Cultivation of Uncultured Bacteria of the Class Ktedonobacteria in the Phylum Chloroflexi

The class Ktedonobacteria in the phylum Chloroflexi is known to contain a large number of uncultured, environmental 16S rRNA gene clones, and cultured representatives are a limited number. In this review, recent findings on the taxonomical and ecological significance of the class Ktedonobacteria in the phylum Chloroflexi are discussed based on the findings from both the characteristics of the cultured Ktedonobacteria and molecular-based analysis. The microorganisms characterized so far include four species in three genera, Ktedonobacter, Thermosporothrix and Thermogemmatispora, and were proposed to represent three families, Ktedonobacteraceae, Thermosporotricaceae, and Thermogemmatisporaceae, and two orders, Ktedonobacterales and Thermogemmatisporales. Ktedonobacteria strains showed a common property of gram-positive, aerobic organisms that produce branched vegetative mycelia and form spores by budding.


Introduction
The phylum Chloroflexi, formerly known as the green non-sulfur bacteria, has been recognized as bacterial taxa containing a number of environmental 16S rRNA gene clones with a limited number of cultured representatives [1][2][3][4][5].The phylum Chloroflexi is generally considered to be a deep-branching lineage of the domain bacteria [6][7][8].The phylum has formerly been devided into four subphyla (class-level taxa) on the basis of 16S rRNA gene sequences [1]; subphylum I (now the class Anaerolinea and the class Caldilinea [9]), subphylum II (the class "Dehalococcoidetes"), subphylum III (the class Chloroflexi) and subphylum IV (clone cluster).Later, the cluster of genus Thermomicrobium in the subphylum III has been reclassified as an additional subphylum, the class Thermomicrobia [10], and more recently, Yabe et al. [11] proposed to include the class Ktedonobacteria [12] in the phylum Chloroflexi.In addition to these major six subphyla (the classes), other uncultured lineages at the subphylum (class) level, "E05 lineage" and "G04 lineage", are also known in the pylum Chloroflexi [2,13].
The class Ktedonobacteria, which is now belonging to the phylum Chloroflexi, is also known to contains many uncultured environmental clones by the cultivationindependent molecular studies.The finding of the presence of uncultured bacteria in the environments prompted the efforts of cultivation and isolation of such bacterial strains by the use of various methods.The class Ktedonobacteria is such bacterial group which has recently been cultured and characterized.

Present taxonomy of the phylum Chloroflexi
As shown in Table 1, taxonomy of the phylum Chloroflexi at present are summarized.Phylogenetic relationships among all the genera in the phylum Chloroflexi is shown in Figure 1.
The class Thermomicrobia also involves cultured organisms of the genera Thermomicrobium, Sphaerobacter and "Thermobaculum", which are rodshaped, moderately thermophilic or hyperthermophilic, chemolithoheterotrophic and aerobic.The class Anaerolinea is most frequently detected in the various environment and involves cultured organisms of the genera Anaerolinea, Bellilinea, Leptolinea, Levilinea and Longilinea.A new class Caldilineae was proposed by Yamada et al. [9] for the genus Caldilinea.The class  (14), and according to the reports by Costello et al. [13] and Davis et al. [15], the table was modified.The morphology, physiology and genetic properties of cultivated genera of the phylum Chloroflexi are shown in Table 2.These strains are obligately aerobic, mesophilic or moderately thermophilic, multicellular filamentous or rod-formed or cocci, chemolithohetero-trophic organisms degrading carbohydrates.Thus, the phylum Chloroflexi is highly heterogeneous with respect to phylogenetic, morphological and physiological characteristics.Thus, the phylum contains a large number of yet-to-be cultured organisms from natural and artificial environments.Further studies to isolate and cultivate these organisms are required.

