Isolation and identification of Ktedonobacteria using 16S rRNA gene sequences data

The aims of this study were to isolate and identify the members of Ktedonobacteria from soil samples in Cisolok geysers area, West Java, Indonesia. To isolate the bacteria, sample was spread on 10-fold-diluted Reasoner’s 2A medium and the plates were incubated at 30 °C until the Ktedonobacteria-like colonies appeared. Ktedonobacteria-like colonies showed the characteristics of a firm pale orange colony. The colonies were purified and colony PCR was performed using Ktedonobacteria specific and universal eubacterial primers for 16S rRNA gene. Almost full-length of 16S rRNA gene sequences were obtained in order to know the identity of the taxonomic affiliation of the isolates. Three Ktedonobacteria-like isolates, designated S3.2.1.5, S3.2.1.6 (isolated from soil under Bamboo tree) and S3.2.2.5 (isolated from soil in decayed Bamboo stems) were obtained. The detection using Ktedonobacteria specific primers showed positive results, which confirmed that all of three isolates belong to class Ktedonobacteria. The sequence homology search using EzBioCloud showed that these isolates (S3.2.1.5, S3.2.1.6, and S3.2.2.5) were belonged to the family Dictyobacteraceae and closely related to Bacterium SOSP1-79 (99.20%, 98.83%, 99.34%, respectively) and Dictyobacter aurantiacus S-27T (97.41%, 97.41%, 98.00%, respectively). The low percentage of sequence homology to type strains indicates that they could be a candidate of novel taxa of the genus Dictyobacter. Further characterizations such as morphology, physiology, and chemotaxonomy are needed in order to clarify the taxonomic position of these potential novel species of Dictyobacter.


Introduction
The class Ktedonobacteria was proposed by Cavaletti et al. in 2006 for actinomycete-like bacteria and placed in the phylum Chloroflexi, which known as a deep branching lineage in the domain Bacteria [1]. This class consists of the environmental 16S rRNA gene clones, uncultured, and cultivated isolates with a valid names and cultivated isolates which have not been proposed yet [2]. To date, this class contains

Isolation
The sampling was conducted in November 2018 at forest around Cisolok geysers, West Java, Indonesia. Two samples, designated S3.2.1 and S3.2.2 (06 o 55'991"S, 106 o 27'187"E), were collected from soil under bamboo tree and soil in decayed bamboo stems in forest around Cisolok geysers, respectively. The Reasoner's 2A medium (1:10 dilution) with the addition of gellan gum as a solidifying agent was used for isolation medium of Ktedonobacteria [10]. A soil sample was spread directly on the surface of the medium. The plates were incubated at 30 o C until the Ktedonobacteria-like colony appeared, which showed a firm pale orange colony [4]. The Ktedonobacteria-like colony was purified at least three times to obtain pure cultures.

