Potentially Mobile Denitrification Functional Genes Identified in Azospirillum sp. Strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification functional gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested the potential occurrences of horizontal gene transfer (HGT) for the denitrification functional genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 Azospirillum sp. denitrifying strains, and searched for mobile genetic elements that contain denitrification functional genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons) with their size ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549-941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir - nor gene arrangement, especially between TSH58, Sp7T and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir - nor gene cluster and the novel IS 6 family insertion sequences (IS Az581 and IS Az582 ). The nirK within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing nir - nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside of the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers. IMPORTANCE Evolutionary history of denitrification is complex. While occurrence of horizontal gene transfer has been suggested for denitrification functional genes, most studies report circumstantial evidences such as disagreement between denitrification functional gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification functional genes including nirK and norCBQD located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely received benefit by acquiring denitrification functional genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge in the ecology and evolution of denitrification.