Bioactive Metabolites from UV-Resistant Streptomyces alkaliterrae CH-8 in Desert Soil: In-Vitro Study on Their Potent Antioxidant and Antibiofilm Activities

Bioactive Metabolites from UV-Resistant Streptomyces alkaliterrae CH-8 in Desert Soil: In-Vitro Study on Their Potent Antioxidant and Antibiofilm Activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioactive Metabolites from UV-Resistant Streptomyces alkaliterrae CH-8 in Desert Soil: In-Vitro Study on Their Potent Antioxidant and Antibiofilm Activities Tayyaba Alam, Salah Ud Din, Afaq Ahmad, Mahwish Ali, Malik Badshah, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7264315/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract The exploration of extremotolerant microorganisms from arid ecosystems offers promising avenues for novel bioactive compound discovery. In this study, a radioresistant actinobacterial strain, CH-8, was isolated from the hyper-arid soil of the Cholistan Desert, Pakistan. Phylogenetic analysis of the 16S rRNA gene revealed 99.88% similarity with Streptomyces alkaliterrae . Strain CH-8 demonstrated high oxidative and UV-B tolerance, with 78% and 60% survival under 10 mM H₂O₂ and 2712 J/m² UV-B exposure, respectively. Intracellular metabolites were extracted using methanol and purified via silica gel chromatography. The purified fraction showed strong antioxidant activity (78% DPPH scavenging), and was rich in phenolics (149 mg GAE/g) and flavonoids (2.082 mg QE/g). Crude extracts exhibited significant anti-biofilm activity against Gram-positive pathogens, achieving up to 85.4% inhibition and 88.1% eradication. Cytotoxic potential was confirmed via brine shrimp lethality assay (IC₅₀ = 19.23 µg/mL), and DNA damage protection indicated radioprotective capability. LC-MS analysis identified several bioactive compounds, including a glycosylated polyketide (m/z 575.6) resembling dynemicin L, as well as lankacidin C and lynamicin D. These findings position S. alkaliterrae CH-8 as a promising source of therapeutic secondary metabolites, and highlight the Cholistan Desert as a reservoir of untapped microbial and biochemical diversity. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Microbiology Radioresistant Streptomyces alkaliterrae Antibiofilm Polyketide Bioactive metabolites Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Points 1. CH-8, a radioresistant actinobacterium, was isolated from the Cholistan Desert, Pakistan. 2. Its methanolic extract exhibited potent antioxidant, anti-biofilm, cytotoxic, and radioprotective activities. 3. LC-MS profiling revealed known and potentially novel glycosylated polyketides, macrolides, and indole alkaloids. 4. CH-8 demonstrates high biotechnological potential as a source of therapeutic secondary metabolites. Introduction Recent scientific advancements have significantly expanded our understanding of the boundaries of life, revealing that microbial life can thrive in environments once considered uninhabitable. Organisms have been discovered in extreme habitats such as deep-sea hydrothermal vents, hot springs, nuclear waste sites, soda lakes, and polar as well as arid deserts. These microorganisms, collectively termed “extremophiles,” exhibit remarkable adaptations that enable survival under harsh physicochemical conditions. Extremophiles are broadly categorized based on the nature of the extreme environment they inhabit, including thermophiles, psychrophiles, acidophiles, alkaliphiles, halophiles, barophiles, metallophiles, and radiophiles. The study of extremophiles not only deepens our understanding of the limits of life on Earth but also holds promise for the discovery of novel biomolecules with potential biotechnological and pharmaceutical applications 1 . One of the key extreme environmental stressors is electromagnetic radiation, particularly in the form of ultraviolet (UV) radiation 2 . It is believed that UV exposure had been a challenge to cell repair processes and overall survival on the Early Archaean Earth 3 . Several bacterial strains including Bacillus subtilis and Deinococcus radiodurans have been isolated, thriving under such extreme conditions [4, 5] . Ultraviolet radiation (UVR) has been identified as a harmful abiotic factor that significantly impacts microorganisms at various levels, causing damage to essential cellular biomolecules such as DNA, proteins, and lipids 6 . UVR can either directly or indirectly affect the cellular structures leading to formation of reaction oxygen species (ROS) [7, 8] . ROS interact with key cellular constituents, including DNA, proteins, and lipids. Given that these biomolecules are critical for maintaining cellular membrane structure, their oxidative damage can compromise membrane integrity, impair permeability, and ultimately lead to bacterial cell death 9 . Bacterial strains have evolved diverse physiological and biochemical mechanisms to mitigate the detrimental effects of UV radiation, including efficient DNA repair systems and defense strategies against UV-induced ROS 10 . Among these protective mechanisms, several key cellular systems play critical roles. The most prominent is the enzymatic antioxidant defense system, which includes catalase, superoxide dismutase (SOD), and peroxidases enzymes essential for maintaining metabolic homeostasis under oxidative stress. In addition to these enzymatic defenses, non-enzymatic antioxidants such as vitamins C, E, and B-complex, glutathione, and cysteine act as free radical scavengers, collectively mitigating the deleterious effects of UV-induced oxidative damage [11, 12] . Extremolytes are low molecular weight organic osmolytes that accumulate in extremophilic microorganisms, enabling their survival under harsh environmental conditions. These compounds confer protection by stabilizing vital cellular structures and facilitating the formation of hydration shells, thereby preserving macromolecular integrity 13 . Extremolytes constitute almost 25% of dry cell weight 14 . In the quest for radiation-resistant biomolecules, diverse extremolytes have been identified and isolated from radiotolerant organisms. Notable examples include scytonemin, mycosporine-like amino acids (MAAs), melanin, bacterioruberin, carotenoids (including lycopene and astaxanthin). The biosynthesis of these protective compounds exemplifies the remarkable biochemical adaptations evolved by extremophiles to withstand elevated radiation exposure [15, 16] . These extremolytes contribute significantly to the overall stress resistance of extremophiles, each playing distinct protective roles. Some of them function by forming a hydration shell around cellular components, thereby shielding critical structures from the damaging effects of extreme environmental conditions encountered in their native habitats 17 . Several extremolytes additionally function as potent antioxidants, directly scavenging reactive oxygen species to protect critical cellular components from UV radiation and oxidative damage 18 . Notably, many extremolytes involved in UV-radiation resistance exhibit multifunctional properties, conferring simultaneous protection against multiple environmental stressor 19 . One of the most important extremolytes that are efficiently involved in antioxidant activities are flavonoid metabolites. These metabolites either interact with reactive radicals and result in the formation of stable and inactive radicals or directly scavenge free radicals 20 . Extremolytes represent a largely untapped resource with significant biotechnological potential, often referred to as an 'unexplored gold mine'. These compounds offer promising applications across multiple industries, including cosmetics, medicine, and food production. Of particular interest are mycosporine-like amino acids (MAAs), which exhibit unique photoprotective properties. When exposed to UV radiation, MAAs demonstrate remarkable stability and protective capacity, making them valuable candidates for UV-protective sunscreens in cosmetic formulations. Furthermore, their potential as therapeutic agents for preventing UV-induced skin carcinogenesis has attracted considerable scientific attention 21 . The primary aim of this study was the isolation of a UV radiation (UVR)-resistant bacterial strain from the Cholistan Desert, Pakistan, followed by in vitro evaluation of its bioactive metabolites. The Cholistan Desert is a largely unexplored ecological niche with considerable potential to harbor extremotolerant and stress-resistant microorganisms. The region is characterized by a harsh subtropical climate, marked by minimal annual precipitation, low humidity, high evaporation rates, and prolonged exposure to intense solar radiation 22 . Historically, approximately 5000 years ago, the area received substantial monsoonal rainfall; however, climatic shifts over time diverted monsoon systems away, drastically reducing precipitation and transforming the region into an arid desert. A unique feature of the Cholistan Desert is the frequent occurrence of consecutive dry years, often spanning 4 to 6 years, resulting in significant thermal fluctuations. During summer months (May to June), average temperatures range from 35°C to 50°C, while in winter (December to February), they fall to 15°C to 20°C 23 . In this context, we report the isolation of a rare and highly UVR-resistant actinobacterial strain, designated CH-8, from the hyper-arid soils of the Cholistan Desert, has been scarcely reported in literature. To the best of our knowledge, this is the first report of S. alkaliterrae exhibiting such pronounced UV and oxidative stress resistance from a desert environment. The extreme habitat and physiological resilience of this strain suggest it may possess unique bioactive compounds with promising biotechnological potential, highlighting the Cholistan Desert as a valuable but underexplored source of novel microbial resources. Material and method Chemicals and Reagents The Luria Bertani (LB) medium used in this study for the growth of bacterial strain was purchased from Merk (KGaA Darmstadt, Germany). Double distilled water was used for broth and buffers preparations. Methanol, acetone, ethyl acetate, chloroform, diethyl ether, ethanol, dimethylsulfoxide (DMSO) and all other solvents were obtained from Sigma-Aldrich, USA. All the solvents are of analytical grade with 99% purity. Muller Hinton Agar medium used for the antibacterial activities and chemicals used for bioassays were purchased from Merk (KGaA Darmstadt, Germany). Isolation of UV resistant Bacteria Sand samples were collected aseptically from Cholistan desert, Pakistan by following standard protocols of sampling 24 and UV resistant bacterial strains were isolated using serial dilutions and cultured on sterile basic TGY medium containing (g/L): Trypton, 10; glucose, 1 and yeast extract, 5; using spread plate method. Test plates were irradiated with ultraviolet-B (UV-B) radiation using a UV chamber equipped with a 280 nm wavelength lamp (20 W output) positioned 30 cm above the samples. Exposure duration was standardized to 5 min, with the UV fluence rate (energy dose per unit area) J/m² as measured by a calibrated radiometer. Following UV treatment, plates were immediately transferred to red light conditions (λ > 600 nm) to prevent photo-reactivation of microbial isolates. UV-resistant colonies were subsequently selected and sub-cultured on fresh TGY agar plates for further study. Identification of UVResistant Bacterial Strain Morphological and biochemical identification of strain CH-8 was done by performing Gram staining, sugar utilization tests and various enzymes production tests 25 . Genomic DNA was extracted from strain CH-8 using the Thermo Scientific GeneJET Genomic DNA Purification Kit following manufacturer's protocols. The 16S rRNA gene was amplified and sequenced via Sanger sequencing (Macrogen Inc., Geumcheon-gu, Seoul, South Korea). The obtained sequence was analyzed using the NCBI BLAST algorithm for preliminary taxonomic identification and deposited in GenBank under accession number (PP757466). Phylogenetic analysis was performed using MEGA-X software. A neighbor-joining tree was constructed based on 16S rRNA gene sequences from closely related type strains, with bootstrap values calculated from 1000 replicates to assess nodal support 26 . Growth Conditions Optimization The optimal growth conditions for S. alkaliterrae CH-8 were determined by evaluating its growth under varying physicochemical parameters, Temperature (30–45°C) increments of 5°C; pH: 4.0–10.0 (adjusted using 0.1 M NaOH/HCl); Media, Nutrient broth (NB), Luria-Bertani broth (LB), and Tryptone-Glucose-Yeast extract (TGY) broth; Carbon sources, Glucose, xylose, mannitol, lactose, and dextrose (1% w/v each); NaCl tolerance, 1–8% (w/v). Growth was monitored spectrophotometrically by measuring optical density (OD600) at 24 h intervals using a UV-Vis spectrophotometer (Analytik Jena Specord 200 Plus). All experiments were performed in triplicate under aerobic conditions with shaking at 150 rpm. Growth curves were plotted as optical density (OD) 600 versus time, and optimal conditions were identified based on maximum biomass production. Bacterial Survival Curves under UV Radiation and Oxidative Stress The survival rate of the isolated strain S. alkaliterrae CH-8 under ultraviolet (UV) radiation and oxidative stress was assessed. The strain was cultured in TGY broth and incubated at 37°C in a shaking incubator at 130 rpm for 48 h. Following incubation, the culture was serially diluted using autoclaved normal saline until the optical density at 600 nm (OD₆₀₀) reached approximately 0.08–0.10. A 20 µL aliquot of the diluted culture was aseptically spread onto TGY agar plates using the spread plate technique. For UV radiation tolerance assessment, inoculated plates were exposed to varying durations of UV radiation (254 nm), ranging from 2 to 12 min, under a UV chamber 27 . To evaluate oxidative stress resistance, a bacterial cell suspension of S. alkaliterrae CH-8 was treated with varying concentrations of hydrogen peroxide (H₂O₂), ranging from 10 to 60 mM. After treatment, a 20 µL aliquot from each concentration was aseptically spotted onto TGY agar plates. The plates were incubated at 37°C for 48 h. Following incubation, colony formation was recorded, and the survival rate was determined by comparing the number of colony-forming units (CFUs) on H₂O₂-treated plates with those on untreated control plates. Extraction of Intracellular Bacterial Extract S. alkaliterrae CH-8 was cultured in 1 L TGY broth under optimal conditions (37°C, 130 rpm) until late exponential phase. Cells were harvested by centrifugation (8,000 rpm, 20 min, 4°C), washed twice with PBS buffer (pH 7.4), and subjected to a sequential extraction protocol. Pellets were resuspended in chilled methanol (1:5 w/v) for cold shock treatment (4°C, 1 h) followed by heat shock (60°C, 30 min) with periodic vortexing. Mechanical lysis was enhanced using glass beads (0.1 mm) via vortexing (15 min) and ultrasonication (20 kHz, 20 min with 30 sec pulse/1 min cooling intervals). The lysate was clarified by centrifugation (8,000 rpm, 15 min, 4°C), and the pigmented supernatant was transferred to glass vials. Samples were air-dried under sterile laminar flow (25 ± 2°C) and stored at − 80°C. All steps were performed in triplicate with appropriate controls. Antibacterial Activity of Intracellular Extract The antibacterial potential of the intracellular crude extract of S. alkaliterrae CH-8 was evaluated using the disk diffusion method on Mueller-Hinton agar (MHA) plates. The assay was performed against a panel of clinically relevant and ATCC reference strains, including Gram-positive bacteria ( Staphylococcus aureus , Staphylococcus epidermidis , and Bacillus subtilis ) and Gram-negative bacteria ( Pseudomonas aeruginosa and Escherichia coli ). Sterile filter paper disks (6 mm diameter) were impregnated with the crude extract and placed onto the surface of freshly inoculated MHA plates. The plates were incubated at 37°C for 24 h. Antibacterial activity was assessed by measuring the diameter of the zones of inhibition in millimeters (mm) around each disk, and the results were recorded accordingly 28 . Anti-biofilm potential of Intracellular Extract Inhibition of Biofilms Formation The biofilm inhibition assay was conducted with slight modifications to previously described protocols in order to evaluate the effect of the intracellular extract from S. alkaliterrae CH-8 on the initial attachment of bacterial cells 29 . About 1 × 10⁸ CFU/mL suspension of each test strain ( Staphylococcus aureus , Staphylococcus epidermidis , Bacillus subtilis , and Enterococcus faecalis ) was prepared by inoculating overnight-grown cultures into sterile nutrient broth. A total of 150 µL of the bacterial suspension was dispensed into each well of a sterile 96-well microtiter plate, in the presence of varying concentrations (20–100 µg/mL) of the intracellular extract. Wells containing bacterial suspension without extract served as negative controls. Following incubation at 37°C for 24 h, non-adherent planktonic cells were removed by washing the wells three times with sterile phosphate-buffered saline (PBS). Adherent cells were fixed by adding 99% methanol for 20 min. The wells were then stained with 1% (w/v) crystal violet for 20 min, followed by three additional PBS washes to remove excess dye. After air drying, the bound dye was solubilized using 33% glacial acetic acid, and optical density was measured at 595 nm using a microplate reader. The percentage of biofilm inhibition was calculated using the Eq. 1: \(\:\text{%}\:\text{o}\text{f}\:\text{a}\text{n}\text{t}\text{i}-\text{b}\text{i}\text{o}\text{f}\text{i}\text{l}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}=\frac{1\:-\:\text{A}\text{b}\text{s}\:\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\:\times\:\:100\) Eq. 1. Where Abs sample represents the absorbance of the test sample (containing bacterial suspension with extract), and Abs control denotes the absorbance of the bacterial suspension alone (without extract. Eradication of Performed Biofilms Biofilms were cultivated in 96-well microtiter plates by incubating bacterial suspensions at 37°C for 8 h under static conditions. Following initial biofilm formation, 100 µL aliquots of intracellular extract (concentration range: 20–100 µg/mL) were added to respective wells containing pre-formed biofilms of test strains. The plates were then incubated for an additional 18 h at 37°C to assess biofilm inhibition. Post-incubation, biofilm biomass was quantified using the standardized crystal violet staining protocol as detailed above. All experiments were performed with appropriate controls and in biological triplicates. Phytochemical Screening of total Phenolic and Total Flavonoids Contents of Intracellular Extract The total phenolic content of the intracellular extract from S. alkaliterrae CH-8 was determined using the Folin-Ciocalteu method, 30 with gallic acid as the calibration standard. The assay was performed according to the referenced protocol with minor modifications. To determine total phenolic contents 1.5 mL Fc reagent and 100 µL of intracellular extract was added to test tube. The reaction mixture was incubated for 15 min and then 1 mL of 20% Na₂CO 3 solution was added. Test tubes were then left in dark conditions and after 1 h of incubation, absorbance was measured at 710 nm against the blank which contained all the chemical ingredients except sample extract. Total phenolic content was calculated from the calibration curve and the outcomes were presented as milligrams of gallic acid equivalent per gram of dry weight. The total flavonoid content of S. alkaliterrae CH-8 crude bacterial extract was determined by using aluminum colorimetric chloride assay 31 with some modifications using quercetin as a standard. 