Culture Supernatant of the Polar Actinomycete Nocardioides antarcticus CCTCC AB 2014053T Resuscitates VBNC Cells, Promotes Bacterial Growth, and Alters Bacterial Diversity in Antarctic Soil

Culture Supernatant of the Polar Actinomycete Nocardioides antarcticus CCTCC AB 2014053T Resuscitates VBNC Cells, Promotes Bacterial Growth, and Alters Bacterial Diversity in Antarctic Soil | 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 Research Article Culture Supernatant of the Polar Actinomycete Nocardioides antarcticus CCTCC AB 2014053T Resuscitates VBNC Cells, Promotes Bacterial Growth, and Alters Bacterial Diversity in Antarctic Soil Chol Song Kim, Jin Hyok Kim, Chan Hyon Han, Tong Ryul Kim, Hye Gyong Mun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8668372/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Resuscitation-promoting factor (Rpf) is a secreted protein of some Gram-positive bacteria that exerts significant resuscitative effects on viable but non-culturable (VBNC) microorganisms. Culture supernatants of Rpf-secreting strains, like Rpf itself, can recruit VBNC bacteria and modify soil bacterial diversity. This study investigated the resuscitative and growth-promoting effects of the culture supernatant from the polar actinomycete Nocardioides antarcticus CCTCC AB 2014053T (harboring the rpf gene) on VBNC and normal bacterial cells in Antarctic soil, as well as its impact on soil bacterial diversity. Three VBNC bacterial species—Brachybacterium paraconglomeratum, Staphylococcus edaphicus, and Pseudarthrobacter psychrotolerans—were successfully isolated from 10⁻¹⁰-fold serially diluted Antarctic soil treated with the culture supernatant. The culture supernatant, particularly its high-molecular-weight fraction, significantly promoted the growth of B. paraconglomeratum and P. psychrotolerans (growth rates: 125.0–191.7% relative to the untreated control). In contrast, the low-molecular-weight fraction inhibited the growth of S. edaphicus (growth rates: 52.9–91.7% relative to the control). High-throughput sequencing revealed that treatment with the high-molecular-weight fraction resulted in the classification of 782 soil bacterial genera, an 8.8% increase compared to the control, while the low-molecular-weight fraction reduced the number of genera to 90.9% of the control. Additionally, the culture supernatant altered the soil bacterial community composition, with distinct differences between the high- and low-molecular-weight fractions. These findings demonstrate that the N. antarcticus culture supernatant, containing Rpf, holds potential for isolating novel bacterial taxa by resuscitating VBNC cells and promoting bacterial growth in extreme environments. Nocardioides antarcticus Culture supernatant Antarctic soil Resuscitation-promoting factor (Rpf) Viable but non-culturable (VBNC) bacteria Bacterial diversity Figures Figure 1 Figure 2 Figure 3 Introduction The Antarctic and Arctic regions are characterized by extreme environmental conditions, including low temperatures, oligotrophy, intense solar radiation, aridity, and polar day-night cycles [1–5]. These stresses force most indigenous microorganisms into the viable but non-culturable (VBNC) state, a physiological condition where cells remain metabolically active but cannot be cultured on conventional growth media [1]. The VBNC state poses a major challenge to the isolation and exploitation of novel microbial resources from polar environments. Resuscitation-promoting factor (Rpf) is a secreted protein first identified in the actinomycete Micrococcus luteus in 1998 [6]. It functions as a "bacterial cytokine" that stimulates the resuscitation of VBNC cells and promotes the growth of culturable bacteria [6, 28]. Rpf has been extensively studied for its applications in pathogen control [7], vaccine development [8, 14, 15], diagnostic testing [9, 13, 16], and environmental remediation [10–12, 19–25]. For example, Rpf can convert VBNC Mycobacterium tuberculosis to an active state, enhancing antibiotic sensitivity [13], and induce protective immune responses in mice when used as a subunit vaccine [14]. In environmental applications, Rpf has been used to resuscitate VBNC pathogenic bacteria in drinking water for detection [17], restore spoilage bacteria in beer [18], and enhance the biodegradation of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and phenol [11, 12, 22–25]. Most studies on Rpf have focused on M. luteus and M. tuberculosis , with applications limited to isolating pollutant-degrading VBNC bacteria from contaminated environments or detecting pathogens [6, 11, 12, 17, 22–26]. However, polar microorganisms possess unique metabolic pathways and survival strategies adapted to extreme conditions, making them valuable resources for biotechnological applications [29, 32]. To date, few studies have explored the effects of culture supernatants from Rpf-secreting polar microorganisms on VBNC cells and soil bacterial diversity. Nocardioides antarcticus CCTCC AB 2014053T is a polar actinomycete identified to harbor the rpf gene through whole-genome analysis. In this study, we aimed to: (1) verify the presence of Rpf in the culture supernatant of N. antarcticus ; (2) evaluate the resuscitative effect of the culture supernatant on VBNC bacteria in Antarctic soil; (3) assess its growth-promoting activity on normal bacterial cells; and (4) investigate changes in soil bacterial diversity and community composition following treatment with the culture supernatant. Materials and Methods Strains and Soil Samples The polar actinomycete Nocardioides antarcticus CCTCC AB 2014053T was obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). Antarctic soil samples were collected from Asman Hill (69°22′685″S, 