Results and Discussion
The firm pale orange colonies were observed in plates after incubation at 30 o C for a week. Three isolates designated S3.2.1.5, S3.2.1.6, and S3.2.2.5 as shown in Figure 1, were obtained from soil samples under bamboo tree and in decayed bamboo stems from forest around Cisolok geysers. As shown in Figure 1, three isolates were forming branched substrate mycelia with aerial mycelia borne directly from substrate mycelia. The aerial mycelia were harbouring a sporangium-like structures ( Figure 1). The result showed that these three isolates shared similar morphological features to Ktedonobacteria, such as forming firm pale orange colonies, branched substrate mycelia, and aerial mycelia with sporangium-like structure. Based on the previous study, the Ktedonobacteria showed a firm pale orange colony [4], branched vegetative mycelia with budding [6] or sporangium-like structures in their aerial mycelia [7]. The use of 1/10 R2A gellan gum as isolation medium was demonstrated by the previous study of Yabe et al. [10] for isolation of Ktedonobacteria.
Three Ktedonobacteria-like isolates showed an amplified 16S rRNA gene fragments after the amplification using Ktedonobacteria-specific primers by colony PCR method (data not shown). The amplification of 16S rRNA gene by colony PCR for three Ktedonobacteria-like isolates in this study showed PCR bands after amplification using both Ktedonobacteria specific and universal eubacteria 16S rRNA gene primers, indicating that these isolates are members of the class Ktedonobacteria. According to Yabe et al. [8], the KTED161F is proposed as Ktedonobacteria specific primer due to the dissimilarities to the 3' end of non-target sequences in each class within the phylum Chloroflexi, while the GNSB941R is a Chloroflexi-specific primer. Yabe et al. [8] was successfully demonstrated the use of primer set of KTED161F and GNSB941R which shows high specificity for detecting class Ktedonobacteria within the employed samples.   [1], the bacterium SOSP1-79 is a Ktedonobacteria isolates from soil collected in cereal field in Egypt. Their closest related species is Dictyobacter aurantiacus S-27 T sp. nov. As reported by Yabe et al. [10], this type strain was isolated from paddy soil collected from waterlogged rice paddy field on the mountainside of Gunung Salak, West Java, Indonesia. This study confirmed that the use of Ktedonobacteria specific primers as designed by Yabe et al. [11], was able to successfully and accurately detect the Ktedonobacteria by colony PCR method. Yabe et al. [8] reported the numerous members of Ktedonobacteria were detected from forest soil by using Ktedonobacteria specific primers, with most of the representative sequences were belonged to unknown Ktedonobacterales group.
As shown in Figure 2, the 16S rRNA gene sequence-based NJ phylogenetic tree (as supported by ME and ML trees) depicted that the three isolates were belonged to the genus Dictyobacter within family Dictyobacteraceae within the class Ktedonobacteria, as supported by 90% of bootstrap value. The phylogenetic tree showed that these isolates were clustering together with bacterium SOSP1-79 with 99% of bootstrap value, and they were formed a distinct lineage from their closest related species Dictyobacter aurantiacus S-27 T as supported by 76% bootstrap values. The tree indicated that most of the members of Dictyobacteraceae belong to undescribed taxa. These undescribed taxa are challenging and interesting for taxonomic study and prospecting. Further analysis, such as the analysis of morphological, physiological, and biochemical are required in order to determine the identity of these three isolates. The previous study [4] showed that the family Dictyobacteraceae consists of two genera, namely Dictyobacter and Tengunoibacter, four valid species name, and 13 cultivated isolates which have not been proposed yet.
The three Ktedonobacteria-like isolates found in this study shared similar colony character to bacterium SOSP1-79 strain, such as the formation of orange colour of colony, which indicated the SOSP1-79 strain was the closely-related taxon to these three isolates. According to Cavaletti et al. [1], the colony of SOSP1-79 bacterium on ISP 3 agar after 3 weeks of incubation showed orange colour. Additionally, Cavaletti et al. [1] reported the antibiotic resistance profiles of strain SOSP1-79, which showed that the bacterium was sensitive to 5 μg/ml of novobiocin or ramoplanin and 20 mg/ml of apramycin and glycopeptide A40926, meanwhile resistant to 20 μg/ml of rifampin. Further study is needed to elucidate the antibiotic profiles of the three Ktedonobacteria-like isolates (S3.2.1.5, S3.2.1.6, and S3.2.2.5) obtained in this study.

Conclusions
Three bacterial isolates that shared similar morphological features to Ktedonobacteria were obtained from soil samples collected under bamboo tree and in decayed bamboo stems in forest around Cisolok geysers, West Java, Indonesia. Three Ktedonobacteria-like isolates showed an amplified 16S rRNA gene fragments by using Ktedonobacteria-specific primers that performed based on colony PCR method. Based on phylogenetic tree analyses, three isolates were affiliated with the genus Dictyobacter within the family Dictyobacteraceae, with Dictyobacter aurantiacus as the closest related species. Interestingly, they showed low homology percentage (97.41-98.00%) to Dictyobacter aurantiacus and they were formed a distinct lineage from this species. Therefore, the full characterization of morphological, physiological, biochemical, and whole genome analysis for these isolates are needed to elucidate the taxonomic position of these potential novel species. The results of this study confirmed that colony PCR method is useful method for the detection of the members of class Ktedonobacteria. This study also indicated that the forest soil around Cisolok geysers is a potential source for isolating novel taxa of Ktedonobacteria.