Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 13 May 2019 20:40:33 Frankia saprophytica sp. nov., an atypical, non-infective (Nod–) and non-nitrogen fixing (Fix–) actinobacterium isolated from Coriaria nepalensis root nodules Imen Nouioui, 1 Faten Ghodhbane-Gtari, 2 Hans-Peter Klenk 1 and Maher Gtari 3, * Abstract Strain CN3 T ,a Coriaria nepalensis isolate, appears to form hyphae and sporangia typical of members fo the genus Frankia. However, it failed to form vesicles, to reduce acetylene and to induce nodules on its original host plant. A polyphasic approach was used here to determine the taxonomic status of strain CN3 T . The 16S rRNA gene sequence of strain CN3 T showed the highest sequence identity with Frankia asymbiotica type strain M16386 T (99.4 %). Digital DNA–DNA hybridization between strains CN3 T and M16386 T was 25.7 %, which is clearly below the accepted cut-off point of 70 %. The G+C content of DNA was 71.8 mol%. Whole-cell hydrolysates of strain CN3 T were rich in meso-diaminopimelic acid. Cell-wall sugars were composed of galactose, glucose, mannose, rhamnose and traces of ribose. The polar lipid profile contained phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphoglycolipids, phospholipid, six uncharacterized glycolipids and two uncharacterized lipids. The predominant menaquinone (>25 %) was MK-9(H 6 ). Major fatty acids (>15 %) of strain CN3 T consisted of iso-C 16 : 0 ,C 17 : 1 !8c and C 15 : 0 . Based on 16S rRNA gene phylogeny, genome sequence analysis and phenotypic results, strain CN3 T (=DSM 105290 T =CECT 9314 T ) is proposed to represent the type strain of a novel species, Frankia saprophytica sp. nov. The genus Frankia encompasses soil actinobacteria distin- guishable by their ability to fix nitrogen and to establish symbiotic relationships with a wide range of dicotyledonous host plants [1]. Isolates of the genus Frankia are able to re- induce nitrogen-fixing root nodules on their host plant (ful- filling Koch’s postulates) and generally fall into one of the three phylogenetic clusters defined regardless of the molecu- lar marker used [2]. Five species are currently recognized by validly published names: Frankia alni and Frankia casuari- nae [3] belonging to cluster 1, Frankia coriariae [4] from cluster 2, and Frankia discariae [5] and Frankia elaeagni [3] falling within cluster 3. However, a number of strains lack- ing one or more morphological and/or symbiotic features have also been isolated from actinorhizal root nodules [6– 10]. Molecular studies indicated that these isolates belong to the genus Frankia, representing a distinct cluster (cluster 4) [8–10]. Some of these atypical strains have the ability to induce ineffective, non-nitrogen-fixing root nodules on their host plants and are classified within Frankia inefficax [11]. Frankia asymbiotica includes strains able to fix nitro- gen but unable to induce root nodules on their original host or any of the actinorhizal plants tested [12]. Strain CN3 T , belonging to the genus Frankia, was isolated from root nodules of Coriaria nepalensis (Muree, northern Pakistan) [13] and was found to be a non-nitrogen fixing (Fix) and non-infective (Nod) actinobacterium. The taxo- nomic status of strain CN3 T was determined here on the basis of a polyphasic approach and is proposed as the type strain of a novel species, namely Frankia saprophytica sp. nov. Strain CN3 T was maintained in basic propionate (BAP) medium [14] supplemented with 0.5 mM NH 4 Cl as nitrogen source and incubated at 28  C. Chemotaxonomic analyses were performed using freeze-dried biomass from 4-week-old cultures, while wet biomass was used for other phenotypic analyses. The ability of strain CN3 T to oxidize carbon and nitrogen sources and to grow in the presence of a broad range of Author affiliations: 1 School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, UK; 2 Laboratoire Microorganismes et Biomol ecules Actives, Facult e des Sciences de Tunis, Universit e Tunis El Manar, 2092 Tunis, Tunisia; 3 Institut National des Sciences Appliqu ees et de Technologie, Universit e Carthage, Centre Urbain Nord BP 676-1080 Tunis Cedex, Tunisia. *Correspondence: Maher Gtari, maher.gtari@fst.rnu.tn Keywords: polyphasic taxonomy; actinobacteria; symbiosis; actinorhizas. Abbreviations: dDDH, digital DNA-DNA Hybridization; GGDC, Genome-to-Genome Distance Calculator; GTR, General Time-Reversible; ML, maximum- likelihood; MP, maximum-parsimony; MRE, maximal-relative-error; MUSCLE, MUltiple Sequence Comparison by Log-Expectation; PAUP, Phylogenetic Analysis Using Parsimony; RAxML, Randomized Axelerated Maximum Likelihood; TNT, Tree analysis New Technology. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and draft genome sequence reported are MF960832 and AGJN00000000, respectively. One supplementary table and one supplementary figure are available with the online version of this article. TAXONOMIC DESCRIPTION Nouioui et al., Int J Syst Evol Microbiol 2018;68:1090–1095 DOI 10.1099/ijsem.0.002633 002633 ã 2018 IUMS 1090