76°22′139″E) in the southwest highlands east of Zhongshan Station. Soil samples were stored at −80 °C until analysis. Genomic DNA Extraction and rpf Gene Annotation Genomic DNA of N. antarcticus was extracted using a Qiagen Genomic DNA Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA purity, quantity, and integrity were assessed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), Qubit (Thermo Fisher Scientific), and 0.7% agarose gel electrophoresis, respectively. Whole-genome sequencing was performed on the PacBio Sequel platform [35]. The genome was annotated against the NR and UniProt databases to identify rpf homologs. The sequence similarity of the predicted Rpf protein with characterized Rpf proteins from other actinomycetes was analyzed using BLASTp. Expression and Purification of Recombinant RpfB Protein The rpfB gene of N. antarcticus was amplified by PCR and cloned into the pET-28a(+) vector (Novagen, Darmstadt, Germany). The recombinant plasmid was transformed into E. coli BL21(DE3) (Novagen), and kanamycin-resistant transformants were selected. Expression of the recombinant RpfB protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h. The protein was purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) and analyzed by 12% SDS-PAGE. Preparation of N. antarcticus Culture Supernatant Fractions N. antarcticus was inoculated into R2A liquid medium (Difco™, BD, Franklin Lakes, NJ, USA) and cultured at 20 °C with shaking (160 rpm) until the mid-exponential growth phase (36 h). The culture was centrifuged at 13,000 rpm for 5 min at room temperature, and the supernatant was filtered through a 0.22 μm Millex®-GP filter (Merck KGaA, Darmstadt, Germany) to obtain sterile supernatant. The supernatant was fractionated into high-molecular-weight (>10 kDa) and low-molecular-weight (<10 kDa) fractions using a 10 kDa ultracentrifugal filter (Merck KGaA). The protein and polysaccharide contents of the crude supernatant, high-molecular-weight fraction, and low-molecular-weight fraction were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and Phenol-Sulfuric Acid Assay, respectively. All fractions were stored at −20 °C until use. Determination of the Limit Dilution of Soil Samples A 10⁻¹ soil suspension was prepared by adding 1 g of soil to 9 mL of sterile R2A medium containing 5–7 glass beads (6 mm diameter) and shaking at 160 rpm for 4 h at 20 °C. Serial dilutions (10⁻² to 10⁻¹²) were prepared by transferring 1 mL of the previous dilution to 9 mL of sterile R2A medium. Each dilution was incubated at 20 °C with shaking for 5–7 days, and the optical density at 600 nm (OD₆₀₀) was measured using a BioTex microplate reader (BioTex, Winooski, VT, USA). The limit dilution was defined as the highest dilution at which no bacterial growth was observed (OD₆₀₀ ≈ 0.045, consistent with the control). Resuscitation of VBNC Bacteria from Antarctic Soil A mixture of 1 mL of N. antarcticus culture supernatant and 4 mL of the limit dilution (10⁻¹⁰) soil suspension was incubated at 10 °C for 30 days. When bacterial growth was visually observed, serial dilutions were prepared and spread on R2A agar plates. Plates were incubated at 20 °C until single colonies appeared. Isolates were identified by 16S rRNA gene sequencing using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Sequences were compared to the NCBI GenBank database using BLASTn, and similarity values ≥97% were considered conspecific. Growth-Promoting Activity Assay Isolated bacteria were inoculated (5% v/v) into fresh R2A liquid medium supplemented with different concentrations (1%, 5%, 10%, 15%, 20%, 25% v/v) of crude supernatant, high-molecular-weight fraction, or low-molecular-weight fraction. The control group contained no supernatant. Cultures were incubated at 20 °C for 30 days, and OD₆₀₀ values were measured at regular intervals. Growth promotion rates were calculated as [(OD₆₀₀ of treated group − OD₆₀₀ of control) / OD₆₀₀ of control] × 100%. Analysis of Soil Bacterial Diversity A 10⁻¹ soil suspension was prepared as described above. Aliquots (9 mL) of the suspension were mixed with 1 mL of crude supernatant, high-molecular-weight fraction, low-molecular-weight fraction, or sterile water (control), with three replicates per group. Mixtures were incubated at 0 °C for 7 days. Total genomic DNA of soil microorganisms was extracted using the DNeasy PowerSoil® Pro Kit (Qiagen) following the manufacturer’s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTABT-3′) with sample-specific barcodes [49]. PCR reactions were performed in 20 μL volumes containing 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu DNA Polymerase (TransGen Biotech, Beijing, China), and 10 ng of template DNA. The PCR program was: initial denaturation at 95 °C for 2 min; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min. Amplicons were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, Union City, CA, USA) and quantified using the QuantiFluor™ dsDNA System (Promega, Madison, WI, USA). Sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by Shanghai Biomedical Science and Technology Co., Ltd. (Shanghai, China). Raw reads were filtered to remove low-quality sequences, and operational taxonomic units (OTUs) were clustered at a 97% similarity cutoff using USEARCH UPARSE [50]. Alpha diversity indices (Shannon, Simpson, Sobs) were calculated using QIIME 2 (version 2022.2). Beta diversity was analyzed using principal coordinate analysis (PCoA) based on the Bray-Curtis distance matrix. Taxonomic classification was performed using the SILVA database (version 138). Statistical Analysis All experiments were performed in triplicate. Data were expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test using SPSS 22.0 (IBM, Armonk, NY, USA). P < 0.05 was considered statistically significant. Results Identification of the rpfB Gene in N. antarcticus Genome annotation revealed a rpfB gene (1215 bp) in N. antarcticus CCTCC AB 2014053T, encoding a predicted RpfB protein. BLASTp analysis showed that the RpfB protein shared 36.3% sequence similarity with the characterized RpfB protein of Mycobacterium smegmatis (Table 1). Recombinant RpfB protein was successfully expressed in E. coli BL21(DE3) and purified to homogeneity, appearing as a single band at ~47 kDa on SDS-PAGE (Fig. S1). Table 1 Annotation of the rpfB gene in N. antarcticus CCTCC AB 2014053T Organism and Strain Name Gene Name Length (bp) NR Description Swiss-Prot Description Identities (%) Reference species Nocardioides antarcticus AB 2014053 T rpfB 1215 resuscitation promoting factor rpfB OS = Mycoli . smegmatis 36.3 Nocardioides rubriscoriae Characterization of N. antarcticus Culture Supernatant Fractions The protein and polysaccharide contents of the high-molecular-weight fraction were 5.6-fold and 4.4-fold higher than those of the crude supernatant, respectively. In contrast, the low-molecular-weight fraction contained only 50% of the protein and 10% of the polysaccharide present in the crude supernatant (Table S1). SDS-PAGE analysis showed multiple protein bands in the crude supernatant and high-molecular-weight fraction, including a band corresponding to the ~47 kDa RpfB protein. No protein bands were detected in the low-molecular-weight fraction (Fig. S2), confirming that RpfB is concentrated in the high-molecular-weight fraction. Limit Dilution of Antarctic Soil Samples Serial dilution experiments showed that bacterial growth (OD₆₀₀ > 0.045) was observed up to the 10⁻⁹ dilution, while no growth was detected at dilutions ≥10⁻¹⁰ (Table S2). Thus, the limit dilution of the soil samples was determined to be 10⁻¹⁰. Resuscitation of VBNC Bacteria Three bacterial strains were isolated from the 10⁻¹⁰ dilution soil suspension treated with N. antarcticus culture supernatant: Brachybacterium paraconglomeratum (97.75% 16S rRNA gene similarity), Staphylococcus edaphicus (100% similarity), and Pseudarthrobacter psychrotolerans (98.98% similarity) (Table 2). In contrast, nine strains were isolated using conventional methods (10⁻³ to 10⁻⁵ dilutions), including Rhodococcus qingshengii , Amoebinatus massiliensis , and P. psychrotolerans (99.24% similarity) (Table 2). B. paraconglomeratum and S. edaphicus were only isolated from the supernatant-treated limit dilution, indicating they were in the VBNC state in the original soil. P. psychrotolerans was isolated by both methods, suggesting it existed in both active and VBNC states. The low similarity values of B. paraconglomeratum (97.75%) and Devosia naphthalenivorans (98.19%, isolated by conventional methods) suggest they may represent novel taxa. Table 2 Bacterial strains isolated from Antarctic soil samples Classification No isolated bacterial species similarity (%) Soil Extreme Dilution (10 -10 ) + Culture Supernatants 1 Brachybacterium paraconglomeratum 97.75* 2 Staphylococcus edaphicus 100 3 Pseudarthrobacter psychrotolerans 98.98 Conventional strain isolation methods (10 -3 , 10 -4 and 10 -5 dilutions of soil samples) 1 Rhodococcus qingshengii 100 2 Amoebinatus massiliensis 100 3 Pseudarthrobacter psychrotolerans 99.24 4 Kocuria rhizophila 99.86 5 Nocardioides astragali 98.81 6 Rhizobacter fulvus 99.23 7 Sphingomonas asaccharolytica 98.76 8 Polaromonas ginsengisoli 100 9 Devosia naphthalenivorans 98.19* Indicates potential novel taxa (similarity < 98%). Growth-Promoting and Inhibitory Effects The crude supernatant and high-molecular-weight fraction significantly promoted the growth of B. paraconglomeratum and P. psychrotolerans (P < 0.05), with growth promotion rates ranging from 125.0% to 191.7% at the optimal concentration (10–15%) (Figs. 1 and S3). In contrast, the low-molecular-weight fraction had no significant effect on these two strains but inhibited the growth of S. edaphicus in a concentration-dependent manner (growth inhibition rates: 8.3–47.1%) (Fig. S4). Changes in Soil Bacterial Diversity High-throughput sequencing generated 5078–7098 OTUs per sample (Table S3). The high-molecular-weight fraction treatment had the highest OTU number (7098), Shannon index (6.7648), and Sobs index (5186.3642), and the lowest Simpson index (0.0041), indicating the highest bacterial diversity and richness (Table S3). In contrast, the low-molecular-weight fraction treatment had lower OTU numbers (4863) and diversity indices than the control (Table S3). At the phylum level, all supernatant treatments increased the number of detected phyla compared to the control, with the high-molecular-weight fraction treatment yielding the highest number (36 phyla) (Fig. 2a). At the genus level, the high-molecular-weight fraction treatment resulted in the classification of 782 genera, an 8.8% increase compared to the control (719 genera), while the low-molecular-weight fraction treatment reduced the number of genera to 654 (90.9% of the control) (Fig. 2b). The bacterial community composition also changed significantly (Fig. 3). The relative abundances of Bacteroidota , Cyanobacteria , Bdellovibrionota , and Armatimonadota were significantly lower in the low-molecular-weight fraction treatment (20.86%, 0.34%, 0.95%, 0.45%) than in the control (33.74%, 3.82%, 3.03%, 1.35%) (P < 0.05). In contrast, the relative abundances of Acidobacteriota , Actinobacteriota , and Gemmatimonadota were higher in both the high- and low-molecular-weight fraction treatments compared to the control (P < 0.05). Discussion This study demonstrated that the culture supernatant of the polar actinomycete N. antarcticus CCTCC