200 µL of intracellular extract was diluted with 800 µL of methanol and mixed with 1 mL of 5% NaNO₂ in test tube. After 5 min, 1 mL AlCl 3 (10%) was added which was followed by addition of 2 mL of NaOH (1 M). Reaction mixture was then given an incubation time of 45 min in the dark at room temperature. After incubation time, absorbance of the reaction mixture was measured at 510 nm. Methanol was used as blank in the process. Total flavonoid content of the extracts was expressed as percentage of quercetin equivalent per 100 g dry weight of sample. Purification of Intercellular Bacterial Extract The intracellular crude extract of S. alkaliterrae CH-8 was fractionated using silica gel (Merck®, 100–200 mesh) column chromatography. A stepwise gradient elution was performed with solvents of increasing polarity: hexane, chloroform, diethyl ether, ethyl acetate, acetone, ethanol, and methanol. The mobile phase composition was systematically varied from non-polar to polar to facilitate the separation of metabolites based on their differential affinities. The eluent was collected in glass vials with the flow rate of 1 mL/min and by the fraction size of 10 mL each. The collected fractions were subjected to rotary evaporator (BÜCHI Rotavapor R-200, Switzerland) and dried powder was kept in vials. The dried chromatographic fractions were reconstituted in dimethyl sulfoxide (DMSO) for preliminary antioxidant evaluation. Screening was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Among the tested fractions, the methanol-eluted (100%) fraction exhibited the highest antioxidant activity and was selected for further analysis. For subsequent in vitro bioassays, the active fraction was serially diluted in DMSO. All solutions were freshly prepared prior to experimentation and protected from light to prevent degradation. In vitro Bioassays of Purified Cellular Extract Radical Scavenging Activity by DPPH Assay Antioxidant potential of active intracellular metabolic fraction obtained from S. alkaliterrae CH-8 was determined by DPPH assay 32 . Various concentrations of active purified fraction (20–100 µg/mL) were treated with DPPH (0.2 mM) solution in 96-well micro titer plate. The plate was incubated in dark at 37°C for 30 min. After incubation absorbance was measured at 517 nm using micro titer plate reader (Multiskan FC Microplate Photometer, USA). Ascorbic acid was used as a positive control in this assay. DPPH radical scavenging ability of the extracts was calculated by the Eq. 2. \(\:\text{%}\:\text{D}\text{P}\text{P}\text{H}\:\text{r}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{p}\text{o}\text{t}\text{e}\text{n}\text{t}\text{i}\text{a}\text{l}\:=\frac{\text{A}\text{b}\text{s}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:-\:\text{A}\text{b}\text{s}\:\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\:\times\:\:100\) Eq. 2. DNA Damage Prevention Assay DNA damage prevention potential of S. alkaliterrae CH-8 intracellular extract was assessed using DNA nicking assay as described by Lee et al. (2002), with some modifications 33 . The DNA protection activity of the purified intracellular extract was evaluated using the pUC19 plasmid DNA. Plasmid DNA was extracted from Escherichia coli competent cells using a standard plasmid isolation protocol. The reaction mixture (total volume: 25 µL) was prepared by combining 3 µL of pUC19 plasmid DNA, 10 µL of the purified extract, 4 µL of FeSO₄ (2 mM), 4 µL of H₂O₂ (30%), and 4 µL of sodium nitroprusside (1 M). The mixture was incubated at 37°C for 1 h to induce oxidative DNA damage. Following incubation, the reaction mixtures were loaded onto a 0.8% agarose gel prepared in 1× TAE buffer. Electrophoresis was performed at 90 V for 1 h using a horizontal gel electrophoresis system (Bio-Rad, USA). The gel was subsequently visualized under a UV transilluminator (Biometra TI 2, Analytik Jena, Germany). Plasmid DNA alone served as the positive control, indicating intact DNA. DNA treated with hydrogen peroxide and sodium nitroprusside without extract served as the negative control, representing oxidative DNA damage. Protective effects of the extract were assessed by comparing the integrity and migration pattern of DNA bands. Cytotoxic Assay Cytotoxicity of bacterial metabolic intracellular extract was determined using brine shrimp lethality test (BST) by following protocol described by Meyer et al. (1982), with slight modifications 34 . 10 Nauplii were collected using a micropipette and were transferred to glass tube containing 5 mL artificial seawater and different concentrations of prepared S. alkaliterrae CH-8 bacterial intracellular extract ranging from 20–100 µL. Cytotoxic effect of extracts was observed after 24 h by counting number of alive and dead nauplii. Nauplii were termed as alive if they exhibited any movement during 15 sec of observation. Mitomycin C was used as positive control and DMSO was taken as negative control. The experiment was run in triplicate and mean values were calculated. Anti-hemolysis Assay The anti-hemolytic activity of the S. alkaliterrae CH-8 intracellular extract was evaluated using the hemolysis inhibition assay, following the method described by Karim et al. (2020), with minor modifications 35 . A total of 10 mL of peripheral blood was collected from a healthy individual into an EDTA-coated tube and centrifuged at 3000 rpm for 10 min to separate erythrocytes. The resulting erythrocyte pellet was washed twice with 0.2 M PBS buffer (pH 7.4) and resuspended in 0.9% normal saline. To assess anti-hemolytic activity, 0.4 mL of the erythrocyte suspension was treated with varying concentrations (20–100 µg/mL) of the intracellular crude extract and purified fractions of S. alkaliterrae CH-8. The mixtures were incubated at 37°C for 10 min, after which 0.2 mL of hydrogen peroxide (H₂O₂) was added to induce oxidative stress. Samples were further incubated at 37°C for 3 h. Following incubation, all mixtures were centrifuged at 3000 rpm for 10 min, and the absorbance of the supernatant was measured at 540 nm using a UV–Vis spectrophotometer (Analytik Jena, Specord 200 Plus, USA) to determine the degree of hemolysis. Ascorbic acid was used as a positive control, while DMSO served as the negative control. A combination of PBS and H₂O₂ without extract was used as a blank control. Characterization of Cellular Extract FTIR Analysis The partially purified active fraction was analyzed using Fourier Transform Infrared (FT-IR) spectroscopy to determine the chemical moieties in the extract responsible for the biological activities using FTIR spectrometer (Perkin Elmer Spectrum 65, UK). The sample was analyzed in triplicates within the spectral range of 400 to 4000 cm − 1 using the 4 cm − 1 resolution FT-IR spectrometer. The IR transmittance of partially purified fraction was graphed by plotting intensity against wave number. LC-MS of Partially Purified Fraction The most active partially purified fraction of S. alkaliterrae CH-8 bacterial strain was subjected to mass spectrometry using an LC-MS system (Agilent 6310 Ion Trap LC-MS, USA). The partially purified dried fraction, dissolved in methanol (1 mg/ml) was allowed separated on C-18 column (100 × 3.0 mm, 2.7 µm) by maintaining gradient elution at the flow rate of 1 mL/min. water and acetonitrile was employed as mobile phase. Water (Line A) and acetonitrile (Line B) were used as the mobile phases. The column was pre-equilibrated with a solvent composition of 90% A and 10% B. Following sample injection, this ratio was maintained for 1 min, after which a linear gradient was applied, transitioning to 100% B (0% A) over the course of 1 h. Mass spectrometric analysis was performed using electrospray ionization (ESI) in negative ion mode, targeting [M − H] ⁻ m/z transitions. Statistical Analysis All quantitative data are presented as mean ± standard deviation (SD) of triplicate independent experiments. Statistical comparisons were performed using Student's paired t-test, with p-values < 0.05 considered statistically significant. Dose-dependent responses (cytotoxicity, UV-B radiation survivability, and oxidative stress resistance) were evaluated through linear regression analysis between treatment groups and their respective controls. For anti-biofilm and anti-hemolytic assays, both one-way ANOVA (for between-group comparisons) and two-way ANOVA (for assessing interactions among multiple variables) were employed, followed by appropriate post-hoc tests where significant differences were detected. Results Isolation and Identification of Strain CH-8 Initial screening of Cholistan desert soil samples yielded eight bacterial strains demonstrating resistance to 5 min UVB (280 nm) exposure. Through subsequent rigorous selection involving extended 10 min UVB exposure, only two isolates designated CH-1 and CH-8 exhibited sustained viability. Strain CH-8 was prioritized for further investigation due to its superior survival rate under prolonged UVB stress, as quantified through colony-forming unit (CFU) analysis. Strain CH-8 was identified as a non-motile, Gram-positive rod that exhibited blackish pigmentation when cultured on TGY agar plates. Biochemical characterization revealed positive results for citrate utilization, catalase activity, amylase, and protease production. Molecular identification was carried out by sequencing the 16S rRNA gene amplicon. Phylogenetic analysis was performed using the Neighbor-Joining method 36 in MEGA version 11, which revealed 99.88% sequence similarity with Streptomyces alkaliterrae . Pairwise sequence alignment using the EzTaxon database further confirmed the close phylogenetic relationship between strain CH-8 and other validly described Streptomyces species. The 16S rRNA gene sequence of strain CH-8 has been deposited in the NCBI GenBank database under the accession number PP757466. The phylogenetic tree illustrating its position is presented in Fig. 1 . Growth Conditions Optimization of S. alkaliterrae CH-8 strain The growth parameters of S. alkaliterrae CH-8 were systematically optimized. The strain exhibited growth within a narrow temperature range of 30–40°C, with optimal growth observed at 40°C. It also tolerated a pH range of 7.0–10.0, showing maximum growth at pH 8.0. When tested in various growth media, including nutrient broth, LB broth, and TGY broth, S. alkaliterrae CH-8 demonstrated the highest optical density and pigment production in LB broth. To evaluate carbon source utilization, the strain was cultured in media supplemented with glucose, mannitol, xylose, lactose, and dextrose. Among these, lactose supported the most favorable growth, indicating it as the preferred carbon source. Additionally, salt tolerance was assessed by exposing the strain to NaCl concentrations ranging from 1–8%. Growth decreased with increasing salt concentration, with the maximum tolerance observed at 4% NaCl. Detailed results are provided in Supplementary information Fig. S1 (A-E). Bacterial Survival against UVB radiations and H 2 O 2 Survival rate was observed in comparison with E. coli (ATCC 10536). In results it was found that S. alkaliterrae CH-8 showed survivability of 60% at 2712 J/m 2 under UVB radiations (Fig. 2 A). Similarly, in response to oxidative stress, induced by H 2 O 2 it was found that strain CH-8 had 78% survivability at 10 mM concentration of H 2 O 2 . In contrast, UV sensitive control strain ( E. coli ) showed less than 50% survivability at such concentrations (Fig. 2 B). Extraction of Intracellular Bacterial Extract The intracellular crude extract of S. alkaliterrae CH-8 was obtained through sonication-assisted cell lysis, followed by solvent extraction. Approximately 1 g of the crude extract was obtained and subsequently used for antibacterial activity assessment and photochemical screening. Antibacterial Activity of Cellular Extract Antibacterial activity of intracellular crude extract of S. alkaliterrae CH-8 was performed against Gram positive bacteria ( Staphylococcus aureus , Staphylococcus epidermidis and Bacillus subtilis ) and Gram-negative bacteria ( Pseudomonas aeruginosa and Escherichia coli ). Among all the strains, crude extract was more active against Staphylococcus aureus , Staphylococcus epidermidis and Bacillus subtilis and details of zones of inhibition in comparison with antibiotics gentamicin and vancomycin are given in Table 1 . However, no activity was observed against Gram-negative strains (Fig. 3 ). Table 1. Zones length of antibacterial activity of intracellular crude metabolic extract and positive control. Types of Organism Name of Pathogenic Strains Zone of Inhibition Diameter (cm) Crude Extract of CH-8 Strain Positive control (Antibiotic discs) Negative control (Solvent) Gram negative bacteria Escherichia coli - 3±0.02 cm - Pseudomonas aeruginosa - 3±0.01 cm - Gram positive bacteria Staphylococcus aureus 2.4±0.22 cm 3±0.07 cm - Staphylococcus epidermidis 2.7±0.4 cm 3±0.01 cm - Bacillus subtilis 1.2±0.15 cm 3±0.04 cm - Gentamicin was used as positive control against Gram negative bacteria; Vancomycin was used as positive control against Gram positive bacteria and DMSO was used as negative control. Values were represented as mean ± SD (n=3) Anti-biofilm potential of Intracellular Extract The anti-biofilm potential of the crude intracellular extract from S. alkaliterrae CH-8 was evaluated using the crystal violet assay against several biofilm-forming Gram-positive pathogens. The extract significantly inhibited the initial attachment phase of S. aureus , S. epidermidis , B. subtilis and E. faecalis in a concentration-dependent manner, with inhibition percentages ranging from 24.7–85.4%. Among the tested strains, E. faecalis displayed the least susceptibility to biofilm inhibition. The highest inhibition was observed at 100 µg/mL of extract, with B. subtilis and S. aureus showing inhibition rates of 85.4% and 76.3%, respectively (Fig. 4 ). In the biofilm eradication assay, the extract demonstrated the ability to disrupt established biofilms of the same pathogens. Again, E. faecalis showed the least response, whereas substantial eradication was observed against B. subtilis (88.1%) and S. epidermidis (87.5%) at the 100 µg/mL concentration, indicating the strong anti-biofilm efficacy of the CH-8 extract (Fig. 4 ). Total Phenolic and Total Flavonoids Contents of Cellular Extract The total phenolic content (TPC) and total flavonoid content (TFC) of the intracellular crude extract from S. alkaliterrae CH-8 were quantified using standard calibration curves of gallic acid and quercetin, respectively. TPC was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g DW), while TFC was reported as milligrams of quercetin equivalent per gram of dry weight (mg QE/g DW). The extract exhibited a TPC of 149 mg GAE/g DW and a TFC of 2.082 mg QE/g DW, indicating a substantial presence of bioactive phenolic and flavonoid compounds. Purification of Intracellular Bacterial Extract The intracellular crude extract from S. alkaliterrae CH-8 was extracted after cell lysis through ultra-sonication and purified by using silica gel column chromatography. Among various