型和II型MOB的pmoA1序列以及亚硝化单胞菌 - 亚硝化螺菌属群的amoA序列。使用paml模型进行的系统发育分析表明，pmoA2受到强烈的纯化选择，因此具有重要的细胞功能。我们检测了甲基孢囊菌属菌株SC2的总RNA提取物中pmoA的基因表达。在Northern杂交中观察到pmoA1有强烈信号，而pmoA2的结果不明确。然而，逆转录PCR证实pmoA2表达了，尽管表达水平低于pmoA1。这提供了实验证据，表明pmoA2的基因产物可能是一种功能活性酶。
