INTRODUCTION:

The discovery of antibiotics is one of the greatest stories of human success¹, but this must be hampered by the problem of antibiotic resistance²^,³^,⁴. So the solutions must be sought to overcome it. Streptomyces has become an important source of bioactive compounds with high commercial value and continues to be routinely screened to look for new bioactive substances⁵^,⁶. Streptomycetes have the capacity mainly to produce various kinds of bioactive compounds that have broad spectrum activities⁷. Streptomyces species until 2005 were reported to have produced more than 7600 compounds representing the largest group (45%) of bioactive microbial metabolites⁸.

Many types of bacteria that have been isolated from various sources such as in this study are bacteria originating from the peat soil².

The extent of peatland in Kalimantan Tengah can be a great source of finding bacteria such as Streptomyces which have produced numerous compounds to overcome antibiotics that have been resistant so far. Research on peatland habitat in Kalimantan Tengah, which is largely unexplored and being explored, is expected to find Streptomyces strains that produce active products as antibacterial candidates⁹. This antibacterial especially for ESBL-producing bacteria which is quite worrying because the increasing incidence of infections¹⁰^,¹¹.

Various types of bacteria isolated from various sources must be identified. Macroscopic, microscopic, physiological and biochemical characteristics identification are still inaccurate. Identification techniques using 16S rRNA gene sequences to study bacterial phylogeny and taxonomy have become the most commonly used genetic markers, the reason is, 1) almost all bacteria have this gene, always appearing in multigenic families, or operons, 2) the function of this gene is not change almost all the time, and 3) gene size (1500 bp) is informative enough to identify a bacterium¹¹^,¹².

RNA coding genes are used to determine taxonomy, phylogeny (evolutionary relationships) and estimate the range of species divergence of bacteria. Comparison of rDNA sequences can show evolutionary relationships between organisms. The use of the 16S rRNA sequence was pioneered by Carl Woese, who also found the classification of the 3 largest domains of living things, namely bacteria, archaea and eukaria. rRNA coding genes are genes that are able to maintain their sustainability for millions of years of evolutionary diversity. Most prokaryotes have 3 types of rRNA, namely 5S, 16S and 23S. The use of 5S rRNA has also been studied but this gene is too small to be used in phylogenetic determination. 16S and 23S rRNA genes have sufficient size to analyze. The 16S rRNA gene measuring around 1550 base pairs and about 500 bases at the end of the sequence is an area called the hypervariable region¹³. This area is a part that distinguishes between organisms. Primers used in amplification sequences will recognize a sustainable area and amplify the hypervariable region, thereby obtaining a sequence that is unique to the organism¹⁴.

The sequencing analysis step begins by isolating DNA from bacterial cultures, both solid and liquid cultures. The DNA obtained will be used as a mold in the amplification stage by PCR. The primer used in PCR is a universal 16S rRNA primer measuring around 1500 bp, so that it can amplify the 16S rRNA region of all bacteria. PCR products are purified first by using commercial kits to remove primary remnants and nucleotide fragments¹⁵^,¹⁶.

Purified PCR products are determined by their nucleotide sequencing method. In the sequencing stage certain size PCR products are used as molds. Primers in the PCR stage are also used in sequencing, except that each primer is used separately in one sequencing cycle (forward or reverse only). Unlike PCR, the products produced from sequencing have different sizes. This is because ddNTP (dideoxyribonuclease triphosphat) is added to sequencing or the dNTP terminator labeled with dyes. This termator in one cycle will bind randomly and stop the reading process. At each base terminator (ddATP, ddGTP, ddCTP, or ddTTP), there are fluorescent dyes that can absorb different wavelengths so that the terminator base will be readable with fluorometry¹⁷.