fractions collected with different solvents, the fraction dissolved in 100% methanol exhibited the highest ROS scavenging activity based on preliminary DPPH assay and was further subjected to in-vitro bioassays. In-vitro Bioassays of Intracellular Crude Extract and Purified Fraction Radical Scavenging Activity by DPPH Assay The antioxidant potential of the intracellular crude extract and purified fractions from S. alkaliterrae CH-8 was evaluated using the DPPH radical scavenging assay, as illustrated in Fig. 5 A. The crude extract demonstrated a DPPH radical scavenging activity of 73.62%, while the purified fraction exhibited an enhanced activity of 78%, indicating strong antioxidant capacity and suggesting enrichment of active constituents in the purified form. Cytotoxic Assay The cytotoxic potential of the intracellular crude extract and purified fractions of S. alkaliterrae CH-8 was assessed using the brine shrimp lethality assay and compared with the standard anticancer agent Mitomycin C. A concentration-dependent increase in toxicity was observed for both crude and purified extracts. At the highest tested concentration (100 µg/mL), survival rates were reduced to 20% and 10% for the crude extract and purified fraction, respectively. The half-maximal inhibitory concentration (IC₅₀) values, determined through linear regression analysis of the dose response curve (percentage survivability vs. concentration), were 28.3 µg/mL for the crude extract and 19.23 µg/mL for the purified fraction (Fig. 5 B), indicating notable cytotoxic efficacy. DNA Damage Prevention Assay The protective effect of the intracellular crude extract and purified fraction of S. alkaliterrae CH-8 against oxidative DNA damage was assessed using plasmid pUC18. In the assay, hydroxyl radicals (•OH) generated via the Fenton reaction induced plasmid DNA strand breaks, as evidenced by the degraded DNA pattern in the negative control lane. In contrast, the presence of both the crude extract and purified fraction effectively preserved the integrity of plasmid DNA, suggesting their potential to mitigate oxidative stress-induced genotoxicity. The positive control lane, containing untreated plasmid DNA, retained the native supercoiled form, validating the assay conditions (Supplementary information Fig. S2). Anti-hemolysis Assay The protective effect of crude extract and purified fractions of S. alkaliterrae CH-8 on erythrocytes was evaluated by exposing human red blood cells (RBCs) to oxidative agents. In results it was observed that crude extract had 43% inhibitory activity on oxidative stress-induced lysis while 60% inhibitory activity of purified fraction was observed at 100 µg concentration. Ascorbic acid was used as positive control and was giving inhibitory activity of 72.3% at 100 µg (Fig. 6 ). Characterization of Cellular Extract FTIR Analysis The FTIR spectrum of the purified methanolic fraction from S. alkaliterrae CH-8 revealed several characteristic peaks indicative of diverse functional groups associated with bioactive secondary metabolites. A broad absorption band in the analytical region corresponded to O–H stretching vibrations from methanol used as the solvent. Prominent peaks at 2997 cm⁻¹ and 2914 cm⁻¹ were attributed to aliphatic C–H stretching. A distinct band observed at 1666 cm⁻¹ suggested the presence of C = C stretching vibrations, indicative of aromatic or alkene moieties. In the fingerprint region (1500–500 cm⁻¹), sharp absorption peaks between 1450–1350 cm⁻¹ indicated O–H bending of carboxylic acids and methylene groups. A band at 1310 cm⁻¹ corresponded to C–N stretching of aromatic amines, while the peak at 1019 cm⁻¹ was attributed to C = O stretching vibrations, associated with ethers, polyphenols, and flavonoids. Additionally, peaks below 900 cm⁻¹ indicated C–H bending in substituted aromatic compounds. Collectively, these spectral features support the presence of complex bioactive moieties commonly found in microbial secondary metabolites (Supplementary information Fig. S3). LC-MS of Partially Purified Fraction The purified methanolic fraction of S . alkaliterrae CH-8 was analyzed using liquid chromatography–mass spectrometry (LC-MS) equipped with electrospray ionization in negative ion mode (ESI-MS) to identify potential bioactive metabolites. The scan range was set from m/z 100 to 1000, and chromatographic separation was performed over a 1 h gradient run. The resulting total ion chromatogram (TIC) displayed multiple peaks, corresponding to various molecular ions and their characteristic fragmentation patterns (Fig. 7 A), which were further interpreted through comprehensive literature comparisons and database cross-referencing. A prominent molecular ion peak was observed at m/z 575.6 [M − H] ⁻, eluting at a retention time of 4.4 min, corresponding to a neutral molecular mass of 576.6 Da. The presence of a significant fragment ion at m/z 411.4 [M − H] ⁻, indicating a mass loss of approximately 164 Da, suggested glycosidic bond cleavage, likely representing a hexose sugar unit (Fig. 7 B). This fragmentation pattern is characteristic of glycosylated polyketides. Based on structural analogy and spectral alignment, the compound was putatively identified as a glycosylated enediyne-type aromatic polyketide similar to dynemicin L, a class of metabolites known for their potent bioactivities (Fig. 7 C). Notably, such compounds are typically synthesized via type I polyketide synthase (PKS-I) gene clusters, which are prevalent in Streptomyces genomes. While the whole genome of S. alkaliterrae is not yet sequenced, close phylogenetic relatives harbor PKS-I gene clusters, supporting this biosynthetic potential. Another significant molecular ion peak at m/z 480.6 [M − H] ⁻ was detected at a retention time of 11.0 min. The corresponding fragment ion profile was consistent with indole alkaloids, aligning closely with lynamicin D, a bioactive compound known for its antibacterial and antitumor properties (Fig. 7 D). In addition, a peak at m/z 459.4 [M − H] ⁻ was observed at 44.3 min, tentatively identified as lankacidin C, a macrolide polyketide with known antibiotic properties (Fig. 7 E). At 52.6 minutes, a late-eluting compound with a molecular ion at m/z 543.5 [M − H] ⁻ showed a characteristic neutral loss of 35 Da, indicating the presence of chlorine. The resulting base peak at m/z 508.1 [M − H] ⁻ strongly supports the presence of a halogenated macrolide or polyketide (Fig. 7 F). Importantly, several detected compounds showed no exact matches in existing metabolomic databases, suggesting that S. alkaliterrae CH-8 may produce novel or structurally rare secondary metabolites given in the Table 2 , with potential pharmaceutical relevance. These findings highlight the strain’s untapped biosynthetic repertoire and underscore the significance of extremophilic actinomycetes as reservoirs of chemically diverse natural products. Table 2 Metabolic profiling of methanolic fraction of S. alkaliterrae CH-8 by LC-MS – negative ionization mode No. Retention time (min) Base peak [M-H]⁻ (m/z) Parent peak [M-H]⁻ (m/z) Fragment Ions [M-H]⁻ (m/z) Tentative Compound Class Closest Match / Analog 1 3.2 495.6 557.6 378.7, 435.8, 470.8, 557.6 Polyhydroxy polyketide core Urdamycin-type precursor 2 3.7 575.9 575.9 260.4, 327.8, 411.4, 462.3 Glycosylated polyketide Angucycline-like glycoside (No exact match in databases) 3 4.4 575.6 575.6 324.9, 411.4, 538.0 Glycosylated macrolide or enediyne core Dynemicin L 4 11.0 480.6 480.6 395.9, 453.7 Indole Alkaloid Lynamicin D 5 44.3 401.8 459.4 231.1, 314.1, 459.4 Polyketide macrolide Lankacidin C 6 47.2 241.5 467.2 210.2, 340.5, 392.0, 467.2 Polar aromatic fragment or phenolic acid Degradation product/shunt metabolite 7 52.6 508.3 543.5 543.5 Anguclinone (Aromatic Polyketide) Monacyclinone J 8 54.5 508.0 699.1 359.6, 392.0, 581.4, 617.4, 661.2, 699.1 Amphiphilic glycosylated polyketide Possibly a polyene–macrolide hybrid (No exact match in databases) Discussion This study investigates the biotechnological potential of Streptomyces alkaliterrae CH-8, a novel UV-B-resistant bacterium isolated from the extreme environment of Pakistan's Cholistan desert. Desert ecosystems present unique physiological challenges for microbial survival, characterized by oligotrophic conditions, extreme temperature fluctuations, minimal precipitation, and prolonged solar UV exposure. These selective pressures drive the evolution of specialized adaptations, making desert-derived microorganisms like strain CH-8 particularly valuable sources of stress-resistant bioactive metabolites with potential multifunctional applications [37, 38, 39, 40] . Microorganisms in the desert either protect themselves via antioxidant systems or bring forth sophisticated repairing mechanisms. Repairing system does not directly prevent the access of radiation to cellular structures and biomolecules but participate in repairing the damage that is done by incoming UVR 41 . Moreover, another similar study proposed three mechanisms including prevention, tolerance and repairing mechanisms which work collectively to cope up with UVR 42 . Several radio resistant bacterial strains have been reported previously from deserts e.g. Radiobacillus deserti TKL69 T from Taklimakan Desert 43 , Geodermatophilus tzadiensis from Saharan Desert 44 , Kocuria sp.TMM11 from Thal Desert 45 , Deinococcus deserti from Sahara Desert 46 , Streptomyces huasconensis D23 from Kumtagh Desert 47 and Promicromonospora panici PT9 T Tunisian Sahara desert 48 highlighting these extreme habitats as natural reservoirs. In term of taxonomy, strain CH-8 was identified as Gram positive actinobacterium belonging to Streptomyces genus, showing 99.88% similarity with Streptomyces alkaliterrae 49 . Genome of genus Streptomyces with high GC content, typically contains 25–70 biosynthetic gene clusters (BGCs) 50 encoding approximately 10,000 bioactive compounds, including antibiotics, antifungals, anticancer agents, and herbicides 51 . Numerous studies have previously reported isolation of Streptomyces strains from desert environments, such as the 49 strains reported from savanna soil in Sudan Desert 52 , confirming deserts as promising hosts for actinobacterial diversity. Despite its ecological significance, S. alkaliterrae remains largely unexplored, with no existing literature documenting its UV-B resistance mechanisms or metabolic potential. This study presents the first report of S. alkaliterrae CH-8 isolated from Pakistan's Cholistan desert, demonstrating remarkable UV-B tolerance (> 50% survival at 2712 J/m²) and producing bioactive intracellular metabolites. The strain's radiation resilience appears mediated by multiple structural adaptations: a thick peptidoglycan cell wall, sporulation capacity, and protective pigments that collectively shield against photodamage. These survival strategies are further enhanced by DNA-binding proteins and secondary metabolites that scavenge reactive oxygen species, providing comprehensive protection against radiation-induced cellular damage 53 . Initially, S. alkaliterrae CH-8 was exposed to extended dose of UVR and H 2 O 2 and was found resilient against these stresses, suggesting the existence of extensive preventive mechanisms. Moreover, strain CH-8 was found as catalase positive and hence its survivability against ROS and H 2 O 2 can also be attributed to enzymes catalase and peroxidase 54 . A study has reported that HspR target genes conserved in Streptomyces are involved in the control of the H 2 O 2 stress response 55 . Moreover, it has been reported in various studies that reactive oxygen species (ROS), induced due to exposure of bacterial strain to UVR have drastic impacts on the overall regulatory machinery of a bacterial cell and affects bacterial cell division, repairing mechanisms and cell recovery 56 . Researchers have been trying to seek for counter measures that organisms adopt to counterbalance ROS 57 . In this study, strain CH-8 has been observed to produce black colored, diffusible pigmented compounds. Colored compounds that have been extracted from various bacterial strain are reported to have the potential to scavenge superoxide anions [58, 59] . Another study revealed that colored metabolites uplift the chances of microbial survival by either providing them protection from external damaging factors or mediating the regulating machinery of the cell [60, 61, 62] . The intracellular crude extract and partially purified fractions of S. alkaliterrae CH-8 were evaluated for multiple bioactive properties, including cytotoxicity, antihemolytic activity, antioxidant capacity, and antibacterial effects. The crude extract demonstrated significant radical scavenging activity (73.62%), which increased to 78% in the purified fraction. For comparative analysis, we observed that under similar experimental conditions, Streptomyces sp. strain MUSC 14 exhibited only 24.71% DPPH scavenging activity at a higher concentration (4 mg/mL) 63 . The purified fraction exhibited concentration dependent cytotoxic effects, suggesting its potential as anticancer agents. These investigations align with the previous report of cytotoxic extract from Streptomyces . Thus, further evaluation of purified extracts is required to evaluate their anticancer activities in cell lines 64 . RBCs are very reactive to oxidative stress due to their high polyunsaturated fatty acids content and pro-oxidative nature of hemoglobin 65 . Hemolysis is therefore considered as a visible sign of damage that can be done to the RBCs by the free radicals 66 . In this study, crude extract prevented 43% hemolysis while purified fraction prevented 60% as compared to positive control ascorbic acid, which prevented 72.3% hemolysis. In parallel, some compounds have been reported to have the potential of scavenging free radicals and thus preventing hemolysis 67 . Phenolic compounds are well known for their distinctive redox qualities including their ability to scavenge free radicals and anti-inflammatory effects 68 . Total phenolic content of intracellular crude extract of strain CH-8 was found as 149 mg/g suggesting the involvement of phenolic compounds in staunch resistance against oxidative stress. A similar study has linked phenolic compounds in Streptomyces sp. to antioxidant and antimicrobial activity 69 . Flavonoids contents of crude extract were found as 2.082 mg/g. It was reported in a study that flavonoids bind with DNA and forms DNA duplex which protects DNA from oxidative damage 70 . The crude intracellular extract of S. alkaliterrae CH-8 demonstrated significant antibacterial activity against Gram-positive pathogens. These findings align with recent reports of antimicrobial compounds from related Streptomyces species, including bacteriocins from Streptomyces pluripotent that exhibit potent activity against methicillin-resistant Staphylococcus aureus (MRSA) strains 71 . Another study has reported the ethyl acetate extract of Streptomyces sp. strain ACTN 2, isolated from mangroves sediments exhibited antimicrobial potential against B. subtilis and P. aeruginosa 72 . Biofilm forming bacterial pathogens pose a significant challenge in a clinical scenario. It is becoming more difficult to impede the development and growth of mature biofilms in contrast to initially formed biofilms. The resistant of sessile cells in mature biofilms to antimicrobial agents is due to their production of exo-polysaccharide matrix and mutation in bacteria dwelling in biofilms 73 . It was found that crude extract of strain CH-8 exhibited above 80% eradication of mature biofilm of Gram-positive pathogens. The observed antibacterial efficacy aligns with documented bioactivity profiles of marine Streptomyces spp. extracts against multidrug-resistant clinical isolates, as reported in recent literature 74 . The LC-MS analysis of the S. alkaliterrae CH-8 extract revealed a diverse array of bioactive secondary metabolites, several of which showed close spectral resemblance to known compounds from Streptomyces species with established therapeutic relevance. Notably, one of the early eluting peaks was putatively identified as a precursor of the urdamycin-type compounds angucycline-class antibiotics. These metabolites have previously been reported from Streptomyces fradiae and S. cellulosae and are recognized for their potent antibacterial and antifungal activities. The presence of such a metabolite in CH-8 underscores its biosynthetic potential and supports the hypothesis that extremophilic actinomycetes are prolific producers of clinically valuable natural products [75, 76] . While the compound corresponded to Lynamicin D, an indole alkaloid derived from Streptomyces sp. SCSIO 03032 77 have been reported to exhibit a spectrum of bioactivities, including antimicrobial and antioxidant effects, although specific studies on its antibiofilm activity remain limited. A compound exhibiting a parent ion peak at m/z 459.4 and a corresponding base peak at m/z 401.8 was tentatively identified as Lankacidin C, a polyketide macrolide known for its potent antibacterial properties. The biosynthesis of this compound has previously been attributed to Streptomyces rochei , further highlighting the antimicrobial potential of S. alkaliterrae CH-8. 