Frankia strain Ag45/Mut15T was isolated from a root nodule of Alnus glutinosa growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on A. glutinosa, in which it produces hyphae and clusters of N2-fixing vesicles. N2-fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15T contained meso-diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H6) and MK-9(H4). The major fatty acid profile (>10 %) consisted of iso-C16:0, C17 : 1 ω8c and C17 : 0. Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15T was most closely related to Frankia torreyi CpI1T and Candidatus Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on atpD, dnaA, ftsZ, pgk and rpoB amino acid sequences positioned the strain within cluster 1 of Alnus- and Myrica-nodulating species, close to Candidatus F. nodulisporulans AgTrST and F. canadensis ARgP5T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15T and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation. Candidatus F. nodulisporulans AgTrST, which cannot be cultivated in vitro, was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15T with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15T was not able to sporulate in nodule tissues like strain AgTrST.Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15T (=DSM 114737T=LMG 326O1T) to a novel species, with Ag45/Mut15T as type strain, for which the name Frankia umida sp. nov. is proposed.
Keywords: nitrogen fixing; polyphasic taxonomy; root nodule; symbiosis.