Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.
Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.
复制标题
DOI：
10.3389/fmicb.2012.00303
复制
发表时间：
2012
影响因子：
5.2
通讯作者：
Dunfield PF
中科院分区：
文献类型：
--
作者：
Sharp CE;Stott MB;Dunfield PF
对甲烷营养型疣状微菌“Methylacidiphilum infernorum”菌株V4的基因组分析表明，赋予其甲烷营养型生活方式的大多数途径与在蛋白细菌甲烷营养型中发现的那些途径相似。然而，由于其甲烷单加氧酶编码基因（pmo）的序列差异很大，“通用”pmoA聚合酶链反应（PCR）引物并不靶向这些细菌。与嗜甲烷的变形菌不同，“嗜甲基酸菌”以自养的方式固定碳，并仅将甲烷用于能源生产。因此，用于检测环境中的甲烷氧化菌的技术，如13 CH 4-稳定同位素探测（SIP）和pmoA-靶向PCR不能检测到疣微生物甲烷氧化菌，它们可能在以前的环境研究中被忽略了。我们开发了一种改进的SIP技术，以确定活性甲烷Verrucomicrobia在环境中的标记与13 CO2和13 CH 4，单独和组合。在“M. infernorum”菌株V4同化13 CO2而不同化13 CH 4，证实了其自养生活方式。为了通过13 CO2-SIP特异性检测甲烷营养菌（与其他自养菌相反），开发了对疣状微生物-pmoA基因特异性的定量PCR（qPCR）测定，并与SIP组合使用。孵化的酸性，高温地热土壤与13 CH 4 + 12 CO2引起的疣微生物-pmoA基因的密度分布相对于对照组的变化不大。然而，标记与13 CO2与12 CH 4或13 CH 4的组合诱导的分布verrucomicrobial-pmoA基因向重DNA馏分的强烈转变。改良的SIP技术表明，土壤中主要的甲烷氧化菌是自养菌，属于疣微菌。这是第一个示范的自养，非变形菌甲烷营养原位，并提供了一个工具，以检测在其他生态系统中的疣微生物甲烷营养菌。
Genomic analysis of the methanotrophic verrucomicrobium “Methylacidiphilum infernorum” strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), “universal” pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, “Methylacidiphilum” fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as 13CH4-stable isotope probing (SIP) and pmoA-targeted PCR do not detect verrucomicrobial methanotrophs, and they may have been overlooked in previous environmental studies. We developed a modified SIP technique to identify active methanotrophic Verrucomicrobia in the environment by labeling with 13CO2 and 13CH4, individually and in combination. Testing the protocol in “M. infernorum” strain V4 resulted in assimilation of 13CO2 but not 13CH4, verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via 13CO2-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-pmoA genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with 13CH4 + 12CO2 caused little shift in the density distribution of verrucomicrobial-pmoA genes relative to controls. However, labeling with 13CO2 in combination with 12CH4 or 13CH4 induced a strong shift in the distribution of verrucomicrobial-pmoA genes towards the heavy DNA fractions. The modified SIP technique demonstrated that the primary methanotrophs active in the soil were autotrophs and belonged to the Verrucomicrobia. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy in situ, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.
登录
查看更多内容
影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
影响因子：
4.2
作者：
Benson, Courtney A.;Bizzoco, Richard W.;Kelley, Scott T.
通讯作者：
Kelley, Scott T.
影响因子：
4.4
作者：
Kolb, S;Knief, C;Conrad, R
通讯作者：
Conrad, R
影响因子：
5.5
作者：
Hou S;Makarova KS;Saw JH;Senin P;Ly BV;Zhou Z;Ren Y;Wang J;Galperin MY;Omelchenko MV;Wolf YI;Yutin N;Koonin EV;Stott MB;Mountain BW;Crowe MA;Smirnova AV;Dunfield PF;Feng L;Wang L;Alam M
通讯作者：
Alam M
影响因子：
18.3
作者：
Kip, Nardy;van Winden, Julia F.;Op den Camp, Huub J. M.
通讯作者：
Op den Camp, Huub J. M.