AB 2014053T, which contains the RpfB protein, can resuscitate VBNC bacteria, promote the growth of normal bacterial cells, and alter soil bacterial diversity. Genome annotation and protein purification confirmed the presence of the rpfB gene in N. antarcticus and the secretion of RpfB into the culture supernatant. The RpfB protein shared low sequence similarity (36.3%) with characterized Rpf proteins from other actinomycetes, suggesting it may represent a novel Rpf homolog with unique functions. The high-molecular-weight fraction of the supernatant contained higher levels of protein and polysaccharide, including RpfB, while the low-molecular-weight fraction lacked detectable proteins, consistent with the size of RpfB (~47 kDa). Three VBNC bacterial species— B. paraconglomeratum , S. edaphicus , and P. psychrotolerans —were successfully resuscitated from the limit dilution (10⁻¹⁰) of Antarctic soil treated with the culture supernatant. This is consistent with previous studies showing that Rpf can resuscitate VBNC cells from various environments [6, 16, 26]. P. psychrotolerans was isolated by both conventional and supernatant-treated methods, indicating it exists in both active and VBNC states, which may be an adaptive strategy for surviving extreme polar conditions. The potential novel taxa ( B. paraconglomeratum and D. naphthalenivorans ) isolated in this study highlight the utility of Rpf-containing supernatants for discovering new microbial resources. The high-molecular-weight fraction of the supernatant significantly promoted the growth of B. paraconglomeratum and P. psychrotolerans , likely due to the presence of RpfB and other growth-promoting factors (e.g., polysaccharides, amino acids). In contrast, the low-molecular-weight fraction inhibited the growth of S. edaphicus , which may be attributed to the presence of antibiotics (e.g., vancomycin) or toxins secreted by N. antarcticus [35]. The resistance of B. paraconglomeratum and P. psychrotolerans to these inhibitors suggests they have evolved adaptive mechanisms to coexist with N. antarcticus in the polar environment. Treatment with the high-molecular-weight fraction increased soil bacterial diversity and richness, while the low-molecular-weight fraction had the opposite effect. This is likely because the high-molecular-weight fraction contains RpfB and other functional molecules that resuscitate VBNC cells and promote bacterial growth, leading to the expansion of the bacterial community. In contrast, the low-molecular-weight fraction contains inhibitory compounds that reduce the abundance of sensitive taxa (e.g., Bacteroidota , Cyanobacteria ). The increased relative abundances of Acidobacteriota , Actinobacteriota , and Gemmatimonadota in both fractions may be due to their resistance to inhibitory compounds and ability to utilize nutrients released by other bacteria. Conclusion This study is the first to report the resuscitative and growth-promoting effects of the culture supernatant from a polar actinomycete ( N. antarcticus ) on VBNC and normal bacterial cells in Antarctic soil. The results demonstrate that the supernatant, particularly its high-molecular-weight fraction containing RpfB, can resuscitate VBNC bacteria, promote bacterial growth, and enhance soil bacterial diversity. These findings provide a novel approach for isolating novel microbial taxa from extreme environments and highlight the potential of polar actinomycetes and their secreted factors for biotechnological applications. Future studies should focus on identifying additional growth-promoting/inhibitory factors in the supernatant, investigating the molecular mechanism of RpfB-mediated resuscitation, and exploring the application of N. antarcticus culture supernatant in environmental remediation and microbial resource exploitation. Declarations Acknowledgements We sincerely thank the China Center for Type Culture Collection (CCTCC) for providing the *N. antarcticus* strain and Antarctic soil samples. Author Contributions Chol Song Kim and Jin Hyok Kim designed the study, analyzed the data, and wrote the manuscript. Chan Hyon Han, Tong Ryul Kim, Hye Gyong Mun, Ju Hyok Kim, Il Hyang Pang, and Ju Hyang Hyon performed the experiments and collected data. All authors reviewed and approved the final manuscript. Funding The authors declare no funding sources. Competing Interests The authors declare no competing interests. References Dong K, Pan H, Yang D, et al. Induction, detection, formation, and resuscitation of viable but non-culturable state microorganisms. Compr Rev Food Sci Food Saf. 2020;19:149–83. Rozema J, Boelen P, Blokker P, et al. Depletion of stratospheric ozone over the Antarctic and Arctic: responses of plants of polar terrestrial ecosystems to enhanced UV-B, an overview. Environ Pollut. 2005;137:428–42. Elnitsky MA, Benoit JB, Denlinger DL, et al. Desiccation tolerance and drought acclimation in the Antarctic collembolan Cryptopygus antarcticus . 