Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs).
Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.
Miyashita
Ai
A
Subsurface Geobiology Advanced Research Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, Kanagawa, Japan.
Mochimaru
Hanako
H
Kazama
Hiromi
H
Ohashi
Akiyoshi
A
Yamaguchi
Takashi
T
Nunoura
Takuro
T
Horikoshi
Koki
K
Takai
Ken
K
Imachi
Hiroyuki
H
D000693:Anaerobiosis; D001105:Archaea / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D052818:Autotrophic Processes; D017931:DNA Primers / Q000235:genetics*; D019641:DNA, Archaeal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004783:Environmental Microbiology; D008697:Methane / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D008697:Methane