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Three Gram-negative, non-pigmented, rod-shaped, facultatively aerobic bacterial strains, designated d8-1T, d8-2 and IMCC1716, were isolated from a freshwater spring sample and a eutrophic freshwater pond. Based on characterization using a polyphasic approach, the three strains showed highly similar phenotypic, physiological and genetic characteristics. All of the strains harboured the nitrogenase gene nifH, but nitrogen-fixing activities could not be detected in nitrogen-free culture media. The three strains shared 99.6-99.7 % 16S rRNA gene sequence similarity and showed 89-100 % DNA-DNA relatedness, suggesting that they represent a single genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains d8-1T, d8-2 and IMCC1716 formed a monophyletic branch in the periphery of the evolutionary radiation occupied by the genus Azonexus. Their closest neighbours were Azonexus caeni Slu-05T (96.7-96.8 % similarity) and Azonexus fungiphilus BS5-8T (96.3-96.6 %). The DNA-DNA relatedness of the novel strains to these two species of the genus Azonexus was less than 70 %. The isolates could also be differentiated from recognized members of the genus Azonexus on the basis of phenotypic and biochemical characteristics. It is evident, therefore, that the three strains represent a novel species of the genus Azonexus, for which the name Azonexus hydrophilus sp. nov. is proposed. The type strain is d8-1T (=LMG 24005T=BCRC 17657T).

Azonexus hydrophilus sp. nov., a nifH gene-harbouring bacterium isolated from freshwater