Streptomyces iakyrus TA 36 as First-Reported Source of Quinone Antibiotic γ–Rubromycin

A wide range of bioactive compounds with potential medical applications are produced by members of the genus Streptomyces. A new actinomycete producer of the antibiotic γ-rubromycin, designated TA 36, was isolated from an alpine soil sample collected in Peru (Machu Picchu). Morphological, physiological and biochemical characteristics of the strain, together with data obtained via phylogenetic analysis and MALDI-TOF MS, were used for the correct identification of the isolate. The isolate TA 36 showed morphological characteristics that were consistent with its classification within the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences showed that the TA 36 strain was most similar to S. iakyrus and S. violaceochromogenes with 99% similarity. Phylogenetic analysis together with the profile of whole cell proteins indicated that the strain tested could be identified as S. iakyrus TA 36. The crude extract Ext.5333.TA 36 showed various effects against the tested organisms with strong antimicrobial activity in the growth of Staphylococcus aureus (Newman) (MIC value of 0.00195 µg/µL). HPLC fractionation and LC/MS analysis of the crude extract led to the identification of the quinone antibiotic γ-rubromycin, a promising antitumour and antibacterial antibiotic. To the best of our knowledge, there is currently no report on the production of γ-rubromycin by S. iakyrus. Therefore, this study suggests S. iakyrus TA 36 as the first-reported source of this unique bioactive secondary metabolite.


Introduction
Antibiotic resistance, as well as the need to find new antibiotics or new sources of existing antibiotics, is a very serious problem of this century [1,2]. In addition to antibiotic resistance, one of the leading causes of death in the world is cancer [3], and although progress has been made in the last two decades in the prevention of this disease and in the care of cancer patients, the number of cancer patients is still high [4,5]. The search for new anticancer drugs and antibiotics is therefore necessary, but the drug development process faces many obstacles (regulatory approval or high costs) [6]. For pharmaceutical companies, developing new products is therefore not a priority. Finding new sources of existing antibiotics or cancer drugs is therefore important [7].
Currently, there is a considerable amount of microbiological and pharmaceutical research being conducted for the control of human pathogens and the discovery of new species of microorganisms for the isolation of their antimicrobial agents [8,9]. Microorganisms, in particular those isolated from soil, are one of the most important natural sources of antimicrobial agents that are capable of inhibiting human pathogens [10,11].
Some of these microorganisms are also being explored for their production of antimicrobials with anticancer activities [12]. Among these, actinomycetes are a valuable group of prokaryotes that produce a number of secondary metabolites with a wide range of biological activities. These secondary metabolites include antibiotics, antitumour agents or immunosuppressants. For this reason, they are of economic and biotechnological importance [13,14]. Several thousand antibiotics that occur naturally in the environment have been isolated from actinomycetes [15], mainly from the genus Streptomyces [16]. Recently, fewer actinomycetes have been isolated from the general environment [17]. This is mainly due to the fact that unusual environments, such as forest and alpine soils, are still poorly understood and hold great promise for the discovery of bioactive compounds [18,19] A remarkable amount of research has been devoted to the study of the bioactive quinone compounds produced by streptomycetes. Among related strains, S. corcho-rusii has been reported to produce resistomycin, an HIV-1 protease inhibitor [19,20]. Himalomycins A and B, two new quinone antibiotics with potent antibacterial activity, were isolated from the Streptomyces isolate B6921 [21,22], and whole komodoquinone A, a neuritogenic anthracycline, was isolated from the fermentation broth of Streptomyces sp. KS3 [23,24]. Because of their interesting biological activities and complex molecular architectures, the naturally occurring quinone rubromycin family has attracted the attention of a number of research groups over the last sixty years [25]. More recently, the authors Boumehira et al. [7] discovered that Streptomyces sp. ADR1, isolated from soil collected in the Algerian Sahara, produces two antibiotics: βand γ-rubromycin. Rubromycins are characterised by a challenging molecular structure consisting of a naphthazarin moiety linked to an isocoumarin ring by a bis-benzannulated 5, 6-spiroketal system [26,27]. It has broad biological activities including antibacterial and anticarcinogenic activity [26,[28][29][30] and activity against human telomerase-16 [31] and HIV-1 reverse transcriptase [32]. Studies show that there is a direct link between cancer and human telomerase, and that the spiroketane moiety of rubromycin is important in inhibiting it [7,33]. The results of the above-mentioned studies clearly demonstrate the high potential of soil reservoirs as an important source for isolating actinomycetes that can produce high-value antibiotic compounds.
Therefore, this study aims to isolate and identify a Streptomyces strain as a new γ-rubromycin producer from alpine soil samples between two prominent peaks, Machu Picchu and Huayna Picchu, Peru. This strain, designated TA 36, was selected for its potential ability to produce antimicrobial molecules. Its antimicrobial and antifungal activities were determined in this study.

