Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis

Abstract

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4. 8 versus 6.8), air/CH(4)/CO(2) headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich 1x NMS selected for type I methanotrophs, while the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

FIG. 1

Ethidium bromide-stained agarose gel (1.5% [wt/vol]) showing PCR amplification products obtained with methanotroph-specific primers. See the text for details on primer design and reaction conditions. Lanes 1 to 6 show results with the type I primer set, MethT1dF and MethT1bR. Lanes 7 to 10 show results with the type II primer set, 27F and MethT2R. Template DNAs are as follows: lane 1, Methylomicrobium album; lane 2, Methylomonas methanica; lane 3, Methylobacter luteus; lane 4, M. capsulatus; lane 5, P. putida; lane 6, E. coli; lane 7, Methylosinus trichosporium; lane 8, Methylocystis parvus; lane 9, A. tumefaciens; and lane 10, S. meliloti.

FIG. 2

Phylogenetic tree showing the relationship of landfill soil type I methanotroph 16S rDNA clone sequences and type I AML isolates to some characterized methanotrophs from the family Methylococcaceae. This tree was constructed by using the fastDNAml program (47), which uses a maximum likelihood algorithm (19). A total of 625 aligned bases corresponding to E. coli positions 141 to 766 were used in this analysis. E. coli served as the outgroup. The scale bar represents 0.10 substitutions per base position. The numbers at the nodes of the tree indicate bootstrap values (18) for each node out of 100 bootstrap resamplings (values below 50 are not shown).

FIG. 3

Phylogenetic tree showing the relationship of landfill soil type II methanotroph 16S rDNA clone sequences and type II AML isolates to some characterized type II methanotrophs. Also included are some partially characterized methane oxidizers, including a recently isolated acidophilic bacterium, strain K, that diverges from the type II methanotrophs (14). This tree was constructed by using the fastDNAml program (47), which uses a maximum likelihood algorithm (19). A total of 623 aligned bases corresponding to E. coli positions 54 to 731 were used in this analysis. A. tumefaciens served as the outgroup. The scale bar represents 0.05 substitution per base position. The numbers at the nodes of the tree indicate bootstrap values (18) for each node out of 100 bootstrap resamplings (values below 50 are not shown).

FIG. 4

Phylogenetic analysis of partial amino acid sequences of the pmoA gene from selected type I and type II methanotrophs, novel isolates and environmental clones (41). This unrooted tree was constructed by using the neighbor-joining method (48) from a matrix of pairwise genetic distances as calculated by the PROTDIST program (20). A total of 110 aligned amino acid positions were used in this analysis. The scale bar represents 10% sequence divergence. Bootstrap analyses (18) for 100 resamplings were performed to provide confidence estimates for tree topologies (values below 50 are not shown).

FIG. 5

Type I methanotroph DGGE analysis. Shown is an ethidium bromide-stained 6.5% polyacrylamide denaturing gradient gel (20 to 70%) showing separation patterns based on sequence difference of the V3 region (approximate E. coli positions 341 to 534) of the 16S rDNA. Lanes 1 and 10, the type I landfill methanotrophic community (250 ng and 1.5 μg, respectively); lane 2, clone T1-08; lane 3, clone T1-01; lane 4, clone T1-14; lane 5, clone T1-09; lane 6, clone T1-02; lane 7, clone T1-04; lane 8, isolate AML-C10; lane 9, isolate AML-D4. Bands A through L are explained in the text.

FIG. 6

DGGE analysis of cloned DNA extracted from type I community bands A and B (Fig. 5). Shown is an ethidium bromide-stained 6.5% polyacrylamide denaturing gradient gel (20 to 70%) showing separation patterns based on sequence differences in the V3 region (approximate E. coli positions 341 to 534) of the 16S rDNA. See Results for experimental details. Lane 1, clone EB-A1; lane 2, clone EB-A2; lane 3, type I landfill methanotrophic community profile; lane 4, clone EB-B1; lane 5, clone EB-B2; lane 6, clone T1-08; lane 7, clone T1-09.

FIG. 7

Type II methanotroph DGGE analysis. Shown is an ethidium bromide-stained 6.5% polyacrylamide denaturing gradient gel (30 to 60%) showing separation patterns based on sequence differences in the V3 region (approximate E. coli positions 341 to 534) of the 16S rDNA. Lanes 1 and 10, the type II landfill methanotrophic community; lane 2, clone T2-06; lane 3, clone T2-07; lane 4, clone T2-03; lane 5, clone T2-02; lane 6, clone T2-01; lane 7, isolate AML-A3; lane 8, isolate AML-F18; lane 9, isolate AML-E13. Bands A through I are explained in the text.