Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs
Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs
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DOI：
10.1128/aem.66.3.966-975.2000
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发表时间：
2000-03-01
影响因子：
4.4
通讯作者：
Murrell, JC
中科院分区：
文献类型：
--
作者：
Gilbert, B;McDonald, IR;Murrell, JC
颗粒甲烷单加氧酶基因簇 pmoCAB 来自 α-变形菌门的两种代表性 II 型甲烷氧化菌（毛孢甲基窦菌 OB3b 和甲基囊藻属 M 菌株），已被克隆和测序。引物延伸实验表明，对于甲基囊藻 M 菌株，pmo 簇可能是从位于第一个基因 pmoC 起始上游 300 bp 处的单个转录起始位点转录的。在假定起始位点的紧邻上游，鉴定了 sigma(70) 启动子的共有序列，这表明这些 pmo 基因被 sigma(70) 识别，并在低铜条件下受到负调控。 pmo 基因被克隆成几个重叠片段，因为这些基因的部分内容似乎对大肠杆菌宿主有毒。甲烷氧化菌含有两个几乎相同的 pmo 基因拷贝，有必要使用 Southern 印迹和 pmo 基因片段探测来区分两个甲烷氧化菌中的两个 pmoCAB 簇。这里报告了来自每个生物体的一份 pmo 基因的完整 DNA 序列。这些基因序列彼此相似度为 84%，与 γ-变形菌门的 I 型甲烷氧化菌（荚膜甲基球菌 Bath）相似度为 75%。衍生蛋白 PmoC 和 PmoA 预计具有高度疏水性，并且主要由跨膜区域组成，而 PmoB 仅具有两个推定的跨膜螺旋。杂交实验表明，毛孢子虫 OB3b 和甲基囊藻 M 菌株中均存在两个 pmoC 拷贝，而不是像在荚膜分枝杆菌 Bath 中发现的三个拷贝。
The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp, strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp, strain M, Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices, Hybridization experiments showed that there are two copies of pmoC in both M, trichosporium OB3b and Methylocystis sp, strain M, and not three copies as found in M. capsulatus Bath.