78 . Various studies also suggested its antitumor and antibacterial activities, particularly against Gram-positive bacteria 79 . The detection of late-eluting metabolites revealed a distinctive chemical profile enriched with polyketide macrolides and amphiphilic glycosylated polyketides. Notably, several peaks lacked exact matches in existing spectral databases, suggesting the presence of previously uncharacterized or novel bioactive metabolites. Such findings underscore the metabolic versatility of S. alkaliterrae CH-8 and its untapped biosynthetic potential. Comparative analyses further support this, as numerous Streptomyces species, including S. africanus , S. pactum , and Streptomyces sp. MUM212, have been well documented for their potent antimicrobial and antioxidant activities, reinforcing the therapeutic relevance of secondary metabolites from this genus 80 , 81 , 82 . These activities are attributed to the presence of high phenolic and flavonoid content within their extracts. Conclusion This study presents the first report on the isolation and characterization of the novel radioresistant bacterial strain Streptomyces alkaliterrae CH-8 from the arid soil of the Cholistan Desert, Pakistan. The strain demonstrated remarkable tolerance to UV-B radiation and produced a crude and partially purified methanolic extract rich in antimicrobial, antioxidant, and anti-biofilm compounds. Metabolite profiling revealed a diverse spectrum of bioactive secondary metabolites, including known and potentially novel glycosylated macrolides, polyketides, and indole alkaloids, underscoring their potential applications as radioprotective agents and therapeutics against multidrug-resistant pathogens. These findings highlight the need for further structural elucidation of these compounds through advanced spectroscopic techniques such as NMR, alongside whole-genome sequencing to identify the biosynthetic gene clusters responsible for their production. Collectively, this work not only establishes the untapped microbial potential of the Cholistan Desert but also emphasizes its promise as a reservoir of novel bioactive metabolites with implications for human health and environmental sustainability. Declarations Compliance with ethical standards Funding This research work was accomplished with funds provided by Quaid-i-Azam University, Islamabad 45320, Pakistan. Conflict of Interest All the authors declare no conflict of interest/competing interest. Ethics approval Not applicable Authors Contributions TA; SUD; AA; AAS: Preparation of the overall research plan as well as protocols for various experiments; TA; SUD; AA: Performed experimental work in lab as per the pre-designed research plan; FH; SK; MB; MA; AAS: Facilitated in interpretation of various analysis and optimization experiments in the current research project; MF: Facilitated the processing and analysis of sample through LC-MS; TA; SUD, AA: Write up of the manuscript; SUD, FH, SK, MB, AAS: Proof reading of the overall manuscript for English comprehension and typing mistakes. Data availability statement All data supporting the findings of this study are available within the paper and its supplementary information. References Cowan, D., Ramond, J.-B., Makhalanyane, T. & De Maayer, P. Metagenomics of extreme environments. Curr. Opin. Microbiol. 25 , 97–102 (2015). 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T. β-Carotene and protein oxidation: effects of ascorbic acid and α-tocopherol. Toxicology 146 , 37–47 (2000). Fiedor, J. & Burda, K. Potential Role of Carotenoids as Antioxidants in Human Health and Disease. Nutrients 6 , 466–488 (2014). Madhusudhan, D. N., Mazhari, B. B. Z., Dastager, S. G. & Agsar, D. Production and Cytotoxicity of Extracellular Insoluble and Droplets of Soluble Melanin by Streptomyces lusitanus DMZ-3. BioMed Res. Int. 2014 , 306895 (2014). Guo, J. et al. High-level production of melanin by a novel isolate of Streptomyces kathirae. FEMS Microbiol. Lett. 357 , 85–91 (2014). Kemung, H. M. et al. Antioxidant Activities of Streptomyces sp. strain MUSC 14 from Mangrove Forest Soil in Malaysia. BioMed Res. Int. 2020 , 6402607 (2020). Tangjitjaroenkun, J., Pluempanupat, W., Tangchitcharoenkhul, R., Yahayo, W. & Supabphol, R. Antibacterial, antioxidant, cytotoxic effects and GC-MS analysis of mangrove-derived Streptomyces achromogenes TCH4 extract. Arch. Biol. 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JBIR-94 and JBIR-125, Antioxidative Phenolic Compounds from Streptomyces sp. R56-07. J. Nat. Prod. 75 , 107–110 (2012). Tiwari, P. & Mishra. Role of Flavonoids in DNA Damage and Carcinogenesis Prevention. J. Carcinog. Mutagen. 08 , (2017). Lee, L.-H. et al. Streptomyces pluripotens sp. nov., a bacteriocin-producing streptomycete that inhibits meticillin-resistant Staphylococcus aureus. Int. J. Syst. Evol. Microbiol. 64 , 3297–3306 (2014). In vitro Antimicrobial Activity of Extracts From Marine Streptomyces Isolated From Mangrove Sediments of Tanzania. J. Biochem. Technol. 3 , 431–435 (2012). Zhang, K., Li, X., Yu, C. & Wang, Y. Promising Therapeutic Strategies Against Microbial Biofilm Challenges. Front. Cell. Infect. Microbiol. 10 , (2020). Dhandapani, R. et al. Potential Bioactive Compounds from Marine Streptomyces sp. and Their In Vitro Antibiofilm and Antibacterial Activities Against Antimicrobial-Resistant Clinical Pathogens. Appl. Biochem. Biotechnol. 194 , 4702–4723 (2022). Fedoryshyn, M. et al. Surprising production of a new urdamycin derivative by S. fradiae Δ urdQ / R . J. Biotechnol. 130 , 32–38 (2007). Xu, X. D. et al. Isolation, Structure Elucidation and Antifungal Activity of Angucycline Antibiotics from Streptomycete cellulosae. Appl. Biochem. Microbiol. 59 , 456–461 (2023). Saurav, K. et al. In silico molecular docking, preclinical evaluation of spiroindimicins A-D, lynamicin A and D isolated from deep marine sea derived Streptomyces sp. SCSIO 03032. Interdiscip. Sci. Comput. Life Sci. 6 , 187–196 (2014). Suzuki, T., Mochizuki ,Susumu, Yamamoto ,Shouji, Arakawa ,Kenji & and KINASHI, H. Regulation of Lankamycin Biosynthesis in Streptomyces rochei by Two SARP Genes, srrY and srrZ. Biosci. Biotechnol. Biochem. 74 , 819–827 (2010). Ayoub, A. T. et al. Antitumor Activity of Lankacidin Group Antibiotics Is Due to Microtubule Stabilization via a Paclitaxel-like Mechanism. J. Med. Chem. 59 , 9532–9540 (2016). Tan, L. T.-H. et al. Streptomyces sp. MUM212 as a Source of Antioxidants with Radical Scavenging and Metal Chelating Properties. Front. Pharmacol. 8 , (2017). Rammali, S. et al. In vitro antimicrobial and antioxidant activities of bioactive compounds extracted from Streptomyces africanus strain E2 isolated from Moroccan soil. Sci. Rep. 14 , 27372 (2024). Mirsonbol, S. Z., Issazadeh, K., Zarrabi, S. & Mirpour, M. Evaluation of antimicrobial activity of Streptomyces pactum isolated from paddy soils and identification of bioactive volatile compounds by GC-MS analysis. World J. Microbiol. Biotechnol. 39 , 63 (2022). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Nov, 2025 Reviews received at journal 20 Sep, 2025 Reviews received at journal 24 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviewers agreed at journal 09 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 06 Aug, 2025 Editor invited by journal 06 Aug, 2025 Editor assigned by journal 04 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7264315","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497957013,"identity":"1f687152-4a72-4fd4-b902-f696690210f2","order_by":0,"name":"Tayyaba Alam","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Tayyaba","middleName":"","lastName":"Alam","suffix":""},{"id":497957014,"identity":"5aebc7eb-c6e9-4d08-8d0a-717cabf531d0","order_by":1,"name":"Salah Ud Din","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Salah","middleName":"Ud","lastName":"Din","suffix":""},{"id":497957015,"identity":"a99ac215-cbf0-4c9b-b1a1-fdff5f018bff","order_by":2,"name":"Afaq Ahmad","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Afaq","middleName":"","lastName":"Ahmad","suffix":""},{"id":497957016,"identity":"6dfde062-5f53-4708-8259-30317f31007b","order_by":3,"name":"Mahwish Ali","email":"","orcid":"","institution":"National University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahwish","middleName":"","lastName":"Ali","suffix":""},{"id":497957017,"identity":"e8768e13-5e94-4157-b9e9-4976236e7115","order_by":4,"name":"Malik Badshah","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Malik","middleName":"","lastName":"Badshah","suffix":""},{"id":497957018,"identity":"90770547-f5d7-45dc-b429-118e959d4f17","order_by":5,"name":"Samiullah Khan","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Samiullah","middleName":"","lastName":"Khan","suffix":""},{"id":497957019,"identity":"938ea936-8ea2-4f58-b2cc-322df4deceab","order_by":6,"name":"Fariha Hasan","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Fariha","middleName":"","lastName":"Hasan","suffix":""},{"id":497957020,"identity":"07b3e977-3120-4cdb-8f02-21acc2d0ceb7","order_by":7,"name":"Muhammad Farman","email":"","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Farman","suffix":""},{"id":497957021,"identity":"3a6cf989-e69f-4bf3-85a8-f786eadfb342","order_by":8,"name":"Aamer Ali Shah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACdsYGZgaGA3IQHhsQ8xDSwgzRYkyKFjA6kNhAtBb+ZubmzwU1d9L7jp8xYPhQdpjBnOcAfi0ShxnbpGcce5Y780yOAeOMc4cZLHsbCFgD1MLMw3Y4d8OBHANm3rbDDAbnCeiQP8zY/Jnn3+F0g/NvDJj/EqPF4DBjgzTQ8ASDG0BbGEFazhJwmCHIL7x9hw1n3nhWcLDnXDqPZc8B/Frkjrc//szz7bA83/nkjQ9+lFnLmfMkEHAZHBwAIwYeA2I1QNSDAAlaRsEoGAWjYIQAAGmlR7mgF4yeAAAAAElFTkSuQmCC","orcid":"","institution":"Quaid-i-Azam University","correspondingAuthor":true,"prefix":"","firstName":"Aamer","middleName":"Ali","lastName":"Shah","suffix":""}],"badges":[],"createdAt":"2025-07-31 16:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7264315/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7264315/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88812907,"identity":"b78e0907-cb12-48e3-892c-66e49804a27d","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282124,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining tree showing phylogenetic analysis of strain CH-8 and related texa based on 16S rRNA gene sequencing. Numbers on the branches indicate bootstrap percentage based on 1000 replicates. Bar, 0.01 indicates nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/d0b49ac27110d125ee815139.png"},{"id":88812908,"identity":"28671d3a-6b0b-4973-add7-7d3ada0b9da6","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111877,"visible":true,"origin":"","legend":"\u003cp\u003eStress tolerance profile of \u003cem\u003eS. alkaliterrae\u003c/em\u003eCH-8 compared to \u003cem\u003eE. coli\u003c/em\u003e (10536). \u003cstrong\u003e(A)\u003c/strong\u003e Percentage survivability under UV-B radiation exposure; \u003cstrong\u003e(B)\u003c/strong\u003e Percentage survivability under oxidative stress induced by H₂O₂.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/83be05ac681ef03f60154fea.png"},{"id":88812911,"identity":"43216188-4751-475a-82be-a64a94e8e53d","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":569303,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of crude metabolic extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 against Gram negative bacteria including: \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa,\u003c/em\u003e and Gram-positive bacteria comprising \u003cem\u003eStaphylococcus aureus,\u003c/em\u003e \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e. Gentamycin was used as positive control and DMSO was used as negative control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/951a1debdd1ad5b50065eaec.png"},{"id":88812909,"identity":"c4fc988e-6e57-44b4-9cfc-a23f0c86286c","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161219,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of crude metabolic extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003eCH-8 on inhibition of biofilm attachment and eradication of mature biofilm against Gram positive pathogens. Cefadroxil (5 µg) was used as positive control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/e03b5a767195434b993ca48e.png"},{"id":88812914,"identity":"bc79f9aa-d735-451f-9bd9-3eed27d4e2a5","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153310,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration-dependent DPPH radical scavenging activity and cytotoxicity analysis of crude and purified extracts from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8.\u003cstrong\u003e (A)\u003c/strong\u003e DPPH scavenging activity; \u003cstrong\u003e(B)\u003c/strong\u003eDose-response curve of brine shrimp lethality bioassay showing percent survivability against increasing extract concentrations.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/f71b523c7f7ebc49e283826c.png"},{"id":88812913,"identity":"72c34022-5294-4e71-ab36-2eeea5c2a466","added_by":"auto","created_at":"2025-08-11 15:52:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":133310,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-hemolytic assay of crude and purified extract from \u003cem\u003eS. alkaliterrae \u003c/em\u003eCH-8.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/c7e16f28d2e66b3e22eebc6a.png"},{"id":88814187,"identity":"01c3c6c4-7299-4b5f-8d77-b456c8164cd3","added_by":"auto","created_at":"2025-08-11 16:08:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2866696,"visible":true,"origin":"","legend":"\u003cp\u003eLC–MS profile of purified fraction of intracellular methanolic extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8. \u003cstrong\u003e(A)\u003c/strong\u003eTotal ion chromatogram (TIC) of intracellular methanolic extract; \u003cstrong\u003e(B)\u003c/strong\u003eMass spectra confirmed the identity of Polyhydroxy polyketide core; \u003cstrong\u003e(C)\u003c/strong\u003eMass spectra identifying Dynemicin L at m/z of 575.9 [M-H] - at base peak; \u003cstrong\u003e(D)\u003c/strong\u003eMass spectra identifying Lynamicin D at the m/z of 480.9 [M-H] - at base peak. \u003cstrong\u003e(E)\u003c/strong\u003eMass spectra identifying Lankacidin C at the m/z of 459.5 [M-H] - at molecular ion peak having fragment ion at the m/z of 401.8 [M-H] – at base peak; \u003cstrong\u003e(F)\u003c/strong\u003eMass spectra identifying Monacyclinone J at m/z of 508.6 [M-H] - at base peak.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/9248634d6d8f4d3cb9171e61.png"},{"id":88816305,"identity":"bdc94c6e-17a1-460a-a92d-204102bd3415","added_by":"auto","created_at":"2025-08-11 16:24:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5824873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/ed0bb348-3483-4d81-9c3b-3e1cd32a6e44.pdf"},{"id":88812941,"identity":"3c279038-ea77-4023-8ee2-5b5cdd189aa1","added_by":"auto","created_at":"2025-08-11 15:52:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2383644,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7264315/v1/17008686a6bb9162ac9fc8f7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioactive Metabolites from UV-Resistant Streptomyces alkaliterrae CH-8 in Desert Soil: In-Vitro Study on Their Potent Antioxidant and Antibiofilm Activities","fulltext":[{"header":"Key Points","content":"\u003cp\u003e1. CH-8, a radioresistant actinobacterium, was isolated from the Cholistan Desert, Pakistan.\u003c/p\u003e\u003cp\u003e2. Its methanolic extract exhibited potent antioxidant, anti-biofilm, cytotoxic, and radioprotective activities.\u003c/p\u003e\u003cp\u003e3. LC-MS profiling revealed known and potentially novel glycosylated polyketides, macrolides, and indole alkaloids.\u003c/p\u003e\u003cp\u003e4. CH-8 demonstrates high biotechnological potential as a source of therapeutic secondary metabolites.