DNA sequences are formed from the alignment of reverse and forward primary readings and are generally referred to as consensus sequences. This consensus sequence is then compared to the sequence data available in the database using certain software. Some systems can determine nucleotide sequences through reading one primer, but reading with two primers gives more accurate results. Some databases that can be used to compare 16S rRNA sequences include GenBank (http://www.ncbi.nlm.nih.gov/), Ribosomal Database Project (RDP-II), (http: //rdp.cme.msu. edu / html /), the European Molecular Biology Laboratory's Ribosomal Database Project (http://www.ebi.ac.uk/embl/), Smart Gene IDNS (http://www.smartgene.ch) and Ribosomal Differentiation of Medical Microorganisms (RIDOM) (http://www.ridom.com/)¹⁸^,¹⁹. In clinical use it is very important to consider whether sequencing of all genes is needed (around 1500 bp). Sequencing the whole gene can be used to distinguish strains from a microorganism. In the discovery of new species, the sequencing of the entire 16S rRNA gene was needed. In most bacterial clinical isolates, a short fragment, which is 500 bp at the beginning of the 16S rRNA gene, was considered to be quite informative in identifying. Kattar et al stated that species from Bordetella sp can be determined from DNA sequences at the beginning of the 16S rRNA gene they have²⁰.

MATERIAL AND METHODS:

Peat soil sample was taken from peat land in Palangka Raya, Kalimantan Tengah with a pH of <6. Isolation of Streptomyces sp. from peat soil composites carried out according to Alexander and Strete². 10 grams of peat soil put in 90 mL phosphate buffer pH 7 and homogenize to obtain suspension. With a sterile 1 mL pippete, 1 mL of the suspension dilute by adding 9 mL of phosphate buffer solution pH 7 (10^-1) to the test tube and diluting to 10^-10. Then, 1 mL of each dilution put into sterile petri dishes using a micropipette and then added 10 mL of ISP-4 agar medium which has been thawed at 45°C. 1 Ose of the colony that grows in isolation media which has the character Streptomyces sp. (macroscopic identification) then planted on solid ISP-4 media on petri dishes and incubated at 28 °C for 4 days. Streptomyces sp. was transferred to the ISP-4 slant agar in order to ensure proper growth. Streptomyces sp. isolate stored at ISP-4 liquid containing 20 % glycerol at -80 °C (to maintenance bacterial morpholagic and physiology).

In this research, the identification of Streptomyces sp. isolated from peat land of Palangka Raya, Kalimantan Tengah employed the PCR technique using 16S rRNA primer, 27F and 1492R. The technical stages of the Streptomyces sp. identification are as follows:

a. DNA isolation technique for the Streptomyces sp. was conducted by employing Genomic DNA extraction using Quick-DNA^TM Fungal/Bacterial Miniprep Kit (Zymo Research)

b. PCR amplification using MyTaq HS Red Mix (Bioline, BIO-25047)

c. PCR products purification using Zymoclean^TM Gel DNA Recovery Kit (Zymo Research) Bi-drectional Sequencing

Amplification 16S rRNA gene employed using primer 27F - 5' AGAGTTTGATCMTGGCTCAG 3' and 1492R - 5' TACGGYTACCTTGTTACGACTT 3'. Then the PCR product analyisis using agarose gel (electrophoresis). PCR products purification with Zymoclean^TMGel DNA Recovery Kit (Zymo Research). Clone to pTA2 Vector with Toyobo TArget Clone (Toyobo). Transform to E. coli Zymo 5α with Mix and Go Competent Cells^TM (Zymo Research). PCR Colony with primer T3 and T7 promotor using KOD FX Neo (Toyobo). Plasmid isolation with ZR Plasmid MiniPrep (Zymo Research). Bi-directional Plasmid Sequencing.

