Diversity of grass-associated Nitrospirillum and proposal of six novel species

In this study, we characterized 86 plant growth-promoting bacterial strains belonging to the genus Nitrospirillum, isolated from diverse host plants and geographic regions. We investigated their evolutionary relationships through phylogenetic analyses of the 16S rRNA and recA genes, complemented by phylogenomic approaches incorporating genomic similarity metrics, such as ANI and dDDH. The classification of type strains was further supported by in silico analyses of chemotaxonomic markers, particularly genes involved in fatty acid biosynthesis and elongation, phospholipid and quinone production, and nitrogen fixation (nifHDK operon). Phenotypic and chemotaxonomic characterization was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid methyl ester (FAME) profiling, and physiological assays. These included evaluations of nitrogen fixation capacity, antibiotic resistance, carbon source utilization, and enzymatic activities. This integrative approach provided detailed insight into the characteristics and diversity of the studied strains. Phylogenetic and genomic analyses revealed six novel taxa within the genus Nitrospirillum, in addition to the previously described species N. amazonense, N. iridis, and N. viridazoti. The distinctiveness of these new lineages was supported by both genomic metrics and phenotypic traits. All novel strains also exhibited activity of nitrogenase enzyme, confirming their nitrogen-fixing ability under in vitro conditions. Based on these findings, we propose the formal description of six novel species: Nitrospirillum bahiense sp. nov. (= BR 11865^T, = UCCCB 233^T), Nitrospirillum guanabarense sp. nov. (= BR 11163^T, = UCCCB 228^T), Nitrospirillum guaranorum sp. nov. (= BR 11164^T, = UCCCB 229^T), Nitrospirillum karajorum sp. nov. (= BR 11752^T, = UCCCB 231^T), Nitrospirillum goiasense sp. nov. (= BR 11828^T, = UCCCB 232^T), and Nitrospirillum pindoramense sp. nov. (= BR 11622^T, = UCCCB 230^T).