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRecent scientific advancements have significantly expanded our understanding of the boundaries of life, revealing that microbial life can thrive in environments once considered uninhabitable. Organisms have been discovered in extreme habitats such as deep-sea hydrothermal vents, hot springs, nuclear waste sites, soda lakes, and polar as well as arid deserts. These microorganisms, collectively termed \u0026ldquo;extremophiles,\u0026rdquo; exhibit remarkable adaptations that enable survival under harsh physicochemical conditions. Extremophiles are broadly categorized based on the nature of the extreme environment they inhabit, including thermophiles, psychrophiles, acidophiles, alkaliphiles, halophiles, barophiles, metallophiles, and radiophiles. The study of extremophiles not only deepens our understanding of the limits of life on Earth but also holds promise for the discovery of novel biomolecules with potential biotechnological and pharmaceutical applications \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. One of the key extreme environmental stressors is electromagnetic radiation, particularly in the form of ultraviolet (UV) radiation \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is believed that UV exposure had been a challenge to cell repair processes and overall survival on the Early Archaean Earth \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Several bacterial strains including \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eDeinococcus radiodurans\u003c/em\u003e have been isolated, thriving under such extreme conditions \u003csup\u003e[4, 5]\u003c/sup\u003e. Ultraviolet radiation (UVR) has been identified as a harmful abiotic factor that significantly impacts microorganisms at various levels, causing damage to essential cellular biomolecules such as DNA, proteins, and lipids \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. UVR can either directly or indirectly affect the cellular structures leading to formation of reaction oxygen species (ROS) \u003csup\u003e[7, 8]\u003c/sup\u003e. ROS interact with key cellular constituents, including DNA, proteins, and lipids. Given that these biomolecules are critical for maintaining cellular membrane structure, their oxidative damage can compromise membrane integrity, impair permeability, and ultimately lead to bacterial cell death \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBacterial strains have evolved diverse physiological and biochemical mechanisms to mitigate the detrimental effects of UV radiation, including efficient DNA repair systems and defense strategies against UV-induced ROS \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Among these protective mechanisms, several key cellular systems play critical roles. The most prominent is the enzymatic antioxidant defense system, which includes catalase, superoxide dismutase (SOD), and peroxidases enzymes essential for maintaining metabolic homeostasis under oxidative stress. In addition to these enzymatic defenses, non-enzymatic antioxidants such as vitamins C, E, and B-complex, glutathione, and cysteine act as free radical scavengers, collectively mitigating the deleterious effects of UV-induced oxidative damage \u003csup\u003e[11, 12]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eExtremolytes are low molecular weight organic osmolytes that accumulate in extremophilic microorganisms, enabling their survival under harsh environmental conditions. These compounds confer protection by stabilizing vital cellular structures and facilitating the formation of hydration shells, thereby preserving macromolecular integrity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Extremolytes constitute almost 25% of dry cell weight \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In the quest for radiation-resistant biomolecules, diverse extremolytes have been identified and isolated from radiotolerant organisms. Notable examples include scytonemin, mycosporine-like amino acids (MAAs), melanin, bacterioruberin, carotenoids (including lycopene and astaxanthin). The biosynthesis of these protective compounds exemplifies the remarkable biochemical adaptations evolved by extremophiles to withstand elevated radiation exposure \u003csup\u003e[15, 16]\u003c/sup\u003e. These extremolytes contribute significantly to the overall stress resistance of extremophiles, each playing distinct protective roles. Some of them function by forming a hydration shell around cellular components, thereby shielding critical structures from the damaging effects of extreme environmental conditions encountered in their native habitats \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSeveral extremolytes additionally function as potent antioxidants, directly scavenging reactive oxygen species to protect critical cellular components from UV radiation and oxidative damage \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Notably, many extremolytes involved in UV-radiation resistance exhibit multifunctional properties, conferring simultaneous protection against multiple environmental stressor \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. One of the most important extremolytes that are efficiently involved in antioxidant activities are flavonoid metabolites. These metabolites either interact with reactive radicals and result in the formation of stable and inactive radicals or directly scavenge free radicals \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Extremolytes represent a largely untapped resource with significant biotechnological potential, often referred to as an 'unexplored gold mine'. These compounds offer promising applications across multiple industries, including cosmetics, medicine, and food production. Of particular interest are mycosporine-like amino acids (MAAs), which exhibit unique photoprotective properties. When exposed to UV radiation, MAAs demonstrate remarkable stability and protective capacity, making them valuable candidates for UV-protective sunscreens in cosmetic formulations. Furthermore, their potential as therapeutic agents for preventing UV-induced skin carcinogenesis has attracted considerable scientific attention \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe primary aim of this study was the isolation of a UV radiation (UVR)-resistant bacterial strain from the Cholistan Desert, Pakistan, followed by in vitro evaluation of its bioactive metabolites. The Cholistan Desert is a largely unexplored ecological niche with considerable potential to harbor extremotolerant and stress-resistant microorganisms. The region is characterized by a harsh subtropical climate, marked by minimal annual precipitation, low humidity, high evaporation rates, and prolonged exposure to intense solar radiation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Historically, approximately 5000 years ago, the area received substantial monsoonal rainfall; however, climatic shifts over time diverted monsoon systems away, drastically reducing precipitation and transforming the region into an arid desert. A unique feature of the Cholistan Desert is the frequent occurrence of consecutive dry years, often spanning 4 to 6 years, resulting in significant thermal fluctuations. During summer months (May to June), average temperatures range from 35\u0026deg;C to 50\u0026deg;C, while in winter (December to February), they fall to 15\u0026deg;C to 20\u0026deg;C \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this context, we report the isolation of a rare and highly UVR-resistant actinobacterial strain, designated CH-8, from the hyper-arid soils of the Cholistan Desert, has been scarcely reported in literature. To the best of our knowledge, this is the first report of \u003cem\u003eS. alkaliterrae\u003c/em\u003e exhibiting such pronounced UV and oxidative stress resistance from a desert environment. The extreme habitat and physiological resilience of this strain suggest it may possess unique bioactive compounds with promising biotechnological potential, highlighting the Cholistan Desert as a valuable but underexplored source of novel microbial resources.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaterial and method\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemicals and Reagents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Luria Bertani (LB) medium used in this study for the growth of bacterial strain was purchased from Merk (KGaA Darmstadt, Germany). Double distilled water was used for broth and buffers preparations. Methanol, acetone, ethyl acetate, chloroform, diethyl ether, ethanol, dimethylsulfoxide (DMSO) and all other solvents were obtained from Sigma-Aldrich, USA. All the solvents are of analytical grade with 99% purity. Muller Hinton Agar medium used for the antibacterial activities and chemicals used for bioassays were purchased from Merk (KGaA Darmstadt, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of UV resistant Bacteria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSand samples were collected aseptically from Cholistan desert, Pakistan by following standard protocols of sampling \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and UV resistant bacterial strains were isolated using serial dilutions and cultured on sterile basic TGY medium containing (g/L): Trypton, 10; glucose, 1 and yeast extract, 5; using spread plate method. Test plates were irradiated with ultraviolet-B (UV-B) radiation using a UV chamber equipped with a 280 nm wavelength lamp (20 W output) positioned 30 cm above the samples. Exposure duration was standardized to 5 min, with the UV fluence rate (energy dose per unit area) J/m\u0026sup2; as measured by a calibrated radiometer. Following UV treatment, plates were immediately transferred to red light conditions (λ\u0026thinsp;\u0026gt;\u0026thinsp;600 nm) to prevent photo-reactivation of microbial isolates. UV-resistant colonies were subsequently selected and sub-cultured on fresh TGY agar plates for further study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of UVResistant Bacterial Strain\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMorphological and biochemical identification of strain CH-8 was done by performing Gram staining, sugar utilization tests and various enzymes production tests \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Genomic DNA was extracted from strain CH-8 using the Thermo Scientific GeneJET Genomic DNA Purification Kit following manufacturer's protocols. The 16S rRNA gene was amplified and sequenced via Sanger sequencing (Macrogen Inc., Geumcheon-gu, Seoul, South Korea). The obtained sequence was analyzed using the NCBI BLAST algorithm for preliminary taxonomic identification and deposited in GenBank under accession number (PP757466). Phylogenetic analysis was performed using MEGA-X software. A neighbor-joining tree was constructed based on 16S rRNA gene sequences from closely related type strains, with bootstrap values calculated from 1000 replicates to assess nodal support \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth Conditions Optimization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe optimal growth conditions for \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 were determined by evaluating its growth under varying physicochemical parameters, Temperature (30\u0026ndash;45\u0026deg;C) increments of 5\u0026deg;C; pH: 4.0\u0026ndash;10.0 (adjusted using 0.1 M NaOH/HCl); Media, Nutrient broth (NB), Luria-Bertani broth (LB), and Tryptone-Glucose-Yeast extract (TGY) broth; Carbon sources, Glucose, xylose, mannitol, lactose, and dextrose (1% w/v each); NaCl tolerance, 1\u0026ndash;8% (w/v). Growth was monitored spectrophotometrically by measuring optical density (OD600) at 24 h intervals using a UV-Vis spectrophotometer (Analytik Jena Specord 200 Plus). All experiments were performed in triplicate under aerobic conditions with shaking at 150 rpm. Growth curves were plotted as optical density (OD) 600 versus time, and optimal conditions were identified based on maximum biomass production.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial Survival Curves under UV Radiation and Oxidative Stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe survival rate of the isolated strain \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 under ultraviolet (UV) radiation and oxidative stress was assessed. The strain was cultured in TGY broth and incubated at 37\u0026deg;C in a shaking incubator at 130 rpm for 48 h. Following incubation, the culture was serially diluted using autoclaved normal saline until the optical density at 600 nm (OD₆₀₀) reached approximately 0.08\u0026ndash;0.10. A 20 \u0026micro;L aliquot of the diluted culture was aseptically spread onto TGY agar plates using the spread plate technique. For UV radiation tolerance assessment, inoculated plates were exposed to varying durations of UV radiation (254 nm), ranging from 2 to 12 min, under a UV chamber \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To evaluate oxidative stress resistance, a bacterial cell suspension of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was treated with varying concentrations of hydrogen peroxide (H₂O₂), ranging from 10 to 60 mM. After treatment, a 20 \u0026micro;L aliquot from each concentration was aseptically spotted onto TGY agar plates. The plates were incubated at 37\u0026deg;C for 48 h. Following incubation, colony formation was recorded, and the survival rate was determined by comparing the number of colony-forming units (CFUs) on H₂O₂-treated plates with those on untreated control plates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction of Intracellular Bacterial Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was cultured in 1 L TGY broth under optimal conditions (37\u0026deg;C, 130 rpm) until late exponential phase. Cells were harvested by centrifugation (8,000 rpm, 20 min, 4\u0026deg;C), washed twice with PBS buffer (pH 7.4), and subjected to a sequential extraction protocol. Pellets were resuspended in chilled methanol (1:5 w/v) for cold shock treatment (4\u0026deg;C, 1 h) followed by heat shock (60\u0026deg;C, 30 min) with periodic vortexing. Mechanical lysis was enhanced using glass beads (0.1 mm) via vortexing (15 min) and ultrasonication (20 kHz, 20 min with 30 sec pulse/1 min cooling intervals). The lysate was clarified by centrifugation (8,000 rpm, 15 min, 4\u0026deg;C), and the pigmented supernatant was transferred to glass vials. Samples were air-dried under sterile laminar flow (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. All steps were performed in triplicate with appropriate controls.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibacterial Activity of Intracellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antibacterial potential of the intracellular crude extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was evaluated using the disk diffusion method on Mueller-Hinton agar (MHA) plates. The assay was performed against a panel of clinically relevant and ATCC reference strains, including Gram-positive bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e) and Gram-negative bacteria (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e). Sterile filter paper disks (6 mm diameter) were impregnated with the crude extract and placed onto the surface of freshly inoculated MHA plates. The plates were incubated at 37\u0026deg;C for 24 h. Antibacterial activity was assessed by measuring the diameter of the zones of inhibition in millimeters (mm) around each disk, and the results were recorded accordingly \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-biofilm potential of Intracellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibition of Biofilms Formation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe biofilm inhibition assay was conducted with slight modifications to previously described protocols in order to evaluate the effect of the intracellular extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 on the initial attachment of bacterial cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. About 1 \u0026times; 10⁸ CFU/mL suspension of each test strain (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e) was prepared by inoculating overnight-grown cultures into sterile nutrient broth. A total of 150 \u0026micro;L of the bacterial suspension was dispensed into each well of a sterile 96-well microtiter plate, in the presence of varying concentrations (20\u0026ndash;100 \u0026micro;g/mL) of the intracellular extract. Wells containing bacterial suspension without extract served as negative controls. Following incubation at 37\u0026deg;C for 24 h, non-adherent planktonic cells were removed by washing the wells three times with sterile phosphate-buffered saline (PBS). Adherent cells were fixed by adding 99% methanol for 20 min. The wells were then stained with 1% (w/v) crystal violet for 20 min, followed by three additional PBS washes to remove excess dye. After air drying, the bound dye was solubilized using 33% glacial acetic acid, and optical density was measured at 595 nm using a microplate reader. The percentage of biofilm inhibition was calculated using the Eq.\u0026nbsp;1:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{%}\\:\\text{o}\\text{f}\\:\\text{a}\\text{n}\\text{t}\\text{i}-\\text{b}\\text{i}\\text{o}\\text{f}\\text{i}\\text{l}\\:\\text{a}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}=\\frac{1\\:-\\:\\text{A}\\text{b}\\text{s}\\:\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{b}\\text{s}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\:\\times\\:\\:100\\)\u003c/span\u003e\u003c/span\u003e Eq.\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eWhere Abs sample represents the absorbance of the test sample (containing bacterial suspension with extract), and Abs control denotes the absorbance of the bacterial suspension alone (without extract.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEradication of Performed Biofilms\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBiofilms were cultivated in 96-well microtiter plates by incubating bacterial suspensions at 37\u0026deg;C for 8 h under static conditions. Following initial biofilm formation, 100 \u0026micro;L aliquots of intracellular extract (concentration range: 20\u0026ndash;100 \u0026micro;g/mL) were added to respective wells containing pre-formed biofilms of test strains. The plates were then incubated for an additional 18 h at 37\u0026deg;C to assess biofilm inhibition. Post-incubation, biofilm biomass was quantified using the standardized crystal violet staining protocol as detailed above. All experiments were performed with appropriate controls and in biological triplicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhytochemical Screening of total Phenolic and Total Flavonoids Contents of Intracellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe total phenolic content of the intracellular extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was determined using the Folin-Ciocalteu method, \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e with gallic acid as the calibration standard. The assay was performed according to the referenced protocol with minor modifications. To determine total phenolic contents 1.5 mL Fc reagent and 100 \u0026micro;L of intracellular extract was added to test tube. The reaction mixture was incubated for 15 min and then 1 mL of 20% Na₂CO\u003csub\u003e3\u003c/sub\u003e solution was added. Test tubes were then left in dark conditions and after 1 h of incubation, absorbance was measured at 710 nm against the blank which contained all the chemical ingredients except sample extract. Total phenolic content was calculated from the calibration curve and the outcomes were presented as milligrams of gallic acid equivalent per gram of dry weight.