Open AccessDetection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA , mmoX , mxaF , and 16S rRNA and Ribosomal DNA, Including pmoA - Based Terminal Restriction Fragment Length Polymorphism Profiling
Author(s) -
Hans-Peter Horz,
Merlin Tchawa Yimga,
Werner Liesack
Publication year - 2001
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.67.9.4177-4185.2001
Subject(s) - methanotroph , methane monooxygenase , biology , 16s ribosomal rna , ribosomal rna , terminal restriction fragment length polymorphism , restriction fragment length polymorphism , alphaproteobacteria , ribosomal dna , methanol dehydrogenase , botany , anaerobic oxidation of methane , microbiology and biotechnology , phylogenetic tree , bacteria , genetics , biochemistry , gene , polymerase chain reaction , catalysis
The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the alpha subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.