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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.

SUBMITTER: Rosado AS

PROVIDER: S-EPMC106771 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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

Rosado A S AS Duarte G F GF Seldin L L Van Elsas J D JD

Applied and environmental microbiology 19980801 8

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 a ...[more]

PMID: 9687429

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Project description:The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

Dataset Information

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

Publications

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

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