| Summary: | Among other attributes, the Betaproteobacterial genus <i>Azoarcus</i> has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The “plant-associated” group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus <i>Aromatoleum</i>) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 <i>Azoarcus-Aromatoleum</i> strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in <i>Azoarcus-Aromatoleum</i> was also investigated by comparing full-length sequences of <i>nif</i> genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) <i>Azoarcus</i> comprising <i>Az. communis</i>, <i>Az. indigens</i> and <i>Az. olearius</i>, and two unnamed species complexes, (ii) <i>Aromatoleum</i> Group 1 comprising <i>Ar. anaerobium</i>, <i>Ar. aromaticum</i>, <i>Ar. bremense</i>, and <i>Ar. buckelii</i>, and (iii) <i>Aromatoleum</i> Group 2 comprising <i>Ar. diolicum</i>, <i>Ar. evansii</i>, <i>Ar. petrolei</i>, <i>Ar. toluclasticum</i>, <i>Ar. tolulyticum</i>, <i>Ar. toluolicum,</i> and <i>Ar. toluvorans</i>. Single strain lineages such as <i>Azoarcus</i> sp. KH32C, <i>Az. pumilus,</i> and <i>Az. taiwanensis</i> were also revealed. Full length sequences of <i>nif</i>-cluster genes revealed two groups of diazotrophs in <i>Azoarcus-Aromatoleum</i> with <i>nif</i> being derived from <i>Dechloromonas</i> in <i>Azoarcus sensu stricto</i> (and two <i>Thauera</i> strains) and from <i>Azospira</i> in <i>Aromatoleum</i> Group 2. Diazotrophy was confirmed in several strains, and for the first time in <i>Az. communis</i> LMG5514, <i>Azoarcus</i> sp. TTM-91 and <i>Ar. toluolicum</i> T<sup>T</sup>. In terms of ecology, with the exception of a few plant-associated strains in <i>Azoarcus</i> (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of <i>nar</i>, <i>nap</i>, <i>nir</i>, <i>nor,</i> and <i>nos</i> genes by most <i>Azoarcus-Aromatoleum</i> strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of <i>bzd</i> genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the <i>Azoarcus-Aromatoleum</i> group should be revisited; retaining the generic name <i>Azoarcus</i> for its entirety, or creating additional genera are both possible outcomes.
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