Morphological, Biochemical and Genetic Strain Characterisation
Actinobacteria are of great biotechnological importance. Therefore, they are constantly being studied. These efforts to find new sources of biologically active substances have revealed that they are present in a wide variety of ecosystem sources, ranging from terrestrial to aquatic [34]. Furthermore, actinomycetes are capable of survival in extreme environments [35]. As a result, a wide variety of soil samples are constantly under investigation and new species or sources of microorganisms with potential antibiotic activity are being sought [36]. In this study, the TA 36 strain, an actinomycete with high inhibitory potential, was isolated from mountain soil (Machu Picchu, Peruvian Andes) as part of our routine screening programme of actinomycetes. This strain, designated TA 36, was found to be Gram-positive, aerobic and non-motile. It had a colony morphology typical of the genus Streptomyces. The strain TA 36 was subjected to polyphasic and molecular taxonomic studies in the present investigation. The strain was found to grow slowly. The strain formed a branched aerial mycelium. The spores were produced in spore chains of the Rectus-flexibilis type. After 3-4 days of incubation, sporulation was observed on the agar media. Strain TA 36 developed well on ISP2, ISP3, ISP4 and ISP7 media, but produced sparse aerial hyphae on ISP5 and ISP6 media ( Figure 1). The TA 36 isolate grew over a temperature range of 25-37 °C, with the optimum temperature for growth recorded as 28-30 °C and no growth observed at 4 °C or above 37 °C. Growth occurred in the presence of 02.5% (w/v) NaCl (optimum range), but not at 5% and above NaCl. The pH range for growth was pH 5 to pH 7 (the optimal range was pH 6-7). Diffusible pigments were observed on the ISP6 medium. The substrate mycelium was olive to beige in colour. A fingerprint of enzymatic activities was obtained using API Coryne and API Zym test strips.
Within the strain tested, significant enzymatic potential was detected. The TA 36 isolate showed high (>40 nmol) alkaline phosphatase, acid phosphatase, leucine arylamidase, beta-galactosidase and N-acetyl-glucoseamidase activity. It also showed moderate to low (3010 nmol) esterase C4, esterase lipase C8, valinarylamidase, trypsin, naphthol-AS-BI phosphohydrolase and alpha-mannosidase activity. Conversely, non-existent enzymes were detected as lipase C14, cystine arylamidase, chymotrypsin, α-galactosidase, betaglucuronidase, α-and β-glucosidase and α-fucosidase. In the Api Coryne test, isolate TA 36 showed positive activity for α-glucosidase, gelatin hydrolysis, alkaline phosphatase and N-acetyl-β-glucosamidase, followed by moderate activity for nitrate reduction, esculin and urease production ( Table 1).  The TA 36 isolate grew over a temperature range of 25-37 • C, with the optimum temperature for growth recorded as 28-30 • C and no growth observed at 4 • C or above 37 • C. Growth occurred in the presence of 02.5% (w/v) NaCl (optimum range), but not at 5% and above NaCl. The pH range for growth was pH 5 to pH 7 (the optimal range was pH 6-7). Diffusible pigments were observed on the ISP6 medium. The substrate mycelium was olive to beige in colour. A fingerprint of enzymatic activities was obtained using API Coryne and API Zym test strips.
In this work, 16S rRNA was sequenced. It was compared with the 16S rRNA sequences of previously described streptomycetes. The results showed that the almost-complete (1467 bp) 16S rRNA gene sequence of strain TA 36 was similar to the other members of the Streptomyces genus, in particular S. violaceochromogenes (NBRC13100T) (99% similarity), and it was identical to the sequence of S. iakyrus (NBRC 13401T). A comparison of the micromorphology of the strains showed that S. violaceochromogenes had Retinaculum apertum sporophores. Streptomyces TA 36 and S. iakyrus had a Rectus flexibilis shape. An extensive literature search revealed that S. iakyrus is capable of producing a wide range of antibiotic and antitumour agents including actinomycin [37] and iakirine [38]. S. violaceochromogenes produces arugomycin [39] and cinerubin [40] with antibacterial and antitumour activities. The phylogenetic tree topology, inferred via the maximum likelihood method based on the 16S rRNA gene sequences of the TA 36 strain and the closest related species of the genus Streptomyces (Figure 2), and the MSP dendrogram, constructed using previously described Streptomyces database spectra (Figure 3), were in agreement. The morphological, physiological and biochemical characteristics of the TA 36 strain, together with the data obtained from the phylogenetic and MSP analyses, indicated that the tested isolate could be identified as S. iakyrus.

