Isolation and 16S rRNA gene sequences analysis of thermophilic Actinobacteria isolated from soil in Cisolok geothermal area, West Java, Indonesia

The thermophilic Actinobacteria are known not only as producers of pharmaceutically important bioactive compounds, but also commercially important enzymes, yet their diversity in geothermal area in Indonesia have been rarely reported. In our previous study, a new thermophilic Actinobacteria genus belonging to the family Pseudonocardiaceae has been isolated from soil in Cisolok geothermal area. This current study reports the taxonomy and findings of potentially new taxa of thermophilic Actinobacteria, based on phylogenetic analysis of 16S rRNA gene sequences. Twenty-five isolates of thermophilic Actinobacteria were isolated from soil samples and maintained using previously described methods. The 16S rRNA gene sequence-similarity search against all related species was performed using EzTaxon-e database. The sequences of 25 isolates showed similarity to member of family Streptomycetaceae (genus Streptomyces; 10 isolates), Thermomonosporaceae (genus Actinomadura; 3 isolates), Streptosporangiaceae (genus Microbispora; 6 isolates), Micromonosporaceae (genus Micromonospora; 2 isolates), Pseudonocardiaceae (genus Amycolatopsis; 3 isolates), and Nocardiaceae (genus Nocardia; 1 isolate). Fifteen out of 25 isolates are belong to non-Streptomycetaceae family, thus regarded as rare Actinobacteria. Nineteen out of 25 isolates showed 16S rRNA gene sequence-similarity values 97 – 99% to their closely related species, suggested the potential for findings novel taxa of thermophilic Actinobacteria from Cisolok geothermal area.


Introduction
Actinobacteria is one of the most dominant phyla in the bacteria domain and has an ecologically significant role in biological processes such as biogeochemical cycles and bioremediation [1]. The The 3rd International Conference on Biosciences IOP Conf. Series: Earth and Environmental Science 457 (2020) 012015 IOP Publishing doi: 10.1088/1755-1315/457/1/012015 2 high abundance of actinobacterial species in various environments was recorded not only in normal but also in extreme environments defined by high temperatures, such as geothermal and volcanic areas, terrestrial hot springs, and geysers [2]. Thermophilic Actinobacteria grow well at quite high temperatures ranging from 40 to 80 o C. They are known not only as producers of pharmaceutically important bioactive compounds such as antibiotics and anti-inflammatory compounds, but also industrially and clinically important enzymes [1,3].
The microbial ecology of Actinobacteria has been diversely reported, yet the ecological diversity, species distribution, and biogeography in hot springs are still poorly described. The difficulties in isolation process and maintenance in pure culture have caused thermophilic Actinobacteria been less explored [4]. Cisolok geothermal area in Sukabumi, West Java, is still less exploited as sources of thermophilic microorganisms, and thus a high-potential area for isolation of novel indigenous thermophilic Actinobacteria. The natural product screening from novel thermophilic Actinobacteria from unexplored and extreme environments will increase the possibility in finding novel secondary metabolites with great potential [5,6].
In previous studies, Yokota et al. [7] successfully isolated new species of thermophilic bacterium namely Paenibacillus cisolokensis sp. nov., from litter of Cisolok geyser. A novel thermophilic Actinobacteria genus and species, Gandjariella thermophila gen. nov., sp. nov., belonging to the family Pseudonocardiaceae has also been isolated from soil forest in Cisolok geothermal area [8]. These studies indicated the high possibility of findings potentially new thermophilic Actinobacteria taxa in Cisolok geothermal area. This current study reports the taxonomy and findings of potentially new taxa of thermophilic Actinobacteria isolated from soil in geothermal area of Cisolok, based on phylogenetic analyses of 16S rRNA gene sequences. 2) soil under Bamboo tree (6 o 57'482"S, 106 o 28'655"E). Soil samples from location 1, 2, and 3, are given code SL1-1 to 2, SL2-1 to 2, and SL3-1 to 2 respectively. Isolation of thermophilic Actinobacteria was performed using method as described by Yabe et al. [9] on Reasoner's 2A (Daigo, Nihon Seiyaku) agar medium with some modifications, incubated at 45 o C for 4 weeks, and purified several times on 1 % (w/v) International Streptomyces Project (ISP) 1 (tryptone 5 g, yeast extract 3 g L -1 ) agar medium [10] to obtain pure isolates. The pure isolates were grown on ISP 1 and ISP 3 (oatmeal 20 g, agar 18 g, trace salt solutions 1 ml L -1 ) agar media [10], incubated at 45 o C for 7 days, and maintained at room temperature as stock cultures. The pure isolates were also stored as agar block in 20 % (v/v) glycerol stock solutions at -80 o C, and preserved as lyophilized cells for long-term preservation [8]. All thermophilic Actinobacteria isolates were deposited at Universitas Indonesia Culture Collection (UICC), Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia.

Bacterial genomic DNA isolation
Genomic DNA for PCR amplification of the 16S rRNA gene was prepared using the method as described by Yabe et al. [11]. The 16S rRNA gene of bacterial isolates was amplified using polymerase chain reaction (PCR) with universal eubacterial primers: 9F (5'-GAGTTTGATCCTGGCTCAG-3') and 1510R (5'-GGCTACCTTGTTACGA-3'). The PCR amplification for the 16S rRNA gene were carried out according to the conditions of MyTaq TM Red Mix (Bioline) which includes initial denaturation at 95 o C for 3 min, followed by 35 cycles including denaturation at 95 o C for 15 s, annealing at 56 o C for 15 s and extension at 72 o C for 1 min. The PCR products were sequenced using 1st BASE DNA sequencing service (http://base-asia.com/dnasequencing-services). The sequences of all type strains used in phylogenetic analyses were retrieved from the DDBJ/EMBL/GenBank databases.

