Expression and lateral transfer of nitrogen fixation genes in the gut of earthworms.

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The contribution of an earthworm species (Amynthas vittatus) to the increase of the nitrogen content of soil was examined. Three specimens of adult earthworms were introduced into 300 g of soil (Gray Lowland soil, silty clay) supplemented with 1% carboxymethyl cellulose in a container and incubated for 32 days at 22℃ in the dark. The contents of total-N, NH_4-N and NO_3-N, and the population of aerobic nitrogen-fixing bacteria in soil significantly increased after incubation with the earthworms, while the natural abundance of ^<15>N (δ^<15>N) in soil decreased. The amount of nitrogen in the earthworms did not decrease during the incubation in the microcosm. Both acetylene reduction activity of the microcosm and incorporation of ^<15>N to soil from atmospheric ^<15>N_2 were significantly enhanced by the introduction of the earthworms into soil, though the observed increment of nitrogen in soil was much higher than the estimated one based on the nitrogen-fixing activity.The community structure of aerobic nitrogen-fixing bacteria in the soils where the earthworms had lived was estimated by both isolating NFBs from the soils and the analysis of PCR-DGGE patterns of nifH from the soils. Physiological characteristics and 16S rDNA sequence analysis showed that the isolated NFBs were classified into Azorhizobium caulinodans, Azospirillum brasilense, A.lipoferum, Klebsiella oxytoca, Pseudomonas sp., Rhizobium etli, Rosemonas faurise, Sinorhizobium morelens, and Xanthobacter sp. The number and the pattern of DGGE bands of nifH from the soil incubated with earthworms were different from those of nifH from earthworm casts and the soil in which earthworms did not live.The results obtained in the present study indicated that the earthworms increased the nitrogen content of soil, presumably due to the enhancement of the nitrogen-fixing activity of the soil from the microcosm by the earthworms.