Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A
Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A
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DOI：
10.1128/jb.00947-09
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发表时间：
2009-11-15
影响因子：
3.2
通讯作者：
Metcalf, William W.
中科院分区：
文献类型：
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作者：
Opulencia, Rina B.;Bose, Arpita;Metcalf, William W.
甲烷八叠球菌物种拥有三个编码甲醇特异性甲基转移酶1（MT 1）同工酶的操纵子（mtaCB 1、mtaCB 2和mtaCB 3）和两个有可能编码甲醇特异性甲基转移酶2（MT 2）的基因（mtaA 1和mtaA 2）。先前的遗传学研究表明，这些基因受到差异调节，编码具有不同水平甲基转移酶活性的酶。在这里，启动子强度对生长和甲烷产生速率的影响通过构建其中mtaCB启动子被交换的菌株来检查。当从强PmtaC 1或PmtaC 2启动子表达时，MtaC和MtaB蛋白中的每一种都支持野生型水平的生长和甲烷产生。相比之下，当PmtaC 3控制它们的表达时，所有mtaCB操纵子表现出较差的生长和较低的甲烷产生速率。因此，之前观察到的表型差异很大程度上可以归因于启动子活性的差异。携带由强四环素调控的PmcrB（tetO 1）启动子表达的mtaC、mtaB和mtaA的各种组合的菌株在甲醇上表现出相似的生长特性，表明所有的MtaC、MtaB和MtaA的组合都可以形成功能性MT 1/MT 2复合物。然而，在体外试验的耦合MT 1/MT 2活性显示菌株之间的显着差异。令人惊讶的是，这些活性变化与蛋白质丰度的差异相关，尽管所有编码基因都是从相同的启动子表达的。定量逆转录PCR和报告基因融合数据表明，mtaCBA成绩单显示不同的稳定性，这是强烈的影响，由生长底物。
Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.