title: An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria.
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.
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Jian-Gong
authors.initials: JG
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xia
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Jemaneh
authors.initials: J
authors.lastname: Zeleke
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zou
authors.affiliation: Department of Microbiology, Chungbuk National University, Cheongju, Korea.
authors.forename: Sung-Keun
authors.initials: SK
authors.lastname: Rhee
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China quanzx@fudan.edu.cn.
authors.forename: Zhe-Xue
authors.initials: ZX
authors.lastname: Quan
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D020562:Betaproteobacteria / Q000235:genetics; D003300:Copper; D017931:DNA Primers / Q000235:genetics; D008697:Methane / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010105:Oxygenases; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers; D000641:Ammonia; D003300:Copper; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010105:Oxygenases; C021555:methane monooxygenase; C045582:ammonia monooxygenase; D008697:Methane
keywords: Comammox bacteria; CuMMO; ammonia-oxidizing bacteria; methane-oxidizing bacteria; tagged highly degenerate primer