\u003c/p\u003e\u003cp\u003eThe total flavonoid content of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 crude bacterial extract was determined by using aluminum colorimetric chloride assay \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e with some modifications using quercetin as a standard. 200 \u0026micro;L of intracellular extract was diluted with 800 \u0026micro;L of methanol and mixed with 1 mL of 5% NaNO₂ in test tube. After 5 min, 1 mL AlCl\u003csub\u003e3\u003c/sub\u003e (10%) was added which was followed by addition of 2 mL of NaOH (1 M). Reaction mixture was then given an incubation time of 45 min in the dark at room temperature. After incubation time, absorbance of the reaction mixture was measured at 510 nm. Methanol was used as blank in the process. Total flavonoid content of the extracts was expressed as percentage of quercetin equivalent per 100 g dry weight of sample.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePurification of Intercellular Bacterial Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intracellular crude extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was fractionated using silica gel (Merck\u0026reg;, 100\u0026ndash;200 mesh) column chromatography. A stepwise gradient elution was performed with solvents of increasing polarity: hexane, chloroform, diethyl ether, ethyl acetate, acetone, ethanol, and methanol. The mobile phase composition was systematically varied from non-polar to polar to facilitate the separation of metabolites based on their differential affinities. The eluent was collected in glass vials with the flow rate of 1 mL/min and by the fraction size of 10 mL each. The collected fractions were subjected to rotary evaporator (B\u0026Uuml;CHI Rotavapor R-200, Switzerland) and dried powder was kept in vials. The dried chromatographic fractions were reconstituted in dimethyl sulfoxide (DMSO) for preliminary antioxidant evaluation. Screening was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Among the tested fractions, the methanol-eluted (100%) fraction exhibited the highest antioxidant activity and was selected for further analysis. For subsequent in vitro bioassays, the active fraction was serially diluted in DMSO. All solutions were freshly prepared prior to experimentation and protected from light to prevent degradation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro Bioassays of Purified Cellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRadical Scavenging Activity by DPPH Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntioxidant potential of active intracellular metabolic fraction obtained from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was determined by DPPH assay \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Various concentrations of active purified fraction (20\u0026ndash;100 \u0026micro;g/mL) were treated with DPPH (0.2 mM) solution in 96-well micro titer plate. The plate was incubated in dark at 37\u0026deg;C for 30 min. After incubation absorbance was measured at 517 nm using micro titer plate reader (Multiskan FC Microplate Photometer, USA). Ascorbic acid was used as a positive control in this assay. DPPH radical scavenging ability of the extracts was calculated by the Eq.\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{%}\\:\\text{D}\\text{P}\\text{P}\\text{H}\\:\\text{r}\\text{a}\\text{d}\\text{i}\\text{c}\\text{a}\\text{l}\\:\\text{s}\\text{c}\\text{a}\\text{v}\\text{e}\\text{n}\\text{g}\\text{i}\\text{n}\\text{g}\\:\\text{p}\\text{o}\\text{t}\\text{e}\\text{n}\\text{t}\\text{i}\\text{a}\\text{l}\\:=\\frac{\\text{A}\\text{b}\\text{s}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:-\\:\\text{A}\\text{b}\\text{s}\\:\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{b}\\text{s}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\:\\times\\:\\:100\\)\u003c/span\u003e\u003c/span\u003e Eq.\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA Damage Prevention Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA damage prevention potential of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 intracellular extract was assessed using DNA nicking assay as described by Lee et al. (2002), with some modifications \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The DNA protection activity of the purified intracellular extract was evaluated using the pUC19 plasmid DNA. Plasmid DNA was extracted from \u003cem\u003eEscherichia coli\u003c/em\u003e competent cells using a standard plasmid isolation protocol. The reaction mixture (total volume: 25 \u0026micro;L) was prepared by combining 3 \u0026micro;L of pUC19 plasmid DNA, 10 \u0026micro;L of the purified extract, 4 \u0026micro;L of FeSO₄ (2 mM), 4 \u0026micro;L of H₂O₂ (30%), and 4 \u0026micro;L of sodium nitroprusside (1 M). The mixture was incubated at 37\u0026deg;C for 1 h to induce oxidative DNA damage. Following incubation, the reaction mixtures were loaded onto a 0.8% agarose gel prepared in 1\u0026times; TAE buffer. Electrophoresis was performed at 90 V for 1 h using a horizontal gel electrophoresis system (Bio-Rad, USA). The gel was subsequently visualized under a UV transilluminator (Biometra TI 2, Analytik Jena, Germany). Plasmid DNA alone served as the positive control, indicating intact DNA. DNA treated with hydrogen peroxide and sodium nitroprusside without extract served as the negative control, representing oxidative DNA damage. Protective effects of the extract were assessed by comparing the integrity and migration pattern of DNA bands.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxic Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCytotoxicity of bacterial metabolic intracellular extract was determined using brine shrimp lethality test (BST) by following protocol described by Meyer et al. (1982), with slight modifications \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. 10 Nauplii were collected using a micropipette and were transferred to glass tube containing 5 mL artificial seawater and different concentrations of prepared \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 bacterial intracellular extract ranging from 20\u0026ndash;100 \u0026micro;L. Cytotoxic effect of extracts was observed after 24 h by counting number of alive and dead nauplii. Nauplii were termed as alive if they exhibited any movement during 15 sec of observation. Mitomycin C was used as positive control and DMSO was taken as negative control. The experiment was run in triplicate and mean values were calculated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-hemolysis Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe anti-hemolytic activity of the \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 intracellular extract was evaluated using the hemolysis inhibition assay, following the method described by Karim et al. (2020), with minor modifications \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. A total of 10 mL of peripheral blood was collected from a healthy individual into an EDTA-coated tube and centrifuged at 3000 rpm for 10 min to separate erythrocytes. The resulting erythrocyte pellet was washed twice with 0.2 M PBS buffer (pH 7.4) and resuspended in 0.9% normal saline. To assess anti-hemolytic activity, 0.4 mL of the erythrocyte suspension was treated with varying concentrations (20\u0026ndash;100 \u0026micro;g/mL) of the intracellular crude extract and purified fractions of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8. The mixtures were incubated at 37\u0026deg;C for 10 min, after which 0.2 mL of hydrogen peroxide (H₂O₂) was added to induce oxidative stress. Samples were further incubated at 37\u0026deg;C for 3 h. Following incubation, all mixtures were centrifuged at 3000 rpm for 10 min, and the absorbance of the supernatant was measured at 540 nm using a UV\u0026ndash;Vis spectrophotometer (Analytik Jena, Specord 200 Plus, USA) to determine the degree of hemolysis. Ascorbic acid was used as a positive control, while DMSO served as the negative control. A combination of PBS and H₂O₂ without extract was used as a blank control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of Cellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFTIR Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe partially purified active fraction was analyzed using Fourier Transform Infrared (FT-IR) spectroscopy to determine the chemical moieties in the extract responsible for the biological activities using FTIR spectrometer (Perkin Elmer Spectrum 65, UK). The sample was analyzed in triplicates within the spectral range of 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using the 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resolution FT-IR spectrometer. The IR transmittance of partially purified fraction was graphed by plotting intensity against wave number.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLC-MS of Partially Purified Fraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe most active partially purified fraction of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 bacterial strain was subjected to mass spectrometry using an LC-MS system (Agilent 6310 Ion Trap LC-MS, USA). The partially purified dried fraction, dissolved in methanol (1 mg/ml) was allowed separated on C-18 column (100 \u0026times; 3.0 mm, 2.7 \u0026micro;m) by maintaining gradient elution at the flow rate of 1 mL/min. water and acetonitrile was employed as mobile phase. Water (Line A) and acetonitrile (Line B) were used as the mobile phases. The column was pre-equilibrated with a solvent composition of 90% A and 10% B. Following sample injection, this ratio was maintained for 1 min, after which a linear gradient was applied, transitioning to 100% B (0% A) over the course of 1 h. Mass spectrometric analysis was performed using electrospray ionization (ESI) in negative ion mode, targeting [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ m/z transitions.\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of triplicate independent experiments. Statistical comparisons were performed using Student's paired t-test, with p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Dose-dependent responses (cytotoxicity, UV-B radiation survivability, and oxidative stress resistance) were evaluated through linear regression analysis between treatment groups and their respective controls. For anti-biofilm and anti-hemolytic assays, both one-way ANOVA (for between-group comparisons) and two-way ANOVA (for assessing interactions among multiple variables) were employed, followed by appropriate post-hoc tests where significant differences were detected.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIsolation and Identification of Strain CH-8\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInitial screening of Cholistan desert soil samples yielded eight bacterial strains demonstrating resistance to 5 min UVB (280 nm) exposure. Through subsequent rigorous selection involving extended 10 min UVB exposure, only two isolates designated CH-1 and CH-8 exhibited sustained viability. Strain CH-8 was prioritized for further investigation due to its superior survival rate under prolonged UVB stress, as quantified through colony-forming unit (CFU) analysis.\u003c/p\u003e\u003cp\u003eStrain CH-8 was identified as a non-motile, Gram-positive rod that exhibited blackish pigmentation when cultured on TGY agar plates. Biochemical characterization revealed positive results for citrate utilization, catalase activity, amylase, and protease production. Molecular identification was carried out by sequencing the 16S rRNA gene amplicon. Phylogenetic analysis was performed using the Neighbor-Joining method \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e in MEGA version 11, which revealed 99.88% sequence similarity with \u003cem\u003eStreptomyces alkaliterrae\u003c/em\u003e. Pairwise sequence alignment using the EzTaxon database further confirmed the close phylogenetic relationship between strain CH-8 and other validly described \u003cem\u003eStreptomyces\u003c/em\u003e species. The 16S rRNA gene sequence of strain CH-8 has been deposited in the NCBI GenBank database under the accession number PP757466. The phylogenetic tree illustrating its position is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth Conditions Optimization of\u003c/b\u003e \u003cb\u003eS. alkaliterrae\u003c/b\u003e \u003cb\u003eCH-8 strain\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe growth parameters of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 were systematically optimized. The strain exhibited growth within a narrow temperature range of 30\u0026ndash;40\u0026deg;C, with optimal growth observed at 40\u0026deg;C. It also tolerated a pH range of 7.0\u0026ndash;10.0, showing maximum growth at pH 8.0. When tested in various growth media, including nutrient broth, LB broth, and TGY broth, \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 demonstrated the highest optical density and pigment production in LB broth. To evaluate carbon source utilization, the strain was cultured in media supplemented with glucose, mannitol, xylose, lactose, and dextrose. Among these, lactose supported the most favorable growth, indicating it as the preferred carbon source. Additionally, salt tolerance was assessed by exposing the strain to NaCl concentrations ranging from 1\u0026ndash;8%. Growth decreased with increasing salt concentration, with the maximum tolerance observed at 4% NaCl. Detailed results are provided in Supplementary information Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (A-E).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial Survival against UVB radiations and H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eSurvival rate was observed in comparison with \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10536). In results it was found that \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 showed survivability of 60% at 2712 J/m\u003csup\u003e2\u003c/sup\u003e under UVB radiations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Similarly, in response to oxidative stress, induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e it was found that strain CH-8 had 78% survivability at 10 mM concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In contrast, UV sensitive control strain (\u003cem\u003eE. coli\u003c/em\u003e) showed less than 50% survivability at such concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction of Intracellular Bacterial Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intracellular crude extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was obtained through sonication-assisted cell lysis, followed by solvent extraction. Approximately 1 g of the crude extract was obtained and subsequently used for antibacterial activity assessment and photochemical screening.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibacterial Activity of Cellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntibacterial activity of intracellular crude extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was performed against Gram positive bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e) and Gram-negative bacteria (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e). Among all the strains, crude extract was more active against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e and details of zones of inhibition in comparison with antibiotics gentamicin and vancomycin are given in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. However, no activity was observed against Gram-negative strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eZones length of antibacterial activity of intracellular crude metabolic extract and positive control.