Table 1: Colonial, cellular, physiologycal, and biochemical characteristic of Streptomyces sp. isolate 2.1

Characateristics	Isolate 2.1
Macroscopic
Colony	white chalk, white round chain colonies cluster and turn black as time goes on and smells like a soil
Microscopic
Gram reaction	+
Shape	rod
Motility	-
Biochemical investigations
Oxidase	-
Nitrate	+
Lysine	-
Ornithine	-
H₂S*	-
Glucose	-
Mannitol	-
Xylose	-
ONPG*	-
Indole	-
Urease	-
VP*	-
Characateristics	Isolate 2.1
Biochemical investigations
Citrate	-
TDA*	+
Gelatin	-
Malonate	-
Inositol	-
Sorbitol	-
Rhamnose	-
Sucrose	-
Lactose	-
Arabinose	-
Adonitol	-
Raffinose	-
Salicin	-
Arginine	-

* H₂S : production of hydrogen sulfide

ONPG : test for b-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-D-galactopyranoside (o-nitrophenyl-β-d-galactophyranoside, ONPG)

VP : the Voges-Proskauer test for the detection of acetoin (acetyl methylcarbinol) produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway

TDA: detection of the enzyme tryptophan deaminase (TDA)

RESULTS AND DISCUSSION:

The isolate (isolat 2.1) obtained from peat soil samples was composted to grow on ISP4 medium. The isolate was able to grow on medium with acidic pH. This acid resistance capability is in accordance with the peat environment conditions that tend to be acidic²^,²¹. Bacteria that are able to grow in acidic environmental conditions in addition to their physiological properties are also due to their thicker wall structure than bacteria that grow at normal pH. Further the Streptomyces sp. isolate was identified by microscopic, morphological and biochemical characters following the Bergey’s Manual of Systematic Bacteriology^22,23 (see Table 1).

This species was founded in Kalimantan Tengah peat soil that was growing well on ISP 4 medium with pH 5-6. This species has white chalk colonies, white round chain colonies cluster and turn black as time goes on and smell like typical soil, microscopically chain rod cells, Gram positive, and biochemically show oxidase (-), not motile, nitrate (+), fermentation test (-), and have tryptophan deaminase activity.

Peat soil generally has a relatively high level of acidity with a pH range of 3-4. Oligotropic peat, like many found in Kalimantan, has very low base cations such as Ca, Mg, K and Na, especially in thick peat. The thicker the peat, the lower bases it contains and the soil reaction becomes more acidic. Oligotropic peat which has quartz sand substratum in Palangka Raya, Kalimantan Tengah has a range of pH 3.25 - 3.75. The acidity level of peat soil is closely related to the content of organic acids (humic acid and fulvic acid). Decomposed organic matter has carboxyl and phenol reactive groups which are as weak acids. It is estimated that 85-95% of the acidity of peat soil is caused by both carboxyl and phenol groups. The acidity of peat soils tends to decrease along with the depth of the peat. The upper layers of shallow peat tend to have a higher pH than thick peat²⁴.

Streptomyces sp. has been isolated from Kalimantan Tengah peat soil. The isolate growth well in ISP-4 slant medium. ISP-4 (International Streptomyces Project-4) composition were starch (soluble), dipotassium phosphate, magnesium sulphate (heptahydrate), sodium chloride, ammonium sulphate, calcium carbonate, ferrous sulphate (heptahydrate), manganous chloride (7H2O), zinc sulphate (7H2O), agar, final pH (at 25°C) adjustment by HCl to 5-6²^,^25,26^,²⁷. Bacteria that are able to grow in acidic environmental conditions in addition to their physiological properties are also due to their thicker wall structure than bacteria that grow at normal pH. The activity of the enzyme tryptophan deaminase (TDA) is positive at 2.1 isolates, indicating the TDA's ability to process the amino acid tryptophan from the bacterial biosynthesis. The molecular mechanisms that regulate the biosynthesis of tryptophan in actinomycetes poorly understood, so did the role of tryptophan in the differentiation program life cycle of microorganisms still unexplored. To reveal the possible regulatory effects of these amino acids on gene expression, an integrated study was based on reverse quantitative transcription-PCR (qRT-PCR) and a proteomic approach carried out on the model of actinomycetes, Streptomyces coelicolor. Comparative analysis of the growth of microorganisms in a minimal medium with or without tryptophan supplementation showed that trp biosynthetic gene expression in S. coelicolor did not experience negative regulation in the presence of end products. In contrast, tryptophan specifically induces trp gene transcription in the biosynthetic gene group of calcium-dependent antibiotics (CDA), a lipopeptide containing tryptophan D-and L residues. In addition, tryptophan stimulates transcription of the CDA regulator CDA gene group and, coherently, produces CDA. Strangely, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure²⁸.