Cultivation of uncultured Ktedonobacteria strains
Among the classes in the phylum Chloroflexi, present paper mainly deals with the cultured strains in the class Ktedonobacteria.Phylotypes affiliated with the Ktedonobacteria are detected in a wide range of environments, and the group now contains quite diverse rRNA gene sequences among known orders in the class Ktedonobacteria (Figure 2).To reveal their physiological and biochemical role, attempts were made to cultivate them.The first pure culture representing the group, i.e., Ktedonobacter racemifer was obtained in 2006 from Italian soil [12].The bacterial strains obtained were filamentous, aerobic and mesophilic, but not thermophilic.Later, the second tangible organism of the group, Thermosporothrix hazakensis was isolated in 2010 from thermophilic atmosphere, hot compost, in Japan [11].Simultaneously, a new thermophilic genus Thermogemmatispora with two new species were successfully cultivated and characterized, which was isolated in 2010 from geothermal soils in Japan [22].To our knowledge, these four species are the only named bacteria of cultivated organisms of the class Ktedonobacteria.However, in addition to these species, other isolates, which has not been taxonomically characterized, are known [12,[24][25] (Figure 2): SOSP strains were isolated from Italian soil [12].iso-C16:0, anteiso-C17:0, iso-C17:0 iso-C17:0 iso-C17:0, iso-C19:0, Stott et al. [25] used media solidified with gellan for the isolation of SOSP strains, and Davis et al. [24] also used media solidified with gellan for the isolation of Ellin7237.Although many improved methods, such as mini-colony formation [26], micromanipulation method [27], diluted nutrient broth method [23], and flow cytometry method [28] are reported for cultivating the uncultured bacteria, the approach by gellan gum media may be useful for further isolating uncultured Ktedonobacteria.
As chemotaxonomic characteristics of Ktedonobacteria strains, they contained menaquinone MK-9(H 2 ) as respiratory quinone, iso-C16:0 and/or iso-C17:0 as major cellular fatty acid.A quite rare fatty acid, 12,17dimethyl-C18:0, was found in the cellular fatty acids of Thermogemmatispora species, and the presence of this fatty acid is taxonomic significance [29].Ktedonobacteria strains contained DNA of 54-60 mol% DNA G+C content, and ornithine, glutamic acid, serine, glycine, and alanine in the cell wall.In Thermogemmatispora species, histidine was additionally included in the cell wall, which indicates the presence of histidine is also taxonomically significant.

Morphological characteristics of the Ktedonobacteria
The morphological properties of cultivated strains in the orders Ktedonobacterales and Thermogemmatisporales in the Ktedonobacteria are shown in Figure 3 [11,22].The strains are gram-positive, aerobic, mesophilic or thermophilic, chemolithoorganoheterotrophic organisms degrading polysaccharides and peptides.Morphological characteristic features of these strains are the formations of branched multicellular filaments and spores.The spores are formed by budding, therefore, the spores formed could be called "blastospores".As far as known, sporulation modes in prokaryotes include formation of endospores exemplified by Firmicutes bacteria, myxospores by myxobbacteria and arthrospores by actinomycetes.Microscopic observations showed that these strains formed multiple exospores per mother cells by budding in branched aerial mycella.Although branched aerial mycelia is a characteristic of actinomycetes, multiple budding sporulation has not been previously described in prokaryotes [30].Life cycle of the strain of Thermosporothrix hazakensis is shown in Figure 4 [31].

Genome sequence of the Ktedonobacteria
Very recently, genome sequence of Ktedonobacter racemifer SOSP1-21 T was reported by Chang et al. [32].
It was revealed that the strain contained DNA with a length of 13.7 Mbp, which is the largest of all completely sequenced so far.K. racemifer stands out because of its enormous genome size of more than 13 Mbp.The genomes of neighboring taxa, Sphaerobacter thermophilus and Thermomirobium roseum are significantly smaller, 3.9 Mbp and 2.9 Mbp, respectively.Our preliminary data on the draft genome sequence of Thermosporothrix hazakensis SK20-1 T , the closest relative of K. racemifer, suggested that it contained 7.

Figure 2 .
Figure 2. Neighbour-Joining Phylogenetic Tree of the Class Ktedonobacteria Derived from Comparative Analysis of 16S rRNA Gene Sequences.Sequences of Clones were Obtained from Greengenes Databases [23]

Table 1 . Taxonomy of the Pylum Chloroflexi
The Table was created based on the report by Euzeby