Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2

Abstract

Methane-oxidizing bacteria (methanotrophs) attenuate methane emission from major sources, such as wetlands, rice paddies, and landfills, and constitute the only biological sink for atmospheric methane in upland soils. Their key enzyme is particulate methane monooxygenase (pMMO), which converts methane to methanol. It has long been believed that methane at the trace atmospheric mixing ratio of 1.75 parts per million by volume (ppmv) is not oxidized by the methanotrophs cultured to date, but rather only by some uncultured methanotrophs, and that type I and type II methanotrophs contain a single type of pMMO. Here, we show that the type II methanotroph Methylocystis sp. strain SC2 possesses two pMMO isozymes with different methane oxidation kinetics. The pmoCAB1 genes encoding the known type of pMMO (pMMO1) are expressed and pMMO1 oxidizes methane only at mixing ratios >600 ppmv. The pmoCAB2 genes encoding pMMO2, in contrast, are constitutively expressed, and pMMO2 oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 and mutants expressing pmoCAB2 but defective in pmoCAB1 consumed atmospheric methane for >3 months. Growth occurred at 10-100 ppmv methane. Most type II but no type I methanotrophs possess the pmoCAB2 genes. The apparent K(m) of pMMO2 (0.11 muM) in strain SC2 corresponds well with the K(m(app)) values for methane oxidation measured in soils that consume atmospheric methane, thereby explaining why these soils are dominated by type II methanotrophs, and some by Methylocystis spp., in particular. These findings change our concept of methanotroph ecology.

Fig. 1.

Methane consumption and growth curves of wild-type Methylocystis sp. strain SC2 and its mutants SC2-P1, SC2-P2, and SC2-P3, shown in relation to the expression of pmoCAB1 and pmoCAB2 mRNA transcripts during exponential growth on methane. (A) Methane consumption (%, vol/vol) over time (solid lines) and cell numbers calculated from OD₆₀₀ measurements: wild-type (black); mutant SC2-P1, defective in a single copy of pmoCAB1 (blue); mutant SC2-P2, defective in both copies of pmoCAB1 (green); and mutant SC2-P3, defective in pmoCAB2 (red). The pmoCAB null mutant SC2-P4 and uninoculated controls were used as negative growth controls; the methane concentration did not decline in these controls. (B) pmoCAB1 and pmoCAB2 expression detected by Northern blot hybridization of total RNA of wild-type strain SC2 (WT) and mutant strains SC2-P2 and SC2-P3. Total RNA was extracted from aliquots of cultures of wild-type strain SC2 and mutant strains in the early exponential growth phase (arrows, Fig. 1A). Gene probes specifically targeted transcripts of either pmoCAB1 (Probe₁) or pmoCAB2 (Probe₂). For target specificity of Probe₁ and Probe₂, see Fig. S1.

Fig. 2.

Detection of pmo mRNA transcripts in wild-type strain SC2 and mutant strains SC2-P1, SC2-P2, and SC2-P3 after a 3-week incubation under ≥700 and ≤600 ppmv CH₄. RT-PCR was carried out to specifically detect mRNA transcripts of either pmoCAB1 or pmoCAB2. The expected size of the mRNA RT-PCR products was 1,075 bp for pmoCAB1 and 1,056 bp for pmoCAB2. Lanes 1, 8, and 14, DNA size markers (Smart Ladder, Eurogentec); lanes 2–7, detection of pmoCAB1 (lanes 2, 3, 5–7) and pmoCAB2 (lane 4) mRNA transcripts after a 3-week incubation under ≥700 ppmv CH₄; lane 2, wild-type strain SC2; lane 3, negative control (same extract of total RNA as used for the positive control in lane 2 but without reverse transcription); lane 4, wild-type strain SC2 (same extract of total RNA from wild-type strain SC2 as in lane 2 but detection of pmoCAB2 mRNA transcripts); lanes 5–7, mutant strains SC2-P1, SC2-P2, and SC2-P3; lanes 9–13, detection of pmoCAB1 (lanes 9 and 11–13) and pmoCAB2 (lane 10) mRNA transcripts after a 3-week incubation under ≤600 ppmv CH₄; lane 9, wild-type strain SC2; lane 10, wild-type strain SC2 (same extract of total RNA from wild-type strain SC2 as in lane 9 but detection of pmoCAB2 mRNA transcripts); lanes 11–13, mutant strains SC2-P1, SC2-P2, and SC2-P3 (as exemplarily shown for wild-type strain SC2, detection of pmoCAB2 was also positive for mutant strains SC2-P1 and SC2-P2 but not for SC2-P3 after a 3-week incubation under ≤600 ppmv CH₄).