The results of molecular determination obtained are as follows:

Amplification :

Sample Name :

2.1. The molecular determination of this species was tested by employing the PCR technique using general 16S rRNA Primer, namely: 27F and 1492R. The nucleic acid (genomic DNA) quantification (Nanodrop) are follows: Conc. 252,1 ng/ μL, A_260/280 1,90, A_260/230 1,80, volume 30 μL. The amplification of 16S rRNA gene was succesfully conducted using the primers 27F and 1492R. 1 μL PCR product (±1400 bp) was analysis using agarose gel. 1 μL PCR products were run on 0,8% TBE agarose gel at 100 Volt for 30 min (see Figure 1).

Cloning :

PCR product was purificated and cloned. PCR Colony with primer T3 and T7 promotor using KOD FX Neo (Toyobo).

Cloning result was analysis using agarose gel. 3 μL PCR products were run on 0,8% TBE agarose gel at 100 Volt for 60 min (see Figure 1.B).

Colony 8 as representative to plasmid isolation and sequencing analysis. Plasmid isolation with ZR Plasmid MiniPrep (Zymo Research) and sequencing analysis with Bi-directional Plasmid Sequencing. Sample name: Sample 2.1 colony (592 col 8), insert lenght: 1605 bp, destination plasmid: pTA2 vector. 16S rRNA sequences was compared to the sequence data available in the database in GenBank (http://www.ncbi.nlm.nih.gov/).

The phylogenetic relationship of the Streptomycetes and related taxa, base on 16S rRNA analysis. The evolutionary history was inferred using the Neighbor-Joining method (see Figure 2).

PCR methods has amplificated the 16S rRNA of isolate 2.1. The result of PCR was purified and cloned to get the best result of sequence. The length of this spesies 16S rRNA gene sequence (1605 bp) was quiet informative in identifying the species²⁹. This species has a genome with GC content 54.6% and BM 492 584 Da. In conclusion, base on tabulation data from BLAST® results that isolates 2.1 belong to the genus Streptomyces sp. with the percentage of identity reaching 99.73% with Streptomyces sp. VEL 17 gene for 16 rRNA, partial sequences with accession code AB914463.2.

CONCLUSIONS :

Identification techniques using 16S rRNA gene sequences to study bacterial phylogeny and taxonomy have become the most commonly used genetic markers. As in this study shows that the identification of antibiotic-producing soil bacteria such as Streptomyces sp. give good result with 16 SrRNA and cloning technique. The result of PCR was purified and cloned to get the best result of sequence to get good sequencing results. . In conclusion, base on tabulation data from BLAST® results that isolates 2.1 belong to the genus Streptomyces sp. with the percentage of identity reaching 99.73% with Streptomyces sp. VEL 17 gene for 16 rRNA, partial sequences with accession code AB914463.2.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Pharmaceutical Chemistry Faculty of Pharmacy, Airlangga University for the facilities. This research in collaboration with the Center for The Study of Medicinal Plants and Traditional Food, Institute of Research and Community Service, Palangka Raya University (LPPM-UPR).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.10.2020 Modified on 30.01.2021

Research J. Pharm. and Tech 2021; 14(12):6639-6644.

DOI: 10.52711/0974-360X.2021.01147