Comparative Genome Analysis of the Nirk-Carrying Azospirillum Sp. Strains.

Azospirillum sp. strains are one of the commonly found denitrifiers in soils. Previously, we have isolated 41 Azospirillum sp. denitrifiers from rice paddy soil in Tokyo, Japan. However, their nitrite reductase gene, a key denitrification functional gene, could not be identified by PCR due most likely to the mismatches in the primer annealing sites. In this study, we have developed a new PCR primers to amplify nirK in the genus Azospirillum. Sequence analysis revealed the diversity within the nirK in the genus Azospirillum. The nirK phylogeny did not match well with the phylogeny of the strains based on the 16S rRNA gene or rep-PCR DNA fingerprinting, suggesting the potential occurrence of the genetic exchange between strains. We performed pulsed-field gel electrophoresis (PFGE) followed by southern-blot hybridization to identify the replicons containing nirK gene. We identified multiple replicons in all Azospirillum sp. strains tested. The nirK gene was identified in the large replicons (640-950 kbp). To further characterize the denitrification functional genes in the Azospirillum sp. strains, the whole genomes of the ten representative strains were sequenced by Illumina HiSeq system. The results obtained in this study suggest the diversity and evolutionary history of nirK in the genus Azospirillum.