Evaluation of Antimicrobial Activity and Determination of Bioactive Compound
The crude extract of S. iakyrus (Ext.5333.TA 36) showed good antibiotic activity against the tested microorganisms. Gram-positive bacteria were more sensitive to the action of the extract compared with Gram-negative bacteria. The highest antimicrobial activity was shown by the extract against S. aureus (Newman), with an MIC value of 0.00195 µg/µL (Table 3).

Evaluation of Antimicrobial Activity and Determination of Bioactive Compound
The crude extract of S. iakyrus (Ext. 5333. TA 36) showed good antibiotic activity against the tested microorganisms. Gram-positive bacteria were more sensitive to the action of the extract compared with Gram-negative bacteria. The highest antimicrobial activity was shown by the extract against S. aureus (Newman), with an MIC value of 0.00195 µg/µL (Table 3). Relatively high antimicrobial activity was registered against B. subtilis and Micrococcus luteus with minimum inhibitory concentrations of 0.0039 and 0.00781 µg/µL, respectively. The strongest antimicrobial activity of actinomycetes extracts against B. subtilis and S. aureus was also observed by Balachandar et al. [41]. The moderate antimicrobial activity was observed against E. coli (TolC) with an MIC value of 0.03125 µg/µL and Chromobacterium violaceum with an MIC value of 0.0625 µg/µL. The low antimicrobial activity was demonstrated against all tested fungal strains. The MIC ranged from 0.25 to 0.125 µg/µL. Our findings are in agreement with Gacem et al. [42]. The significant antibacterial activity of isolate TA 36 makes it a suitable candidate for further investigation of antagonistic activity against human pathogens. However, it was not clear what the nature of the antibacterial activity was. Therefore, the extract was subjected to HPLC fractionation and LC/MS analysis to determine which compound was active.
The results of the antimicrobial activity after the fractionation of Ext. 5333 .TA 36 showed that the fractions had strong antimicrobial activity against S. aureus. The active fractions were found between retention times of 14.0 and 16.5 min based on the Peak Activity Correlation Test (Figure 4)  Relatively high antimicrobial activity was registered against B. subtilis and Micrococcus luteus with minimum inhibitory concentrations of 0.0039 and 0.00781 µg/µL, respectively. The strongest antimicrobial activity of actinomycetes extracts against B. subtilis and S. aureus was also observed by Balachandar et al. [41]. The moderate antimicrobial activity was observed against E. coli (TolC) with an MIC value of 0.03125 µg/µL and Chromobacterium violaceum with an MIC value of 0.0625 µg/µL. The low antimicrobial activity was demonstrated against all tested fungal strains. The MIC ranged from 0.25 to 0.125 µg/µL. Our findings are in agreement with Gacem et al. [42]. The significant antibacterial activity of isolate TA 36 makes it a suitable candidate for further investigation of antagonistic activity against human pathogens. However, it was not clear what the nature of the antibacterial activity was. Therefore, the extract was subjected to HPLC fractionation and LC/MS analysis to determine which compound was active.
The results of the antimicrobial activity after the fractionation of Ext.5333.TA 36 showed that the fractions had strong antimicrobial activity against S. aureus. The active fractions were found between retention times of 14.0 and 16.5 min based on the Peak Activity Correlation Test (Figure 4) of Ext.5333.TA 36 extract.  The active peak, which appeared on the HPLC chromatogram during the retention time, showed UV-VIS maxima at 314 nm and an ESI-HRMS spectrum with significant ion clusters for [M − H 2 O + H] + at m/z 523.0872. According to the available databases, this data set could be assigned to γ-rubromycin. Very similar data have been reported by Boumehira et al. [7], who studied the strain Streptomyces ADR1 as a potential source of βand γ-rubromycin, which are anticancer antibiotic compounds. Two compounds were identified at the UV wavelength of 490 nm: Compound 1 was β-rubromycin (HRESIMS m/z 537.1024) and Compound 2 was γ-rubromycin (HRESIMS m/z 523.0869). For example, authors Harunari et al. [43] studied the culture extract of marine-derived Streptomyces sp. They found that this strain is capable of producing hyaluromicin, a new member of the rubromycin family of antibiotics. In their study, the extract was fractionated via reversed-phase column chromatography, followed by HPLC purification, and the data obtained showed the pseudomolecular ion [M + H] + at m/z 604.1091. Their obtained value was higher than ours and they identified this compound as being hyaluromycin, which consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2enone (C 5 N) unit as an amide substituent of the carboxyl function. γ-Rubromycin belongs to a structurally related group of antibiotics-the rubromycins [38]. Rubromycins are potent antimicrobial agents. It has been reported that γ-rubromycin inhibits the growth of various bacteria and fungi at minimal concentrations (µM and nM) [26,44]. The genomes of the Streptomyces species contain a large number of clusters of genes encoding secondary metabolites. This has made them a rich source of compounds of industrial and clinical relevance [39,45]. The quinone antibiotic rubromycin, discovered by Brockmann and Renneberg [26], has the ability to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity [39]. To our knowledge, only the species S. natalensis, Saccharopolyspora erythraea [23] and S. collinus [26] have been reported as effective producers of rubromycins.

