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FEMS Microbiol Ecol. 2017 Mar;93(3). pii: fiw244. doi: 10.1093/femsec/fiw244. Epub 2016 Dec 8.

An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria.

Author information

1
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
2
Department of Microbiology, Chungbuk National University, Cheongju, Korea.
3
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China quanzx@fudan.edu.cn.

Abstract

The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design.

KEYWORDS:

Comammox bacteria; CuMMO; ammonia-oxidizing bacteria; methane-oxidizing bacteria; tagged highly degenerate primer

PMID:
27940646
DOI:
10.1093/femsec/fiw244
[Indexed for MEDLINE]

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