Genetic diversity of nifH gene sequences in paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments

Abstract

The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifH gene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum, Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two nifH sequence clusters, designated clusters I and II, in P. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH in P. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different nifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.

FIG. 1

Southern hybridization of EcoRI-restricted genomic DNA of P. azotofixans and Paenibacillus sp. strains with a nifH probe (PCR product from strain ATCC 35681 generated with primers NHA1 and NHA2). The figure is a composite of two blots. Lane 1, P. durum DSMZ 1735; lane 2, P. azotofixans SD20; lane 3, BE1; lane 4, P3E20; lane 5, RCPG7; lane 6, SD17; lane 7, 2RC1; lane 8, P. macerans LMD24.3; lane 9, P. polymyxa DSMZ 356; lane 10, ATCC 35681; lane 11, F102; lane 12, C3L4; lane 13, RBN4.

FIG. 2

Alignment of amino acid sequences deduced from the nifH sequence of Paenibacillus sp. and from the sequences of nifH genes of other organisms in the database. The sequences compared correspond to residues 48 to 155 of the Anabaena (Nostoc) sp. strain 7120 sequence (GenBank accession no. A00534). Abbreviations: av, Azotobacter vinelandii; kp, Klebsiella pneumoniae; C3l4, sd20, 2rc1, atcc, be1, rcpg7, and f102, P. azotofixans C3L4, SD20, 2RC1, ATCC 35681, BE1, RCPG7, and F102, respectively; rc, Rhodobacter capsulata; rm, Rhizobium meliloti; LL, Lyngbya lagerheimii; durum, P. durum; p3e20 and frbn4, P. azotofixans P3E20 and RBN4, respectively; cp3, C. pasteurianum nifH3 alternative (anf); cp1, C. pasteurianum nifH1; mtal, Methanococcus thermolithotrophicus alternative (anf); pol, P. polymyxa; mac, P. macerans. For database accession numbers see Fig. 3.

FIG. 3

Phylogenetic tree based on partial nifH product amino acid sequences, including the sequences of P. azotofixans strains and Paenibacillus spp. and other sequences from the database. The location of the nifH fragments used for the analysis corresponds to Anabaena (Nostoc) sp. strain 7120 residues 48 to 155 (GenBank accession no. A00534). The database accession numbers are indicated after the bacterial names. Bootstrap values (percentages) for parsimony (above the lines) and neighbor joining (below the lines) are shown for clusters supported by both analyses; only values greater than 50% are shown.

FIG. 4

Sequence alignment of P. azotofixans-specific stretches of the nifH gene with closely related nifH sequences, showing target regions used to design specific primers. Most of the Paenibacillus sp. sequences were obtained in this study; the only exception was the sequence of P. azotofixans ATCC 35681 (45). DSM, DSMZ. Dots indicate bases identical to those of the target sequence.

FIG. 5

DGGE analysis of PCR-amplified nifH gene fragments from P. azotofixans and P. durum strains: parallel DGGE separation patterns of gene fragments obtained with genomic DNA from strains DSMZ 1735 (lane 1), BE1 (lane 2), SD20 (lane 3), SD17 (lane 4), RCPG7 (lane 5), F102 (lane 6), 2RC1 (lane 7), C3L4 (lane 8), RBN4 (lane 9), P3E20 (lane 10), and ATCC 35681 (lane 11).

FIG. 6

DGGE analysis of PCR-amplified nifH gene fragments generated with DNA from Dutch and Brazilian bulk and rhizosphere soil samples and individual P. azotofixans strains. Lane 1, Ede loamy sand bulk soil; lane 2, Flevo silt loam bulk soil; lane 3, Guaíra bulk soil; lane 4, Cerrado soil, maize rhizosphere, 30 days; lane 5, Cerrado soil, maize rhizosphere, 60 days; lane 6, Várzea soil, maize rhizosphere, 10 days; lane 7, Várzea soil, maize rhizosphere, 30 days; lane 8, Várzea soil, maize rhizosphere, 60 days; lane 9, Várzea soil isolate LV17; lane 10, Várzea soil isolate PV55; lane 11, Várzea soil isolate PV18; lane 12, Várzea soil isolate PV9; lane 13, strain C3L4; lanes M, markers (16S rDNA-based products of [from top to bottom] Listeria innocua ALM105, Arthrobacter sp., and Burkholderia cepacia P2).