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"652\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTypes of Organism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of Pathogenic Strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 322px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZone of Inhibition Diameter (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude Extract of CH-8 Strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive control\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Antibiotic discs)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative control\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Solvent)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eGram negative bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.02\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.01\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eGram positive bacteria\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e2.4\u0026plusmn;0.22\u0026nbsp;cm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.07\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus\u0026nbsp;\u003c/em\u003e\u003cem\u003eepidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e2.7\u0026plusmn;0.4\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.01\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e1.2\u0026plusmn;0.15\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.04\u0026nbsp;cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eGentamicin was used as positive control against Gram negative bacteria; Vancomycin was used as positive control against Gram positive bacteria and DMSO was used as negative control. Values were represented as mean \u0026plusmn; SD (n=3)\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-biofilm potential of Intracellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe anti-biofilm potential of the crude intracellular extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was evaluated using the crystal violet assay against several biofilm-forming Gram-positive pathogens. The extract significantly inhibited the initial attachment phase of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e in a concentration-dependent manner, with inhibition percentages ranging from 24.7\u0026ndash;85.4%. Among the tested strains, \u003cem\u003eE. faecalis\u003c/em\u003e displayed the least susceptibility to biofilm inhibition. The highest inhibition was observed at 100 \u0026micro;g/mL of extract, with \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e showing inhibition rates of 85.4% and 76.3%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the biofilm eradication assay, the extract demonstrated the ability to disrupt established biofilms of the same pathogens. Again, \u003cem\u003eE. faecalis\u003c/em\u003e showed the least response, whereas substantial eradication was observed against \u003cem\u003eB. subtilis\u003c/em\u003e (88.1%) and \u003cem\u003eS. epidermidis\u003c/em\u003e (87.5%) at the 100 \u0026micro;g/mL concentration, indicating the strong anti-biofilm efficacy of the CH-8 extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTotal Phenolic and Total Flavonoids Contents of Cellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe total phenolic content (TPC) and total flavonoid content (TFC) of the intracellular crude extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 were quantified using standard calibration curves of gallic acid and quercetin, respectively. TPC was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g DW), while TFC was reported as milligrams of quercetin equivalent per gram of dry weight (mg QE/g DW). The extract exhibited a TPC of 149 mg GAE/g DW and a TFC of 2.082 mg QE/g DW, indicating a substantial presence of bioactive phenolic and flavonoid compounds.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePurification of Intracellular Bacterial Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intracellular crude extract from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was extracted after cell lysis through ultra-sonication and purified by using silica gel column chromatography. Among various fractions collected with different solvents, the fraction dissolved in 100% methanol exhibited the highest ROS scavenging activity based on preliminary DPPH assay and was further subjected to in-vitro bioassays.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn-vitro Bioassays of Intracellular Crude Extract and Purified Fraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRadical Scavenging Activity by DPPH Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antioxidant potential of the intracellular crude extract and purified fractions from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was evaluated using the DPPH radical scavenging assay, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. The crude extract demonstrated a DPPH radical scavenging activity of 73.62%, while the purified fraction exhibited an enhanced activity of 78%, indicating strong antioxidant capacity and suggesting enrichment of active constituents in the purified form.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxic Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxic potential of the intracellular crude extract and purified fractions of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was assessed using the brine shrimp lethality assay and compared with the standard anticancer agent Mitomycin C. A concentration-dependent increase in toxicity was observed for both crude and purified extracts. At the highest tested concentration (100 \u0026micro;g/mL), survival rates were reduced to 20% and 10% for the crude extract and purified fraction, respectively. The half-maximal inhibitory concentration (IC₅₀) values, determined through linear regression analysis of the dose response curve (percentage survivability vs. concentration), were 28.3 \u0026micro;g/mL for the crude extract and 19.23 \u0026micro;g/mL for the purified fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating notable cytotoxic efficacy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA Damage Prevention Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protective effect of the intracellular crude extract and purified fraction of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 against oxidative DNA damage was assessed using plasmid pUC18. In the assay, hydroxyl radicals (\u0026bull;OH) generated via the Fenton reaction induced plasmid DNA strand breaks, as evidenced by the degraded DNA pattern in the negative control lane. In contrast, the presence of both the crude extract and purified fraction effectively preserved the integrity of plasmid DNA, suggesting their potential to mitigate oxidative stress-induced genotoxicity. The positive control lane, containing untreated plasmid DNA, retained the native supercoiled form, validating the assay conditions (Supplementary information Fig. S2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-hemolysis Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protective effect of crude extract and purified fractions of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 on erythrocytes was evaluated by exposing human red blood cells (RBCs) to oxidative agents. In results it was observed that crude extract had 43% inhibitory activity on oxidative stress-induced lysis while 60% inhibitory activity of purified fraction was observed at 100 \u0026micro;g concentration. Ascorbic acid was used as positive control and was giving inhibitory activity of 72.3% at 100 \u0026micro;g (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of Cellular Extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFTIR Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe FTIR spectrum of the purified methanolic fraction from \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 revealed several characteristic peaks indicative of diverse functional groups associated with bioactive secondary metabolites. A broad absorption band in the analytical region corresponded to O\u0026ndash;H stretching vibrations from methanol used as the solvent. Prominent peaks at 2997 cm⁻\u0026sup1; and 2914 cm⁻\u0026sup1; were attributed to aliphatic C\u0026ndash;H stretching. A distinct band observed at 1666 cm⁻\u0026sup1; suggested the presence of C\u0026thinsp;=\u0026thinsp;C stretching vibrations, indicative of aromatic or alkene moieties. In the fingerprint region (1500\u0026ndash;500 cm⁻\u0026sup1;), sharp absorption peaks between 1450\u0026ndash;1350 cm⁻\u0026sup1; indicated O\u0026ndash;H bending of carboxylic acids and methylene groups. A band at 1310 cm⁻\u0026sup1; corresponded to C\u0026ndash;N stretching of aromatic amines, while the peak at 1019 cm⁻\u0026sup1; was attributed to C\u0026thinsp;=\u0026thinsp;O stretching vibrations, associated with ethers, polyphenols, and flavonoids. Additionally, peaks below 900 cm⁻\u0026sup1; indicated C\u0026ndash;H bending in substituted aromatic compounds. Collectively, these spectral features support the presence of complex bioactive moieties commonly found in microbial secondary metabolites (Supplementary information Fig. S3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLC-MS of Partially Purified Fraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe purified methanolic fraction of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealkaliterrae\u003c/em\u003e CH-8 was analyzed using liquid chromatography\u0026ndash;mass spectrometry (LC-MS) equipped with electrospray ionization in negative ion mode (ESI-MS) to identify potential bioactive metabolites. The scan range was set from m/z 100 to 1000, and chromatographic separation was performed over a 1 h gradient run. The resulting total ion chromatogram (TIC) displayed multiple peaks, corresponding to various molecular ions and their characteristic fragmentation patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which were further interpreted through comprehensive literature comparisons and database cross-referencing. A prominent molecular ion peak was observed at m/z 575.6 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻, eluting at a retention time of 4.4 min, corresponding to a neutral molecular mass of 576.6 Da. The presence of a significant fragment ion at m/z 411.4 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻, indicating a mass loss of approximately 164 Da, suggested glycosidic bond cleavage, likely representing a hexose sugar unit (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This fragmentation pattern is characteristic of glycosylated polyketides. Based on structural analogy and spectral alignment, the compound was putatively identified as a glycosylated enediyne-type aromatic polyketide similar to dynemicin L, a class of metabolites known for their potent bioactivities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notably, such compounds are typically synthesized via type I polyketide synthase (PKS-I) gene clusters, which are prevalent in \u003cem\u003eStreptomyces\u003c/em\u003e genomes. While the whole genome of \u003cem\u003eS. alkaliterrae\u003c/em\u003e is not yet sequenced, close phylogenetic relatives harbor PKS-I gene clusters, supporting this biosynthetic potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnother significant molecular ion peak at m/z 480.6 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ was detected at a retention time of 11.0 min. The corresponding fragment ion profile was consistent with indole alkaloids, aligning closely with lynamicin D, a bioactive compound known for its antibacterial and antitumor properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). In addition, a peak at m/z 459.4 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ was observed at 44.3 min, tentatively identified as lankacidin C, a macrolide polyketide with known antibiotic properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). At 52.6 minutes, a late-eluting compound with a molecular ion at m/z 543.5 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ showed a characteristic neutral loss of 35 Da, indicating the presence of chlorine. The resulting base peak at m/z 508.1 [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ strongly supports the presence of a halogenated macrolide or polyketide (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Importantly, several detected compounds showed no exact matches in existing metabolomic databases, suggesting that \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 may produce novel or structurally rare secondary metabolites given in the Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, with potential pharmaceutical relevance. These findings highlight the strain\u0026rsquo;s untapped biosynthetic repertoire and underscore the significance of extremophilic actinomycetes as reservoirs of chemically diverse natural products.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMetabolic profiling of methanolic fraction of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 by LC-MS \u0026ndash; negative ionization mode\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBase peak [M-H]⁻ (m/z)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eParent peak [M-H]⁻ (m/z)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFragment Ions [M-H]⁻ (m/z)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTentative Compound Class\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eClosest Match / Analog\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e495.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e557.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e378.7, 435.8, 470.8, 557.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePolyhydroxy polyketide core\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUrdamycin-type precursor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e575.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e575.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e260.4, 327.8, 411.4, 462.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGlycosylated polyketide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAngucycline-like glycoside\u003c/p\u003e\u003cp\u003e(No exact match in databases)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e575.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e575.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e324.9, 411.4, 538.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGlycosylated macrolide or enediyne core\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDynemicin L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e480.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e480.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e395.9, 453.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIndole Alkaloid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLynamicin D\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e401.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e459.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e231.1, 314.1, 459.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePolyketide macrolide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLankacidin C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e241.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e467.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e210.2, 340.5, 392.0, 467.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePolar aromatic fragment or phenolic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDegradation product/shunt metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e52.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e508.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e543.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e543.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAnguclinone\u003c/p\u003e\u003cp\u003e(Aromatic Polyketide)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMonacyclinone J\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e508.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e699.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e359.6, 392.0, 581.4, 617.4, 661.2, 699.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAmphiphilic glycosylated polyketide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePossibly a polyene\u0026ndash;macrolide hybrid\u003c/p\u003e\u003cp\u003e(No exact match in databases)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigates the biotechnological potential of \u003cem\u003eStreptomyces alkaliterrae\u003c/em\u003e CH-8, a novel UV-B-resistant bacterium isolated from the extreme environment of Pakistan's Cholistan desert. Desert ecosystems present unique physiological challenges for microbial survival, characterized by oligotrophic conditions, extreme temperature fluctuations, minimal precipitation, and prolonged solar UV exposure. These selective pressures drive the evolution of specialized adaptations, making desert-derived microorganisms like strain CH-8 particularly valuable sources of stress-resistant bioactive metabolites with potential multifunctional applications \u003csup\u003e[37, 38, 39, 40]\u003c/sup\u003e. Microorganisms in the desert either protect themselves via antioxidant systems or bring forth sophisticated repairing mechanisms. Repairing system does not directly prevent the access of radiation to cellular structures and biomolecules but participate in repairing the damage that is done by incoming UVR \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Moreover, another similar study proposed three mechanisms including prevention, tolerance and repairing mechanisms which work collectively to cope up with UVR \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Several radio resistant bacterial strains have been reported previously from deserts e.g. \u003cem\u003eRadiobacillus deserti\u003c/em\u003e TKL69\u003csup\u003eT\u003c/sup\u003e from Taklimakan Desert \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eGeodermatophilus tzadiensis\u003c/em\u003e from Saharan Desert \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eKocuria\u003c/em\u003e sp.TMM11 from Thal Desert \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eDeinococcus deserti\u003c/em\u003e from Sahara Desert \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eStreptomyces huasconensis\u003c/em\u003e D23 from Kumtagh Desert \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ePromicromonospora\u003c/em\u003e panici PT9\u003csup\u003eT\u003c/sup\u003e Tunisian Sahara desert \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e highlighting these extreme habitats as natural reservoirs.