Frankia nepalensis sp. nov., a non- infective non- nitrogen- fixing isolate from root nodules of Coriaria nepalensis Wall

Strains CN4(T), CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N-2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4(T) contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H-4) and MK-9(H-6) are the predominant menaquinones (>15 %), and iso-C-16 : 0 and C-17 : 1 omega 8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4(T) is most closely related to Frankia saprophytica CN 3(T). An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3(T) , Frankia asymbiotica M16386(T) and Frankia inefficax EuI1c(T) with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4(T) and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4(T), CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4(T) has a 10.76 Mb genome. CN4(T) was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4(T) (=DSM 114740(T) = LMG 32595(T)) to a novel species, with CN4(T) as type strain, for which the name Frankia nepalensis sp. nov. is proposed.