The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.

This study was designed to determine the plant growth promoting (PGP) potential of members of the genus Frankia . To this end, the genomes of 21 representative strains were examined for genes associated directly or indirectly with plant growth. All of the Frankia genomes contained genes that encoded for products associated with the biosynthesis of auxins [indole-3-glycerol phosphate synthases, anthranilate phosphoribosyltransferases ( trp D), anthranilate synthases, and aminases ( trp A and B)], cytokinins (11 well-conserved genes within the predicted biosynthetic gene cluster), siderophores, and nitrogenases ( nif operon except for atypical Frankia ) as well as genes that modulate the effects of biotic and abiotic environmental stress (e.g., alkyl hydroperoxide reductases, aquaporin Z, heat shock proteins). In contrast, other genes were associated with strains assigned to one or more of four host-specific clusters. The genes encoding for phosphate solubilization (e.g., low-affinity inorganic phosphate transporters) and lytic enzymes (e.g., cellulases) were found in Frankia cluster 1 genomes, while other genes were found only in cluster 3 genomes (e.g., alkaline phosphatases, extracellular endoglucanases, pectate lyases) or cluster 4 and subcluster 1c genomes (e.g., NAD(P) transhydrogenase genes). Genes encoding for chitinases were found only in the genomes of the type strains of Frankia casuarinae , F. inefficax , F. irregularis , and F. saprophytica . In short, these in silico genome analyses provide an insight into the PGP abilities of Frankia strains of known taxonomic provenance. This is the first study designed to establish the underlying genetic basis of cytokinin production in Frankia strains. Also, the discovery of additional genes in the biosynthetic gene cluster involved in cytokinin production opens up the prospect that Frankia may have novel molecular mechanisms for cytokinin biosynthesis.

A study was made of the ability of four strains of Frankia, representing two distinct host-compatibility groups, to utilize various carbon sources for growth. Isolates EAN1pec and EuI1c, representatives of host-compatibility group II, utilized a variety of sugars and sugar alcohols as carbon sources. Isolates CpI1 and ACN1AG, members of host-compatibility group I, did not use these substrates for growth. All of the isolates studied except for EAN1pec utilized propionate for growth. The only one of the four isolates unable to grow on dicarboxylic acids was EuI1c. Vesicles were detected microscopically during growth of EAN1pec and ACN1AG in media containing ammonia. The numbers and size of vesicles, however, were greater in media without ammonia. Strain ACN1AG did not form vesicles in any of the media when incubated at temperatures greater than 33 °C. The CpI1 isolate formed vesicles only when grown in ammoniafree media. Vesicles were not formed by strain EuI1c in any of the media. Sporulation of the four isolates was carbon-source dependent. Isolate CpI1 formed sporangia when grown in propionate-containing medium at 20 °C but formed none in the medium at 33 °C.

In the last edition of the Bergey's Manual of Systematic Bacteriology, Frankia was the sole genus found in the family Frankiaceae. This single-genus family, together with Geodermatophilaceae, Nakamurellaceae, Sporichthyaceae, Acidothermaceae, and Cryptosporangiaceae, makes up the order Frankiales, an artificial taxon within the phylum Actinobacteria supported only marginally by 16S rDNA similarities (Normand and Benson 2012). Genus Frankia represents a monophyletic assemblage of soil actinobacteria distinguishable by multilocular sporangia-forming branched hyphae and their unique ability to grow in nitrogen-limited substrates due to their ability to fix nitrogen within specialized thick-walled structures, termed vesicles, produced in vitro and in planta. Diversity of Frankia has been noted in several separate reports on the presence of comparable root nodule structures induced in numerous trees and shrubs growing as aggressive pioneers in nitrogenlimited ecosystems such as glacial moraines, gravel slopes, volcanic ashes, mine spoils, or burned forests with a contributed input of 15 % of total biologically fixed nitrogen on earth (Quispel 1990). Despite the absence of apparent close kinship among these plants, they have been termed actinorhizal plants due to the nature of the root nodule-causing agents detected in plant tissues. Based on in planta morphology and the limited cross-infectivity of crushed-nodule inocula, Becking

A major hurdle in the development of a genetic system for Frankia is the lack of genetic markers. To identify potential genetic markers, 12 strains of Frankia were screened for resistance to antibiotics by the use of a growth inhibition assay. All of the strains demonstrated sensitivity to tested antibiotics. Several strains had distinctive patterns of antibiotic resistance that are potentially useful as genetic markers. Novobiocin was the antibiotic to which the most strains were resistant.Key words: genetics, genetic markers, Frankia, actinorhizal, nitrogen fixation, vesicles.