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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-8668372","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598308786,"identity":"e2608d95-21ec-433f-8ebd-779454c27887","order_by":0,"name":"Chol Song Kim","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Chol","middleName":"Song","lastName":"Kim","suffix":""},{"id":598308787,"identity":"76f78a4d-8ff6-4088-aafa-d2e3470fda82","order_by":1,"name":"Jin Hyok Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACNvnDBx9+qPhX38be2PggoaKGsBY+CbZkY4kzBxj7eQ4fNnhw5hhhLXISPGoSvG0HGGfOcEuTfNjCTITDpHsYJCTb7jAb3OAxq0hsYGPgb+9OwK9F5uwBg4Jzz9gMbveY3UjcIcMgcebsBvxaGPISEiTKmHkM7pwBajnDxmAgkUtIS47BAR42ZgmDGzlmBYltzERokcgxbOBpO2wgOSMtjYE4LTzHkpklzqQl8AMDWSLhzDEegn6Rb28+/vNDhU0CGzAqP/6oqJHjb+/FrwUD8JCmfBSMglEwCkYBVgAAsRpN6dr3PisAAAAASUVORK5CYII=","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":true,"prefix":"","firstName":"Jin","middleName":"Hyok","lastName":"Kim","suffix":""},{"id":598308788,"identity":"d7347bf0-4f19-4660-8868-134cf3a1e015","order_by":2,"name":"Chan Hyon Han","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Chan","middleName":"Hyon","lastName":"Han","suffix":""},{"id":598308789,"identity":"7b4aa4f9-dfb2-420a-89fb-1700d76bd34f","order_by":3,"name":"Tong Ryul Kim","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"Ryul","lastName":"Kim","suffix":""},{"id":598308790,"identity":"dce6dffe-9347-48b5-927c-5346303cf236","order_by":4,"name":"Hye Gyong Mun","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Hye","middleName":"Gyong","lastName":"Mun","suffix":""},{"id":598308791,"identity":"19a5de0d-62b3-41f5-93a1-f106e3c724e2","order_by":5,"name":"Ju Hyok Kim","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Ju","middleName":"Hyok","lastName":"Kim","suffix":""},{"id":598308792,"identity":"65a1dabb-8c4d-4ab6-86de-bc19eddabb06","order_by":6,"name":"Il Hyang Pang","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Il","middleName":"Hyang","lastName":"Pang","suffix":""},{"id":598308793,"identity":"a1af780f-0b5c-46c0-a208-d9cfb4ef407b","order_by":7,"name":"Ju Hyang Hyon","email":"","orcid":"","institution":"Kim Il-sung University","correspondingAuthor":false,"prefix":"","firstName":"Ju","middleName":"Hyang","lastName":"Hyon","suffix":""}],"badges":[],"createdAt":"2026-01-22 10:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8668372/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8668372/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105305326,"identity":"71d568c6-68db-4330-a26d-a49f70c07914","added_by":"auto","created_at":"2026-03-24 14:29:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120178,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eN. antarcticus\u003c/em\u003e culture supernatant fractions on the growth of \u003cem\u003eB. paraconglomeratum\u003c/em\u003e. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8668372/v1/776d44ebc231d98c854b93c9.png"},{"id":105305365,"identity":"07b0a606-13b9-4576-b4a1-a18bb373fdc4","added_by":"auto","created_at":"2026-03-24 14:29:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169261,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial diversity of Antarctic soil samples at the phylum (a) and genus (b) levels.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8668372/v1/2552e2fbb739dae121540c51.png"},{"id":105305404,"identity":"e0241862-b342-4c82-8bfc-76691878753c","added_by":"auto","created_at":"2026-03-24 14:29:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134143,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial community composition of Antarctic soil samples at the phylum level.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8668372/v1/fbb0d0795643b4d2d9059e20.png"},{"id":105305488,"identity":"5ee6f96f-520d-440e-b6c8-2b52d50f81e9","added_by":"auto","created_at":"2026-03-24 14:29:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8668372/v1/b989af6c-9f5b-48ae-a39f-21392e51dd3e.pdf"},{"id":105305320,"identity":"77269bf0-fed2-464d-b6cb-f5cff646ad9a","added_by":"auto","created_at":"2026-03-24 14:29:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":235349,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8668372/v1/8de4c40bacbaa5fc7ad3546b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Culture Supernatant of the Polar Actinomycete Nocardioides antarcticus CCTCC AB 2014053T Resuscitates VBNC Cells, Promotes Bacterial Growth, and Alters Bacterial Diversity in Antarctic Soil","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Antarctic and Arctic regions are characterized by extreme environmental conditions, including low temperatures, oligotrophy, intense solar radiation, aridity, and polar day-night cycles [1–5]. These stresses force most indigenous microorganisms into the viable but non-culturable (VBNC) state, a physiological condition where cells remain metabolically active but cannot be cultured on conventional growth media [1]. The VBNC state poses a major challenge to the isolation and exploitation of novel microbial resources from polar environments. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResuscitation-promoting factor (Rpf) is a secreted protein first identified in the actinomycete \u003cem\u003eMicrococcus luteus\u003c/em\u003e in 1998 [6]. It functions as a \"bacterial cytokine\" that stimulates the resuscitation of VBNC cells and promotes the growth of culturable bacteria [6, 28]. Rpf has been extensively studied for its applications in pathogen control [7], vaccine development [8, 14, 15], diagnostic testing [9, 13, 16], and environmental remediation [10–12, 19–25]. For example, Rpf can convert VBNC \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e to an active state, enhancing antibiotic sensitivity [13], and induce protective immune responses in mice when used as a subunit vaccine [14]. In environmental applications, Rpf has been used to resuscitate VBNC pathogenic bacteria in drinking water for detection [17], restore spoilage bacteria in beer [18], and enhance the biodegradation of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and phenol [11, 12, 22–25]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMost studies on Rpf have focused on \u003cem\u003eM. luteus\u003c/em\u003e and \u003cem\u003eM. tuberculosis\u003c/em\u003e, with applications limited to isolating pollutant-degrading VBNC bacteria from contaminated environments or detecting pathogens [6, 11, 12, 17, 22–26]. However, polar microorganisms possess unique metabolic pathways and survival strategies