Sample Collection and Isolation of the TA 36 Strain
Strain TA 36 was isolated from a soil sample collected in Peru (13 • 9 29.4 S 72 • 32 47.1 W) (2438 m above sea level) 70 km from Cusco, Peruvian Andes, using dilution agar plating. The soil sample was collected from a depth of 4-5 cm, placed in clean polyethylene bags and dried at room temperature for 3 days. The slightly acidic pH (5.9) of the soil was determined potentiometrically. One gram of the soil sample was mixed with 10 mL of sterile distilled water and stirred for 10 min to prepare a serial dilution. Streptomycetes were isolated using the spread plate technique on starch and casein medium [46]. Petri dishes were incubated at 30 • C for 7 days. The selected strain was cultured on International Streptomyces Project (ISP2) medium [28] at pH 7.0 at 28 • C for 10 days. It was then preserved in glycerol (30% v/v) and stored at −20 • C.

Morphological Characterisation
The morphological characterisation consisted of a macroscopic characterisation and a microscopic characterisation. After 7-14 days of culture on ISP [28], the colours of the mature sporulating substrate and aerial mycelium of the TA 36 isolate were observed. Synthetic Suter's medium [29] with or without tyrosine was used to detect melanin pigment. For the observation of spore chain morphology via light microscopy (OLYMPUS CX22LED, Japan), a well-grown agar plate containing glucose yeast medium (GYM medium) [47] was used. The observed morphological characteristics of the isolate were compared with the 'Compendium of Actinobacteria' provided by Dr Joachim Wink, Braunschweig, Germany, for presumptive isolate identification (https://www.dsmz.de/collection/catalogue/ microorganisms/special-groups-of-organisms/compendium-of-actinobacteria, accessed on 11 June 2023).

Physiological and Biochemical Tests
Using 12-well plates (BRAND, Washington, DC, USA), the ability of the strain to utilise 10 compounds as sole carbon sources for energy and growth was assessed on ISP9 medium after 7 days at 28 • C. Each source was added to the medium at a final concentration of 1% (w/v). The use of individual carbon sources was investigated according to Shirling and Gotlieb [28]. The effect of salt on growth was determined on the ISP9 medium supplemented with graded doses of sodium chloride (NaCl) (2.5, 5.0, 7.5 and 10% w/v) using six-well plates (BRAND, Washington, DC, USA). The maximum concentration of NaCl in the medium that allowed growth was determined. Growth at different temperatures (4,10,15,25,30,37,40,45 • C) and pH (pH 2.0~10.0) was tested on the ISP2 plates. Commercial kits like ApiZym ® and ApiCoryne ® (bioMérieux, Craponne, France) were used to characterise the strain biochemically [48][49][50]. In order to carry out the Api ® tests, the culture was grown in a shaking flask with the GYM medium for a period of one week. The Api ® strips were incubated for 24 h at a temperature of 30 • C. The required reagents were added to each well after the incubation period. After 5 min, the strips were scored according to the manual criteria.

Genotypic Identification and Phylogenetic Analysis
The 16S rRNA gene of strain TA 36 was sequenced for taxonomic classification in addition to morphological characteristics. The Spin Plant Mini Kit (Invisorb, Berlin, Germany) was used to extract genomic DNA. Nucleo-Spin ® Gel and PCR Clean-up-Kit (Macherey-Nagel, Düren, Germany) were used for purification of PCR products. The analysis procedure is described in Charousová et al. [51,52]. Sequences of type strains for the most similar species were downloaded from the NCBI nucleotide collection database (https://www.ncbi.nlm.nih.gov/nuccore, accessed on 11 June 2023). The phylogenetic tree was constructed via the maximum likelihood method using PhyML [53]. A general time reversible model was used for maximum likelihood analysis with optimised nucleotide equilibrium frequencies, optimised for site variation, and the best of NNI and SPR tree search. The initial tree was generated via BioNJ [54]. The sequence obtained was deposited under accession number OR197579.

