Detection 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

Abstract

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.

FIG. 1

(A) Distance dendrogram constructed for partial pmoA and amoA gene sequences based on 165 derived amino acid sites in relation to pmoA-based T-RFLP (B) and DGGE (C) community patterns. The two patterns and most of the pmoA clone sequences were obtained from sample M84. (A) The dendrogram shows environmental pmoA and amoA (plus other putative monooxygenase) sequences retrieved from roots of submerged rice plants (M84, M90) in relation to pmoA of cultured type I and type II methanotrophs, environmental pmoA clone sequences, and amoA sequences of the β-proteobacterial Nirosomonas and Nitrosospira group. The environmental pmoA sequences used for reference were retrieved from various habitats as follows: beech forest in Denmark (RA14 [AF148521], RA21 [AF148522], Rold1 [AF148523], Rold4 [AF148526], Rold5 [AF148527]), rain forest in Brazil (Pantanal13 [AF148525]), mixed hardwood forest in the United States (Maine6 [AF148528], Maine9 [AF148531]) (33), deciduous forest soil near Marburg, Germany (MR2 [AF200726], MR16 [AF 200729] (29), MR1 [AF200729]) (29), rice soil incubations (He-I [AF126908], He-II [AF126909], He-III [AF126910], He-IV [AF126913], He-VI [AF126911]) (28), and blanket peat bog (PE9 [AF006050], PD2 [AF006047]) (41). The numbers I, II, and III refer to three distinct pmoA sequence clusters of type I methanotrophs, which have been retrieved from rice roots. The numbers at the nodes indicate the percentage of recovery in 500 bootstrap resamplings. Only bootstrap values ≥50 are shown. Scale bar, 0.1 substitution per amino acid site. Database accession numbers of reference organisms are as follows: Methylocystis sp. strain M, U81596; Methylocystis parvus, U31651; Methylosinus trichosporium, U31550; Methylobacter sp. strain BB5.1, AF016982; Methylomicrobium album, U31654; Methylomicrobium pelagicum, U31652; Methylomonas methanica, U31653; Methylocaldum gracile, U89301; Methylocaldum szegediense, U89303; Methylocaldum tepidum, U89304; Methylococcus capsulatus, L40804; strain HB, U89302; Nitrosospira multiformis, U89833; and Nitrosomonas europaea, AF037107. (B) pmoA-based T-RFLP profile. The x axis shows the lengths (in base pairs) of the T-RFs, and the y axis shows the intensities of the fragments in arbitrary units. The numbers in boxes indicate the sizes of T-RFs which could be assigned to phylogenetically defined methanotroph populations or to autotrophic ammonia oxidizers (see arrows). (C) pmoA-based DGGE pattern. For comparative sequence analysis, predominant DGGE bands were excised, reamplified, and reanalyzed by DGGE to verify band purity. Affiliation of these bands to distinct pmoA clusters is indicated (compare with Fig. 1A).

FIG. 2

Comparison of pmoA-based T-RFLP profiles obtained from bulk soil (A) and rice roots (B) of the flooded rice microcosm M84. See Fig. 1 for assignment of the major T-RFs to defined methanotrophic populations and to autotrophic ammonia oxidizers.

FIG. 3

Distance dendrogram constructed for partial mmoX sequences based on 286 derived amino acid sites. The dendrogram shows environmental mmoX sequences retrieved from roots of submerged rice plants (M84, M90) in relation to mmoX sequences of representative type I and type II methanotrophs. The environmental mmoX sequence designated peat bog clone 24 (AF004555) was retrieved from an acidic Sphagnum peat bog (12). The numbers at the nodes indicate the percentage of recovery in 500 bootstrap resamplings. Only bootstrap values ≥50 are shown. Scale bar, 0.1 substitution per amino acid site. Database accession numbers of reference organisms are as follows: Methylocystis sp. strain LR1, Y18440; Methylocystis sp. strain M, U81594; Methylosinus trichosporium, X55394; Methylocella palustris, AF004554; Methylococcus capsulatus, M90050; and Methylomonas sp. strain KSWIII, AB025022.

FIG. 4

Distance dendrogram constructed for partial mxaF sequences based on 172 derived amino acid sites. The dendrogram shows environmental mxaF sequences retrieved from roots of submerged rice plants (M84, M90) in relation to mxaF sequences of representative type I and type II methanotrophs, and Methylobacterium organophilum. The mxaF sequence He-III [AF126296] was retrieved from rice soil incubations (28), while Mo1 [AF283243] and Mo2 [AF283244] were detected in a methanotrophic consortium enriched with a high CH₄/low O₂ mixing ratio (31). The numbers at the nodes indicate the percentage of recovery in 500 bootstrap resamplings. Only bootstrap values ≥50 are shown. Scale bar, 0.1 substitution per amino acid site. Database accession numbers of reference organisms are as follows: Methylocystis sp. strain LR1, Y18441; Methylocystis sp. strain M, U70517; Methylocystis parvus, U70515; Methylosinus sporium, U70514; Methylosinus trichosporium, U70516; Methylocella palustris, AJ27831; Methylomicrobium album, U70513; Methylomonas methanica, U70512; Methylococcus capsulatus, U70511; strain LK6, U86503; and Methylobacterium organophilum, M22629.

FIG. 5

Distance dendrogram showing the 16S rDNA clone sequences retrieved from roots of submerged rice plants (samples M70, M84, and M90) in relation to type I methanotrophs and nonmethanotrophic members of γ-Proteobacteria. The environmental 16S rDNA sequences encompass the clones M70-D2 to M90-D34. Due to limited sequence length (556 bp), the 16S rcDNA clones M70-R5 to M70-R40 (R = 16S ribosomal copy DNA recovered from total RNA of sample M70) have been inserted into the distance dendrogram using parsimony methods. RRI1 (AF179603) was retrieved from rhizosphere soil of a flooded rice microcosm (3). The 16S rDNA sequences of α-proteobacterial type II methanotrophs were used to root the tree. The numbers at the nodes indicate the percentage of recovery in 500 bootstrap resamplings. Only bootstrap values ≥50 are shown. Scale bar, 0.1 substitution per nucleotide sequence position. Database accession numbers of reference organisms are as follows: Methylocystis sp. strain M, U81595; Methylocystis parvus, Y18945; Methylobacter sp. strain BB5.1, AF016981; Methylobacter bovis, L20839; Methylobacter capsulatus, L20843; Methylobacter luteus, M95657; Methylobacter psychrophilus, AF152597; Methylobacter vinelandii, L20841; Methylobacter whittenburyi, X72773; Methylomicrobium agile, X72767; Methylomicrobium album, M95659; Methylomicrobium pelagicum, L35540; Methylomonas aurantiaca, X72776; Methylomonas methanica, AF50806; Methylomonas fodinarum, X72778; Methylomonas rubra, M95662; Methylosphaera hansonii, U77533; Methylocaldum gracile, U89298; Methylocaldum szegediense, U89300; Methylocaldum tepidum, U89297; Methylococcus capsulatus, L20842; Escherichia coli, V00348; Erwinia carotovora, M59149; Vibrio cholerae, O11197; Pseudomonas flavescens, U01916; and Legionella steigerwaltii, X73400.