Molecular characterization of methanotrophic isolates from freshwater lake sediment

Abstract

Profiles of dissolved O(2) and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 micromol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed for mmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.

FIG. 1

Characterization of methane oxidation in Lake Washington sediment. (A) Profiles of methane and/or O₂ with increasing sediment depth for subcores obtained 26 September 1997 and 9 September 1998. The y axis represents sediment depth with 0 mm representing the sediment-water interface. (B) Example of the partitioning of methane to cells and metabolites versus CO₂ from the top 0.5-cm layer.

FIG. 2

Examples of Southern blot analyses for pmoA (A) and mmoX (B) with representative Lake Washington strains. These chromosomal DNA samples were digested with PstI. Chromosomal DNA samples were also digested with EcoRI. MC, M. capsulatus Bath.

FIG. 3

Alignment of multiple DNA sequences of pmoA PCR products from LW8. Eight pmoA PCR products from this strain were cloned and sequenced. Five sequences were identical to LW8A, while three sequences differed in two nucleotide positions and were identical to LW8B. The nucleotide changes (positions 106 and 292 in the PCR products) resulted in amino acid differences (E versus K and I versus F, respectively).

FIG. 4

Alignment of deduced amino acid sequences of the 1.2-kb partial mmoX genes from LW13, LW15, Methylomonas sp. strain KSWIII (accession no. AB025022), Methylomonas sp. strain KSPIII (AB025021), M. capulatus Bath (M90050), M. trichosporium OB3b (X55394), Methylocystis sp. strain WI 14 (AF153282), Methylocystis sp. strain M (U81594), LW3, LW4, LW8, and PW1. Identical residues are in black boxes, and similar residues are in gray boxes.

FIG. 5

Phylogenetic analysis of the derived amino acid sequences of mmoX genes. BOOTSTRAP values of >50% are shown near the clades. The bar represents 10% sequence divergence, as determined by the lengths of the horizontal lines connecting any two species.