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

Abstract

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  ω7c/C1_8 : 1  ω6c), 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. AzHPPD showed less than 61% similarity to previously reported HPPDs and was able to tolerate 30 µM of mesotrione, which is an inhibitor of HPPD.