\u003c/p\u003e\u003cp\u003eIn term of taxonomy, strain CH-8 was identified as Gram positive \u003cem\u003eactinobacterium\u003c/em\u003e belonging to \u003cem\u003eStreptomyces\u003c/em\u003e genus, showing 99.88% similarity with \u003cem\u003eStreptomyces alkaliterrae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Genome of genus \u003cem\u003eStreptomyces\u003c/em\u003e with high GC content, typically contains 25\u0026ndash;70 biosynthetic gene clusters (BGCs) \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e encoding approximately 10,000 bioactive compounds, including antibiotics, antifungals, anticancer agents, and herbicides \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Numerous studies have previously reported isolation of \u003cem\u003eStreptomyces\u003c/em\u003e strains from desert environments, such as the 49 strains reported from savanna soil in Sudan Desert \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, confirming deserts as promising hosts for actinobacterial diversity. Despite its ecological significance, \u003cem\u003eS. alkaliterrae\u003c/em\u003e remains largely unexplored, with no existing literature documenting its UV-B resistance mechanisms or metabolic potential. This study presents the first report of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 isolated from Pakistan's Cholistan desert, demonstrating remarkable UV-B tolerance (\u0026gt;\u0026thinsp;50% survival at 2712 J/m\u0026sup2;) and producing bioactive intracellular metabolites. The strain's radiation resilience appears mediated by multiple structural adaptations: a thick peptidoglycan cell wall, sporulation capacity, and protective pigments that collectively shield against photodamage. These survival strategies are further enhanced by DNA-binding proteins and secondary metabolites that scavenge reactive oxygen species, providing comprehensive protection against radiation-induced cellular damage \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInitially, \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 was exposed to extended dose of UVR and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and was found resilient against these stresses, suggesting the existence of extensive preventive mechanisms. Moreover, strain CH-8 was found as catalase positive and hence its survivability against ROS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can also be attributed to enzymes catalase and peroxidase \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. A study has reported that HspR target genes conserved in \u003cem\u003eStreptomyces\u003c/em\u003e are involved in the control of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stress response \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been reported in various studies that reactive oxygen species (ROS), induced due to exposure of bacterial strain to UVR have drastic impacts on the overall regulatory machinery of a bacterial cell and affects bacterial cell division, repairing mechanisms and cell recovery \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Researchers have been trying to seek for counter measures that organisms adopt to counterbalance ROS \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In this study, strain CH-8 has been observed to produce black colored, diffusible pigmented compounds. Colored compounds that have been extracted from various bacterial strain are reported to have the potential to scavenge superoxide anions \u003csup\u003e[58, 59]\u003c/sup\u003e. Another study revealed that colored metabolites uplift the chances of microbial survival by either providing them protection from external damaging factors or mediating the regulating machinery of the cell \u003csup\u003e[60, 61, 62]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe intracellular crude extract and partially purified fractions of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 were evaluated for multiple bioactive properties, including cytotoxicity, antihemolytic activity, antioxidant capacity, and antibacterial effects. The crude extract demonstrated significant radical scavenging activity (73.62%), which increased to 78% in the purified fraction. For comparative analysis, we observed that under similar experimental conditions, \u003cem\u003eStreptomyces\u003c/em\u003e sp. strain MUSC 14 exhibited only 24.71% DPPH scavenging activity at a higher concentration (4 mg/mL) \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The purified fraction exhibited concentration dependent cytotoxic effects, suggesting its potential as anticancer agents. These investigations align with the previous report of cytotoxic extract from \u003cem\u003eStreptomyces\u003c/em\u003e. Thus, further evaluation of purified extracts is required to evaluate their anticancer activities in cell lines \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. RBCs are very reactive to oxidative stress due to their high polyunsaturated fatty acids content and pro-oxidative nature of hemoglobin \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Hemolysis is therefore considered as a visible sign of damage that can be done to the RBCs by the free radicals \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In this study, crude extract prevented 43% hemolysis while purified fraction prevented 60% as compared to positive control ascorbic acid, which prevented 72.3% hemolysis. In parallel, some compounds have been reported to have the potential of scavenging free radicals and thus preventing hemolysis \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhenolic compounds are well known for their distinctive redox qualities including their ability to scavenge free radicals and anti-inflammatory effects \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Total phenolic content of intracellular crude extract of strain CH-8 was found as 149 mg/g suggesting the involvement of phenolic compounds in staunch resistance against oxidative stress. A similar study has linked phenolic compounds in \u003cem\u003eStreptomyces\u003c/em\u003e sp. to antioxidant and antimicrobial activity \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Flavonoids contents of crude extract were found as 2.082 mg/g. It was reported in a study that flavonoids bind with DNA and forms DNA duplex which protects DNA from oxidative damage \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe crude intracellular extract of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 demonstrated significant antibacterial activity against Gram-positive pathogens. These findings align with recent reports of antimicrobial compounds from related \u003cem\u003eStreptomyces\u003c/em\u003e species, including bacteriocins from \u003cem\u003eStreptomyces\u003c/em\u003e pluripotent that exhibit potent activity against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) strains \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Another study has reported the ethyl acetate extract of \u003cem\u003eStreptomyces\u003c/em\u003e sp. strain ACTN 2, isolated from mangroves sediments exhibited antimicrobial potential against \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Biofilm forming bacterial pathogens pose a significant challenge in a clinical scenario. It is becoming more difficult to impede the development and growth of mature biofilms in contrast to initially formed biofilms. The resistant of sessile cells in mature biofilms to antimicrobial agents is due to their production of exo-polysaccharide matrix and mutation in bacteria dwelling in biofilms \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. It was found that crude extract of strain CH-8 exhibited above 80% eradication of mature biofilm of Gram-positive pathogens. The observed antibacterial efficacy aligns with documented bioactivity profiles of marine \u003cem\u003eStreptomyces\u003c/em\u003e spp. extracts against multidrug-resistant clinical isolates, as reported in recent literature \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe LC-MS analysis of the \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 extract revealed a diverse array of bioactive secondary metabolites, several of which showed close spectral resemblance to known compounds from \u003cem\u003eStreptomyces\u003c/em\u003e species with established therapeutic relevance. Notably, one of the early eluting peaks was putatively identified as a precursor of the urdamycin-type compounds angucycline-class antibiotics. These metabolites have previously been reported from \u003cem\u003eStreptomyces fradiae\u003c/em\u003e and \u003cem\u003eS. cellulosae\u003c/em\u003e and are recognized for their potent antibacterial and antifungal activities. The presence of such a metabolite in CH-8 underscores its biosynthetic potential and supports the hypothesis that extremophilic actinomycetes are prolific producers of clinically valuable natural products \u003csup\u003e[75, 76]\u003c/sup\u003e. While the compound corresponded to Lynamicin D, an indole alkaloid derived from \u003cem\u003eStreptomyces\u003c/em\u003e sp. SCSIO 03032 \u003csup\u003e77\u003c/sup\u003e have been reported to exhibit a spectrum of bioactivities, including antimicrobial and antioxidant effects, although specific studies on its antibiofilm activity remain limited. A compound exhibiting a parent ion peak at m/z 459.4 and a corresponding base peak at m/z 401.8 was tentatively identified as Lankacidin C, a polyketide macrolide known for its potent antibacterial properties. The biosynthesis of this compound has previously been attributed to \u003cem\u003eStreptomyces rochei\u003c/em\u003e, further highlighting the antimicrobial potential of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8. \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Various studies also suggested its antitumor and antibacterial activities, particularly against Gram-positive bacteria \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. The detection of late-eluting metabolites revealed a distinctive chemical profile enriched with polyketide macrolides and amphiphilic glycosylated polyketides. Notably, several peaks lacked exact matches in existing spectral databases, suggesting the presence of previously uncharacterized or novel bioactive metabolites. Such findings underscore the metabolic versatility of \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 and its untapped biosynthetic potential. Comparative analyses further support this, as numerous Streptomyces species, including \u003cem\u003eS. africanus\u003c/em\u003e, \u003cem\u003eS. pactum\u003c/em\u003e, and \u003cem\u003eStreptomyces\u003c/em\u003e sp. MUM212, have been well documented for their potent antimicrobial and antioxidant activities, reinforcing the therapeutic relevance of secondary metabolites from this genus \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. These activities are attributed to the presence of high phenolic and flavonoid content within their extracts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents the first report on the isolation and characterization of the novel radioresistant bacterial strain \u003cem\u003eStreptomyces alkaliterrae\u003c/em\u003e CH-8 from the arid soil of the Cholistan Desert, Pakistan. The strain demonstrated remarkable tolerance to UV-B radiation and produced a crude and partially purified methanolic extract rich in antimicrobial, antioxidant, and anti-biofilm compounds. Metabolite profiling revealed a diverse spectrum of bioactive secondary metabolites, including known and potentially novel glycosylated macrolides, polyketides, and indole alkaloids, underscoring their potential applications as radioprotective agents and therapeutics against multidrug-resistant pathogens. These findings highlight the need for further structural elucidation of these compounds through advanced spectroscopic techniques such as NMR, alongside whole-genome sequencing to identify the biosynthetic gene clusters responsible for their production. Collectively, this work not only establishes the untapped microbial potential of the Cholistan Desert but also emphasizes its promise as a reservoir of novel bioactive metabolites with implications for human health and environmental sustainability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was accomplished with funds provided by Quaid-i-Azam University, Islamabad 45320, Pakistan. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no conflict of interest/competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTA; SUD; AA; AAS: Preparation of the overall research plan as well as protocols for various experiments;\u003c/p\u003e\n\u003cp\u003eTA; SUD; AA: Performed experimental work in lab as per the pre-designed research plan;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFH; SK; MB; MA; AAS: Facilitated in interpretation of various analysis and optimization experiments in the current research project;\u003c/p\u003e\n\u003cp\u003eMF: Facilitated the processing and analysis of sample through LC-MS;\u003c/p\u003e\n\u003cp\u003eTA; SUD, AA: Write up of the manuscript;\u003c/p\u003e\n\u003cp\u003eSUD, FH, SK, MB, AAS: Proof reading of the overall manuscript for English comprehension and typing mistakes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCowan, D., Ramond, J.-B., Makhalanyane, T. \u0026amp; De Maayer, P. 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Rep.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 27372 (2024).\u003c/li\u003e\n\u003cli\u003eMirsonbol, S. Z., Issazadeh, K., Zarrabi, S. \u0026amp; Mirpour, M. Evaluation of antimicrobial activity of Streptomyces pactum isolated from paddy soils and identification of bioactive volatile compounds by GC-MS analysis. \u003cem\u003eWorld J. Microbiol. Biotechnol.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 63 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Radioresistant, Streptomyces alkaliterrae, Antibiofilm, Polyketide, Bioactive metabolites","lastPublishedDoi":"10.21203/rs.3.rs-7264315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7264315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe exploration of extremotolerant microorganisms from arid ecosystems offers promising avenues for novel bioactive compound discovery. In this study, a radioresistant actinobacterial strain, CH-8, was isolated from the hyper-arid soil of the Cholistan Desert, Pakistan. Phylogenetic analysis of the 16S rRNA gene revealed 99.88% similarity with \u003cem\u003eStreptomyces alkaliterrae\u003c/em\u003e. Strain CH-8 demonstrated high oxidative and UV-B tolerance, with 78% and 60% survival under 10 mM H₂O₂ and 2712 J/m\u0026sup2; UV-B exposure, respectively. Intracellular metabolites were extracted using methanol and purified via silica gel chromatography. The purified fraction showed strong antioxidant activity (78% DPPH scavenging), and was rich in phenolics (149 mg GAE/g) and flavonoids (2.082 mg QE/g). Crude extracts exhibited significant anti-biofilm activity against Gram-positive pathogens, achieving up to 85.4% inhibition and 88.1% eradication. Cytotoxic potential was confirmed via brine shrimp lethality assay (IC₅₀ = 19.23 \u0026micro;g/mL), and DNA damage protection indicated radioprotective capability. LC-MS analysis identified several bioactive compounds, including a glycosylated polyketide (m/z 575.6) resembling dynemicin L, as well as lankacidin C and lynamicin D. These findings position \u003cem\u003eS. alkaliterrae\u003c/em\u003e CH-8 as a promising source of therapeutic secondary metabolites, and highlight the Cholistan Desert as a reservoir of untapped microbial and biochemical diversity.\u003c/p\u003e","manuscriptTitle":"Bioactive Metabolites from UV-Resistant Streptomyces alkaliterrae CH-8 in Desert Soil: In-Vitro Study on Their Potent Antioxidant and Antibiofilm Activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 15:52:06","doi":"10.21203/rs.3.rs-7264315/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-13T07:45:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T17:23:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T16:50:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90285564436423175324086385650600150243","date":"2025-08-14T10:43:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T07:47:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316964021142342612009470025669391339767","date":"2025-08-09T05:20:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148133437639688742394654380315623627748","date":"2025-08-09T03:12:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128018122901875029456785765204573151844","date":"2025-08-06T10:43:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-06T10:24:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T09:25:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-04T07:34:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T14:36:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-31T16:29:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d2b5485-b3d2-4912-985b-0cee2940f33d","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52901014,"name":"Biological sciences/Biochemistry"},{"id":52901015,"name":"Biological sciences/Biological techniques"},{"id":52901016,"name":"Biological sciences/Biotechnology"},{"id":52901017,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-12T13:50:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 15:52:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7264315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7264315","identity":"rs-7264315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}