ABSTRACTDenitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification 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 denitrifyingAzospirillumsp. strains and searched for mobile genetic elements that contain denitrification genes. AllAzospirillumsp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase genenirKwas located on large replicons (549 to 941 kbp). Genome sequencing showed thatAzospirillumstrains that had similarnirKsequences also shared similarnir-norgene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity betweennir-norgene clusters among the threeAzospirillumstrains, a composite transposon structure was identified in the genome of strain TSH58, which contains thenir-norgene cluster and the novel IS6family insertion sequences (ISAz581and ISAz582). ThenirKgene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing thenir-norgene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely relatedAzospirillumsp. denitrifiers.IMPORTANCEThe evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses ofAzospirillumsp. denitrifiers, we identified denitrification genes, includingnirKandnorCBQD, located on a mobile genetic element in the genome ofAzospirillumsp. strain TSH58. ThenirKwas actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification.