Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties

Abstract

The similarities and differences in the structures of the nifH gene pools of six different soils (Montrond, LCSA-p, Vernon, Dombes, LCSA-c, and Thysse Kaymor) and five soil fractions extracted from LCSA-c were studied. Bacterial DNA was directly extracted from the soils, and a region of the nifH gene was amplified by PCR and analyzed by restriction. Soils were selected on the basis of differences in soil management, plant cover, and major physicochemical properties. Microenvironments differed on the basis of the sizes of the constituent particles and the organic carbon and clay contents. Restriction profiles were subjected to principal-component analysis. We showed that the composition of the diazotrophic communities varied both on a large scale (among soils) and on a microscale (among microenvironments in LCSA-c soil). Soil management seemed to be the major parameter influencing differences in the nifH gene pool structure among soils by controlling inorganic nitrogen content and its variation. However, physicochemical parameters (texture and total C and N contents) were found to correlate with differences among nifH gene pools on a microscale. We hypothesize that the observed nifH genetic structures resulted from the adaptation to fluctuating conditions (cultivated soil, forest soil, coarse fractions) or constant conditions (permanent pasture soil, fine fractions). We attempted to identify a specific band within the profile of the clay fraction by cloning and sequencing it and comparing it with the gene databases. Unexpectedly, the nifH sequences of the dominant bacteria were most similar to sequences of unidentified marine eubacteria.

FIG. 1

MnlI RFLPs of nifH PCR products obtained from Vernon soil on four sampling dates. Lane 1, 6 April 1998; lane 2, 4 May 1998; lane 3, 10 June 1998; lane 4, 17 July 1998. Migration was performed on a 5% polyacrylamide (19:1) gel, and the molecular size marker (lane 5) was 20 bp.

FIG. 2

Electrophoretogram of MnlI RFLPs of nifH PCR products obtained from the six studied soils. Dashed lines, peaks common to several soils; light arrows, characteristic fragments (see text).

FIG. 3

PCA generated from soil nifH restriction profiles by HaeIII, NdeII, and MnlI. Dark spots, pasture soils; hatched spots, nonpasture soils.

FIG. 4

Polyacrylamide gel electrophoresis of MnlI RFLPs from nifH PCR products obtained from LCSA-c soil fractions. Lane 1, >250-μm fraction; lane 2, 250- to 50-μm fraction; lane 3, 50- to 20-μm fraction; lane 4, 20- to 2-μm fraction; lane 5, <2-μm fraction; lane 6, 20-bp molecular size marker. Asterisk, 250-bp fragment characteristic of the <2-μm fraction.

FIG. 5

PCA generated from nifH restriction profiles from LCSA-c soil microenvironments by HaeIII, NdeII, and MnlI.

FIG. 6

Phylogeny of nifH nucleotide sequences using 21 partial nifH gene sequences from the GenBank database and 9 sequences obtained from the cloning of the 250-bp MnlI band from the LCSA-c clay fraction. GenBank database accession numbers are indicated next to the bacterial names. Locations of the nifH fragments used for the analysis correspond to a sequence fragment of ≈250 bp in positions 214 to 476 (referring to the A. vinelandii nifH coding sequence [M20568]). The tree was constructed by the neighbor-joining method, and bootstrap values above 50 from 1,000 resamplings are shown for each node.