Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils

Abstract

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The narG gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of narG is of special interest because the phylogeny of the narG gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the narG gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified narG fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the narG genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e., Actinobacteria and alpha, beta, and gamma proteobacteria. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the Actinobacteria in an astonishingly high proportion.

FIG. 1.

Comparison of the RISA patterns obtained from the unplanted soil (lane 1, replicate BSA; lane 2, replicate BSB; lane 3, replicate BSC) and from the maize-planted soil (lane 4, replicate PSD; lane 5, replicate PSE; lane 6, replicate PSF) with 16S ribosomal DNA intergenic spacer universal primers. Migration was performed with a 6% polyacrylamide gel, and the molecular size marker was the VIII from Boehringer Mannheim (lane 7).

FIG. 2.

Comparison of the AluI RFLPs of the narG PCR products obtained from the unplanted soil (lane 2, replicate BSA; lane 3, replicate BSB; lane 4, replicate BSC) and from the maize-planted soil (lane 5, replicate PSD; lane 6, replicate PSE; lane 7, replicate PSF). Migration was performed on a 6% polyacrylamide gel, and the molecular size marker was the VIII from Boehringer Mannheim (lane 1).

FIG. 3.

Distribution of the narG RFLP types from the unplanted soil (A) samples BSA (▤), BSB (gray with white dots) and BSC (▨) and distribution of the same RFLP types from the planted soil (B) samples PSD (▥), PSE (▩), and PSF (▧). Only RFLP types containing more than one clone are represented. The asterisks indicate RFLP types belonging to cluster 2.

FIG. 4.

Rarefaction curves of observed diversity of narG RFLP types in the unplanted soil (○) samples BSA (⋄), BSB (x), and BSC (▵) and in the planted soil (•) samples PSD (♦), PSE (+), and PSF (▴).

FIG.5.

Phylogenetic relationship of translated narG gene product. Phylogenetic distances were determined by neighbor-joining analysis. The corresponding RFLP types are indicated in bold and italics after the clone number. Nodes with more than 750 (of 1,000) bootstrap iterations are highlighted by a black circle. The accession numbers or website resources for the narG genes are as follows: Pseudomonas fluorescens C7R12, AF197465; P. fluorescens AK15 U71398; Pseudomonas aeruginosa, Y15252; E. coli, X16181 and X17110; Halobacterium denitrificans, AB076402; Thermus thermophilus, Y10124; Mycobacterium tuberculosis, NC_000962; Ralstonia solanacearum, NC_003296; Bacillus subtilis, X91819; Bacillus stearothermophilus, http://www.genome.ou.edu/bstearo.html; Streptomyces coelicolor, AL512667, AL109989, and AL031515; Staphylococcus carnosus, AF029224; Staphylococcus aureus, NC_002745, Brucella melitensis biovar suis, http://www.tigr.org; B. pseudomallei, http://www.sanger.ac.uk/Projects/; Mycobacterium tuberculosis, Z81360; Corynebacterium diphtheriae, http://www.sanger.ac.uk/Projects/C_diphtheriae/; Ralstonia metallidurans, http://www.jgi.doe.gov/JGI_microbial; R. solanacearum, AL646082; Pyrobaculum aerophilum, NC_003364; Aeropyrum pernix, http://www.bio.nite.go.jp; S. enterica serovar Typhi, NC_003198; S. enterica serovar Typhimurium, NC_003197.