Phylogenetic analyses
The partial sequence of the 16S rRNA gene from thermophilic Actinobacteria isolates was used for a sequence-similarity search against all related species in the database through EzTaxon-e (https://www.ezbiocloud.net/; Yoon et al. [12]), and for phylogenetic analyses. The sequences of 25 thermophilic Actinobacteria isolates were aligned with the sequences of type strains retrieved from the DDBJ/EMBL/GenBank databases and analyzed using MEGA v7.0.26 software package [13]. Phylogenetic trees were reconstructed based on nearly full-length 16S rRNA gene sequences of type strains of the closely related taxa to thermophilic Actinobacteria isolates using the neighbor-joining [14], minimum-evolution [15], and maximum-likelihood [16] methods in the MEGA v7.0.26 software package [13], with bootstrap values based on 1000 replications [17]. Kimura two-parameter method was used to compute the evolutionary distances of each phylogenetic tree [18].

Results and discussion
A total of 25 selected thermophilic Actinobacteria isolates were successfully obtained from soil in geothermal area of Cisolok. The number of isolates selected from soil samples around big geyser (location 1), small geyser (location 2), and forest near big geyser (location 3) are 4, 4, and 17 isolates, respectively. Isolation of the genomic DNA and amplification of the 16S rRNA gene from thermophilic Actinobacteria isolates were performed, then the PCR product samples were sequenced. The result of 16S rRNA gene sequence analysis is shown in table 1.
The sequences of 25 isolates showed similarities to member of families Streptomycetaceae (genus Streptomyces; 10 isolates), Streptosporangiaceae (genus Microbispora; 6 isolates), Thermomonosporaceae (genus Actinomadura; 3 isolates), Pseudonocardiaceae (genus Amycolatopsis; 3 isolates), Micromonosporaceae (genus Micromonospora; 2 isolates), and Nocardiaceae (genus Nocardia; 1 isolate). Similarity values of 19 out of 25 isolates showed 97 -99% to their closely related species (table 1). The result showed that soil of Cisolok geothermal area is potential source for isolating thermophilic Actinobacteria with high possibility of new taxa. According to Panda et. al. [3], based on phylogenetic tree analysis using the 16S rRNA gene sequences, the actinobacterial groups isolated from various hot springs were dominated by orders Streptomycetales (family Streptomycetaceae), Micromonosporales (family Micromonosporaceae), and Streptosporangiales (families Thermomonosporaceae and Streptosporangiaceae). Our results showed that four out of six Actinobacteria families are member of actinobacterial groups that are mostly found in various hot springs. Among 6 Actinobacteria families identified, 5 are regarded as rare Actinobacteria groups, except for family Streptomycetaceae. According to Tiwari and Gupta [6], rare Actinobacteria are usually defined as non-streptomycete strains of class Actinobacteria, with low frequency of isolation compare to the streptomycete strains using common methods. The isolation of rare Actinobacteria, thus becomes an important effort for the discovery of new secondary metabolites and development of clinically important antibiotics and enzymes [6].
Detailed phylogenetic analyses based on 16S rRNA gene sequences using neighbour-joining (NJ), minimum evolution (ME), and maximum-likelihood (ML) methods were performed for two groups of isolates belong to Streptomycetaceae   Figure 1. Neighbour-joining tree showing phylogenetic relationship between isolates belong to Streptomyces genus and the type species of the genus Streptomyces in the family Streptomycetaceae, based on 16S rRNA gene partial sequences. Streptomyces albus NBRC 13014 T was used as an outgroup (BBQG01000088). Bar 0.005 nucleotide substitutions per site.
Waksman and Henrici [19] first proposed the genus Streptomyces on 1943, with Streptomyces albus as the type species. Until recently, there are more than 800 validly published Streptomyces species have been reported (https://www.bacterio.net/streptomyces.html) [20]. The Streptomyces species are widely distributed and abundant in soil and well-known as producers of ~39% of all microbial bioactive compounds [5]. The genus Streptomyces mainly includes mesophilic species with optimum temperature ranging from 25 to 35 o C [21]. However, some thermotolerant (able to grow at 45 o C), and few thermophilic (growth temperatures between 28-55 o C) streptomycetes are presence [21]. In this study, all Streptomyces isolates were able to grow at temperature of 45 o C, which suggested the possibility of thermophilic species. The isolates also have 97.67 -99.55% sequence similarities to their most closely related species, which indicated high possibilities for finding new taxa in the family Streptomycetaceae.
Isolates SL3-1-R-13 and SL3-1-R-14, were located in the clade of the family Micromonosporaceae and most closely related to the type species Micromonospora globbae WPS1-2 T (99.92%) and M. costi CS1-12 T (99.34%), respectively. Meanwhile, phylogenetic analyses showed that both isolates were located in the same cluster only with Micromonospora globbae WPS1-2 T supported by NJ, ME, and ML trees with 99% bootstrap value. The genus Micromonospora is known as sources of new secondary metabolites and novel biosynthetic gene clusters (BGCs) after streptomycetes [24]. Thus potentially new taxa from genus Micromonospora isolated from unique habitat will increase the possibilities of findings new bioactive compounds. Isolate SL2-2-R-15 showed highest similarity (100%) to the member of family Nocardiaceae: Nocardia farcinica NCTC 11134 T , and formed monophyletic cluster with 76% bootstrap value supported by NJ, ME, and ML trees. Isolates SL3-1-R-1, SL3-1-R-3, and SL3-1-R-16 also showed 100% similarities and formed monophyletic clade with