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Quantitative Detection of Methanotrophs in Soil by Novel pmoA -Targeted Real-Time PCR Assays

甲烷单加氧酶 甲烷利用细菌 细菌 α蛋白细菌 蛋白质细菌 土壤微生物学 生物 土壤水分 16S核糖体RNA 化学 微生物学 环境化学 生物化学 甲烷厌氧氧化 生态学 遗传学 催化作用
作者
Steffen Kolb,Claudia Knief,Stephan Stubner,Ralf Conrad
出处
期刊:Applied and Environmental Microbiology [American Society for Microbiology]
卷期号:69 (5): 2423-2429 被引量:449
标识
DOI:10.1128/aem.69.5.2423-2429.2003
摘要

ABSTRACT Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ subclasses of Proteobacteria : the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10 1 and 10 2 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus , Methylomicrobium album , and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus -specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) × 10 6 pmoA molecules g −1 for all methanotrophs. The Methylosinus group was predominant (2.7 × 10 6 ± 1.1 × 10 6 target molecules g −1 ). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 10 6 ± 0.9 × 10 6 target molecules g of soil −1 ). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 × 10 4 target molecules g of soil −1 . Our results showed that pmoA -targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.
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