Stones in arid environments are inhabited by actinobacteria of the family Geodermatophilaceae like the genera Blastococcus and Modestobacter frequent

The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media and fulfilled Koch’s postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation.

The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including ‘molecular tinkering/opportunism’ as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively.

Before the establishment of pure cultures, the species Frankia alni , ‘ Frankia casuarinae ’ and ‘ Frankia elaeagni ’ were proposed to encompass all causal agents of the nitrogen-fixing root nodules of dicotyledonous plants from the genera Alnus, Casuarina or Elaeagnus. The sole Frankia species with a validly published name, the type species F. alni , was described by Woronin (1866) as present in the root of alder. Until now no type strain has been designated for F. alni , even though the absence of a type strain has seriously inhibited the application of modern taxonomic methods to the genus Frankia . Thus, we propose that strain ACN14aT, isolated in pure culture from Alnus viridis ssp. crispa with morphological properties matching the original description of F. alni , be recognized as the type strain of this species according to Rule 18f of the International Code of Nomenclature of Bacteria. We compared ACN14aT to two strains, CcI3T and BMG5.12T, isolated from Casuarina cunninghamiana and Elaeagnus angustifolia, respectively, based on chemotaxonomy, phenotype microarray data and molecular data retrieved from genome sequences. All three tested strains grew as branched hyphae, produced vesicles and multilocular sporangia containing non-motile spores and metabolized short fatty acids, TCA-cycle intermediates and carbohydrates. Chemotaxonomically, the three strains were indistinguishable with respect to phospholipids (phosphatidylinositol, diphosphatidylglycerol, glycophospholipids and phosphatidylglycerol) and cell-sugar composition (glucose, mannose, ribose, rhamnose, galactose and xylose, with the latter two being diagnostic for the genus). The major fatty acids identified in all three strains were iso-C16 : 0, C17 : 1 ω8c, C15 : 0, C17 : 0 and C16 : 0. ACN14aT and BMG5.12T also shared C15 : 1 ω6c, while C18 : 1 ω9c was found to be unique to BMG5.12T. The major menaquinones identified in all three novel type strains were MK-9(H8), MK-9(H6) and MK-9(H4). MK-9(H2) was shared by ACN14aT and BMG5.12T, while MK-10(H4) and MK-8(H4) were only found in BMG5.12T. Analysis of 16S rRNA gene sequences showed 98.1–98.9 % identity between strains ACN14aT, CcI3T and BMG5.12T. Digital DNA–DNA hybridization values between the three type strains were well below 70 %. These results confirm the separation of the strains into three distinct species, Frankia alni , Frankia casuarinae sp. nov. and Frankia elaeagni sp. nov. Thus, we propose ACN14aT (=DSM 45986T=CECT 9034T), CcI3T (=DSM 45818T=CECT 9043T) and BMG5.12T (=DSM 46783T=CECT 9031T) as the respective type strains.