Azospirillum caeni sp. nov., a mesotrione-resistant and nitrogen-fixing bacterium isolated from an activated sludge

A Gram-stain-negative, facultative anaerobic, short rod-shaped, non-spore-forming, mesotrione-resistant bacterium, designated strain A39 T , was isolated from activated sludge treating pesticide-manufacturing wastewater. Strain A39 T grew at 15–42 ℃, pH 6–10 and 0–1.0% NaCl (w/v), tolerated up to 2 mM mesotrione and could fix nitrogen. Strain A39 T showed the highest 16S rRNA gene sequence similarity to Azospirillum halopraeferens Au 4 T (98.1%) and less than 97% sequence similarity with other species of the genus Azospirillum . Phylogenetic analysis based on 16S rRNA gene and genome sequences placed strain A39 T within genus Azospirillum , forming a subclade with A. halopraeferens Au 4 T . The values of digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) between strain A39 T and A. halopraeferens Au 4 T were 25.5% and 82.7%, respectively, which were significantly below the recommended thresholds for species delineation (dDDH <70% and ANI <95–96%). The major fatty acids were summed feature 8 (C 18 : 1 ω7 c / C1 8 : 1 ω6 c), C 16 : 0 and C 18 : 0 . Strain A39 T comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, glycolipid and two aminolipids as the predominant polar lipids; and ubiquinone 10 as the major respiratory quinone. The genome size was 8.1 Mb with a DNA G+C content of 72.1%. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, strain A39 T represented a novel species of the genus Azospirillum , for which the name Azospirillum caeni sp. nov. is proposed. The type strain is A39 T (=MCCC 1K09771 T =KCTC 18385 T ). Additionally, a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD), named AzhppD , was identified in strain A39 T . Az HPPD showed less than 61% similarity to previously reported HPPDs and was able to tolerate 30 µM of mesotrione, which is an inhibitor of HPPD.