GENETIC DIVERSITY OF NIFH GENE-SEQUENCES IN PAENIBACILLUS AZOTOFIXANSSTRAINS AND SOIL SAMPLES ANALYZED BY DENATURING GRADIENT GEL-ELECTROPHORESIS OF PCR-AMPLIFIED GENE FRAGMENTS

The diversity of dinitrogenase reductase gene (nifH) fragments in Paen ibacillus azotofixans strains was investigated by using molecular meth ods. 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, mere amplified by PCR by using degenerate primers and were characterized by DNA sequenc ing. We found that there are two nifH sequence clusters, designated cl usters I and II, in P. azotofixans. The data further indicated that th ere was sequence divergence among the nifH genes of P. azotofixans str ains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH clust er II sequences were similar to the alternative (anf) nitrogenase sequ ence. A nested PCR assay for the detection of nifH (cluster I) of P. a zotofixans was developed by using the degenerate primers as outer prin ters and two specific primers, designed on the basis of the sequence i nformation obtained, as inner primers, The specificity of the inner pr imers 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 di versity of this region of nifH in P. azotofixans strains, as well as i n soil and rhizosphere samples. The results revealed sequence heteroge neity among different nifH genes. Moreover, nifH is probably a multico py gene in P. azotofixans. Both similarities and differences were dete cted in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and pr ovides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environment al samples.