MALDI-TOF MS Analysis of Strain TA 36
Intact proteins were isolated and extracted following Hleba et al. [55] with subsequent MALDI-TOF MS analysis. Extractions were performed on four randomly selected Petri dishes inoculated with purified TA 36 strain. After the extraction, 1 µL of the supernatant was transferred to a MALDI-TOF stainless steel plate (Bruker Daltonics, Bremen, Germany) in triplicate, by means of pipping. The samples were allowed to dry at room temperature.

Bacterial and Fungal Test Organisms Used in the Study
Indicator microorganisms were grown overnight in the Mycosel broth [40]

Primary and Secondary Screening of Antimicrobial Activity
Prior to crude extract preparation, the TA 36 strain was cultured in 5333 medium (starch-15 g, yeast extract-4g, K 2 HPO 4 -1 g, MgSO 4 · 7 H 2 O-0.5 g, distilled water-1000 mL, pH-7). After five days of incubation, 20 mL of the culture was mixed with 20 mL of ethyl acetate (Sigma-Aldrich, USA). The mixture was shaken for 12 min. The sample was then centrifuged (9000 rpm, 10 min) and the supernatant containing the metabolite was transferred to a round bottom flask. A rotary evaporator (Heidolph instruments, Schwabach, Germany) was used to evaporate the ethyl acetate completely at 40 • C. After evaporation, the extract was prepared in accordance with Gacem et al. [42].
The primary screening was carried out according to the agar plug [56,57]. The broth microdilution method [44] in 96-well microplates (BRAND, Wertheim, Germany) was used for the secondary screening of the antimicrobial activity of the TA 36 strain. The dilution steps and the minimum inhibitory concentration (MIC) of the crude extract were observed by means of the inhibited wells (A-H). The more wells that were inhibited, the higher the activity of the tested extract was. For MIC evaluation, the crude extract was prepared to give final concentrations of 0.25 to 0.00195 µg/µL. The prepared 96-well plates were incubated at 37 • C for 24 h and the test fungi were incubated for 48 h. The values were obtained after 24 h via visual observation of the growth [52].

Fractionation of the TA 36 Crude Extract via HPLC and LC/MS Analysis
The selected extract showed high inhibitory potential against the Gram-positive bacterium S. aureus (Newman). Therefore, it was fractionated via HPLC (Agilent 1100 series with an X-Bridge C18 3.5 µm, 2.1 × 100 mm Column (Waters, Milford, CT, USA) and LC/MS analysis (Agilent 1200 series) with DAD detector (200-600 nm) in cooperation with a maXis UHR-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA)). These methods have been described in detail previously in Charousová et al. [52]. Fractions (0.15 mL) from the HPLC column were collected into 96-well plates every 0.5 min. The fractions were then dried with nitrogen at 40 • C in a MiniVap (Porvair Sciences, Wales, UK) for 45 to 60 min. Then, 150 µL of S. aureus (Newman) culture in sufficient growth medium was added to each well. Due to the high inhibitory activity of the extract (visibly inhibited wells), the extract was applied to the LC-MS system. Peak/activity correlations were performed. The results were processed using the data analysis included in Compass software 4.1 (Bruker, Madison, WI, USA). Comparisons of molecular weights, bioactivity, UV spectra and retention times were used to identify the active compound.

Conclusions
The results of this study present more precise identification of strain TA 36 via the analysis of protein spectra using MALDI-TOF MS. The active isolate was identified as S. iakyrus by combining MALDI-TOF MS analysis with 16S rRNA sequencing and by studying the classical physiological, morphological and biochemical properties of the two most closely related strains. The present study demonstrated the strongest antimicrobial activity of Ext. 5333 .TA 36 against S. aureus and good antibacterial activity against B. subtilis and M. luteus. Based on the peak data obtained via HPLC and LC-MS analysis, it was suggested that the active fraction against these pathogens was γ-rubromycin. This work provides evidence for a new producer of this antimicrobial compound, as there are no reports of γ-rubromycin production by S. iakyrus in the available literature. Further studies are now underway in our laboratories to purify the antibiotic γ-rubromycin itself, as well as to identify the other components present in the active Ext. 5333 .TA 36.