adapted to extreme conditions, making them valuable resources for biotechnological applications [29, 32]. To date, few studies have explored the effects of culture supernatants from Rpf-secreting polar microorganisms on VBNC cells and soil bacterial diversity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNocardioides antarcticus\u003c/em\u003e CCTCC AB 2014053T is a polar actinomycete identified to harbor the \u003cem\u003erpf\u003c/em\u003e gene through whole-genome analysis. In this study, we aimed to: (1) verify the presence of Rpf in the culture supernatant of \u003cem\u003eN. antarcticus\u003c/em\u003e; (2) evaluate the resuscitative effect of the culture supernatant on VBNC bacteria in Antarctic soil; (3) assess its growth-promoting activity on normal bacterial cells; and (4) investigate changes in soil bacterial diversity and community composition following treatment with the culture supernatant. \u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and Soil Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe polar actinomycete \u003cem\u003eNocardioides antarcticus\u003c/em\u003e CCTCC AB 2014053T was obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). Antarctic soil samples were collected from Asman Hill (69°22′685″S, 76°22′139″E) in the southwest highlands east of Zhongshan Station. Soil samples were stored at −80 °C until analysis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic DNA Extraction and rpf Gene Annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA of \u003cem\u003eN. antarcticus\u003c/em\u003e was extracted using a Qiagen Genomic DNA Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA purity, quantity, and integrity were assessed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), Qubit (Thermo Fisher Scientific), and 0.7% agarose gel electrophoresis, respectively. Whole-genome sequencing was performed on the PacBio Sequel platform [35]. The genome was annotated against the NR and UniProt databases to identify \u003cem\u003erpf\u003c/em\u003e homologs. The sequence similarity of the predicted Rpf protein with characterized Rpf proteins from other actinomycetes was analyzed using BLASTp. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and Purification of Recombinant RpfB Protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003erpfB\u003c/em\u003e gene of \u003cem\u003eN. antarcticus\u003c/em\u003e was amplified by PCR and cloned into the pET-28a(+) vector (Novagen, Darmstadt, Germany). The recombinant plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (Novagen), and kanamycin-resistant transformants were selected. Expression of the recombinant RpfB protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h. The protein was purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) and analyzed by 12% SDS-PAGE. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of \u003cem\u003eN. antarcticus\u003c/em\u003e Culture Supernatant Fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. antarcticus\u003c/em\u003e was inoculated into R2A liquid medium (Difco™, BD, Franklin Lakes, NJ, USA) and cultured at 20 °C with shaking (160 rpm) until the mid-exponential growth phase (36 h). The culture was centrifuged at 13,000 rpm for 5 min at room temperature, and the supernatant was filtered through a 0.22 μm Millex®-GP filter (Merck KGaA, Darmstadt, Germany) to obtain sterile supernatant. The supernatant was fractionated into high-molecular-weight (\u0026gt;10 kDa) and low-molecular-weight (\u0026lt;10 kDa) fractions using a 10 kDa ultracentrifugal filter (Merck KGaA). The protein and polysaccharide contents of the crude supernatant, high-molecular-weight fraction, and low-molecular-weight fraction were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and Phenol-Sulfuric Acid Assay, respectively. All fractions were stored at −20 °C until use. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the Limit Dilution of Soil Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 10⁻¹ soil suspension was prepared by adding 1 g of soil to 9 mL of sterile R2A medium containing 5–7 glass beads (6 mm diameter) and shaking at 160 rpm for 4 h at 20 °C. Serial dilutions (10⁻² to 10⁻¹²) were prepared by transferring 1 mL of the previous dilution to 9 mL of sterile R2A medium. Each dilution was incubated at 20 °C with shaking for 5–7 days, and the optical density at 600 nm (OD₆₀₀) was measured using a BioTex microplate reader (BioTex, Winooski, VT, USA). The limit dilution was defined as the highest dilution at which no bacterial growth was observed (OD₆₀₀ ≈ 0.045, consistent with the control). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResuscitation of VBNC Bacteria from Antarctic Soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 1 mL of \u003cem\u003eN. antarcticus\u003c/em\u003e culture supernatant and 4 mL of the limit dilution (10⁻¹⁰) soil suspension was incubated at 10 °C for 30 days. When bacterial growth was visually observed, serial dilutions were prepared and spread on R2A agar plates. Plates were incubated at 20 °C until single colonies appeared. Isolates were identified by 16S rRNA gene sequencing using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Sequences were compared to the NCBI GenBank database using BLASTn, and similarity values ≥97% were considered conspecific. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth-Promoting Activity Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated bacteria were inoculated (5% v/v) into fresh R2A liquid medium supplemented with different concentrations (1%, 5%, 10%, 15%, 20%, 25% v/v) of crude supernatant, high-molecular-weight fraction, or low-molecular-weight fraction. The control group contained no supernatant. Cultures were incubated at 20 °C for 30 days, and OD₆₀₀ values were measured at regular intervals. Growth promotion rates were calculated as [(OD₆₀₀ of treated group − OD₆₀₀ of control) / OD₆₀₀ of control] × 100%. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Soil Bacterial Diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 10⁻¹ soil suspension was prepared as described above. Aliquots (9 mL) of the suspension were mixed with 1 mL of crude supernatant, high-molecular-weight fraction, low-molecular-weight fraction, or sterile water (control), with three replicates per group. Mixtures were incubated at 0 °C for 7 days. Total genomic DNA of soil microorganisms was extracted using the DNeasy PowerSoil® Pro Kit (Qiagen) following the manufacturer’s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTABT-3′) with sample-specific barcodes [49]. PCR reactions were performed in 20 μL volumes containing 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu DNA Polymerase (TransGen Biotech, Beijing, China), and 10 ng of template DNA. The PCR program was: initial denaturation at 95 °C for 2 min; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min. Amplicons were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, Union City, CA, USA) and quantified using the QuantiFluor™ dsDNA System (Promega, Madison, WI, USA). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by Shanghai Biomedical Science and Technology Co., Ltd. (Shanghai, China). Raw reads were filtered to remove low-quality sequences, and operational taxonomic units (OTUs) were clustered at a 97% similarity cutoff using USEARCH UPARSE [50]. Alpha diversity indices (Shannon, Simpson, Sobs) were calculated using QIIME 2 (version 2022.2). Beta diversity was analyzed using principal coordinate analysis (PCoA) based on the Bray-Curtis distance matrix. Taxonomic classification was performed using the SILVA database (version 138). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in triplicate. Data were expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test using SPSS 22.0 (IBM, Armonk, NY, USA). P \u0026lt; 0.05 was considered statistically significant. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of the \u003cem\u003erpfB\u003c/em\u003e Gene in \u003cem\u003eN. antarcticus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome annotation revealed a \u003cem\u003erpfB\u003c/em\u003e gene (1215 bp) in \u003cem\u003eN. antarcticus\u003c/em\u003e CCTCC AB 2014053T, encoding a predicted RpfB protein. BLASTp analysis showed that the RpfB protein shared 36.3% sequence similarity with the characterized RpfB protein of \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e (Table 1). Recombinant RpfB protein was successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) and purified to homogeneity, appearing as a single band at ~47 kDa on SDS-PAGE (Fig. S1). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1 Annotation of the \u003cem\u003erpfB\u003c/em\u003e gene in \u003cem\u003eN. antarcticus\u003c/em\u003e CCTCC AB 2014053T\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganism and Strain Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNR Description\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwiss-Prot Description\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentities (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cem\u003eNocardioides antarcticus\u003c/em\u003e AB 2014053\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cem\u003erpfB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eresuscitation promoting factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003erpfB OS = \u003cem\u003eMycoli\u003c/em\u003e.\u003cem\u003e\u0026nbsp;smegmatis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e36.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003eNocardioides rubriscoriae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of \u003cem\u003eN. antarcticus\u003c/em\u003e Culture Supernatant Fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein and polysaccharide contents of the high-molecular-weight fraction were 5.6-fold and 4.4-fold higher than those of the crude supernatant, respectively. In contrast, the low-molecular-weight fraction contained only 50% of the protein and 10% of the polysaccharide present in the crude supernatant (Table S1). SDS-PAGE analysis showed multiple protein bands in the crude supernatant and high-molecular-weight fraction, including a band corresponding to the ~47 kDa RpfB protein. No protein bands were detected in the low-molecular-weight fraction (Fig. S2), confirming that RpfB is concentrated in the high-molecular-weight fraction. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimit Dilution of Antarctic Soil Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerial dilution experiments showed that bacterial growth (OD₆₀₀ \u0026gt; 0.045) was observed up to the 10⁻⁹ dilution, while no growth was detected at dilutions \u0026ge;10⁻\u0026sup1;⁰ (Table S2). Thus, the limit dilution of the soil samples was determined to be 10⁻\u0026sup1;⁰. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResuscitation of VBNC Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree bacterial strains were isolated from the 10⁻\u0026sup1;⁰ dilution soil suspension treated with \u003cem\u003eN. antarcticus\u003c/em\u003e culture supernatant: \u003cem\u003eBrachybacterium paraconglomeratum\u003c/em\u003e (97.75% 16S rRNA gene similarity), \u003cem\u003eStaphylococcus edaphicus\u003c/em\u003e (100% similarity), and \u003cem\u003ePseudarthrobacter psychrotolerans\u003c/em\u003e (98.98% similarity) (Table 2). In contrast, nine strains were isolated using conventional methods (10⁻\u0026sup3; to 10⁻⁵ dilutions), including \u003cem\u003eRhodococcus qingshengii\u003c/em\u003e, \u003cem\u003eAmoebinatus massiliensis\u003c/em\u003e, and \u003cem\u003eP. psychrotolerans\u003c/em\u003e (99.24% similarity) (Table 2). \u003cem\u003eB. paraconglomeratum\u003c/em\u003e and \u003cem\u003eS. edaphicus\u003c/em\u003e were only isolated from the supernatant-treated limit dilution, indicating they were in the VBNC state in the original soil. \u003cem\u003eP. psychrotolerans\u003c/em\u003e was isolated by both methods, suggesting it existed in both active and VBNC states. The low similarity values of \u003cem\u003eB. paraconglomeratum\u003c/em\u003e (97.75%) and \u003cem\u003eDevosia naphthalenivorans\u003c/em\u003e (98.19%, isolated by conventional methods) suggest they may represent novel taxa. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Bacterial strains isolated from Antarctic soil samples\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClassification\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eisolated bacterial species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003esimilarity\u003c/strong\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 28px;\"\u003e\n \u003cp\u003eSoil Extreme Dilution (10\u003csup\u003e-10\u003c/sup\u003e) + Culture Supernatants\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrachybacterium paraconglomeratum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e97.75*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus edaphicus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudarthrobacter psychrotolerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e98.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" style=\"width: 28px;\"\u003e\n \u003cp\u003eConventional strain isolation methods (10\u003csup\u003e-3\u003c/sup\u003e, 10\u003csup\u003e-4\u003c/sup\u003e and 10\u003csup\u003e-5\u003c/sup\u003e dilutions of soil samples)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhodococcus qingshengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eAmoebinatus massiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudarthrobacter psychrotolerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e99.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eKocuria rhizophila\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e99.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eNocardioides astragali\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e98.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizobacter fulvus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e99.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas asaccharolytica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e98.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003ePolaromonas ginsengisoli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cem\u003eDevosia naphthalenivorans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e98.19*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIndicates potential novel taxa (similarity \u0026lt; 98%). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth-Promoting and Inhibitory Effects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe crude supernatant and high-molecular-weight fraction significantly promoted the growth of \u003cem\u003eB. paraconglomeratum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. psychrotolerans\u003c/em\u003e (P \u0026lt; 0.05), with growth promotion rates ranging from 125.0% to 191.7% at the optimal concentration (10\u0026ndash;15%) (Figs. 1 and S3). In contrast, the low-molecular-weight fraction had no significant effect on these two strains but inhibited the growth of \u003cem\u003eS. edaphicus\u003c/em\u003e in a concentration-dependent manner (growth inhibition rates: 8.3\u0026ndash;47.1%) (Fig. S4). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in Soil Bacterial Diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing generated 5078\u0026ndash;7098 OTUs per sample (Table S3). The high-molecular-weight fraction treatment had the highest OTU number (7098), Shannon index (6.7648), and Sobs index (5186.3642), and the lowest Simpson index (0.0041), indicating the highest bacterial diversity and richness (Table S3). In contrast, the low-molecular-weight fraction treatment had lower OTU numbers (4863) and diversity indices than the control (Table S3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the phylum level, all supernatant treatments increased the number of detected phyla compared to the control, with the high-molecular-weight fraction treatment yielding the highest number (36 phyla) (Fig. 2a). At the genus level, the high-molecular-weight fraction treatment resulted in the classification of 782 genera, an 8.8% increase compared to the control (719 genera), while the low-molecular-weight fraction treatment reduced the number of genera to 654 (90.9% of the control) (Fig. 2b). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe bacterial community composition also changed significantly (Fig. 3). The relative abundances of \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eCyanobacteria\u003c/em\u003e, \u003cem\u003eBdellovibrionota\u003c/em\u003e, and \u003cem\u003eArmatimonadota\u003c/em\u003e were significantly lower in the low-molecular-weight fraction treatment (20.86%, 0.34%, 0.95%, 0.45%) than in the control (33.74%, 3.82%, 3.03%, 1.35%) (P \u0026lt; 0.05). In contrast, the relative abundances of \u003cem\u003eAcidobacteriota\u003c/em\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e, and \u003cem\u003eGemmatimonadota\u003c/em\u003e were higher in both the high- and low-molecular-weight fraction treatments compared to the control (P \u0026lt; 0.05). \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that the culture supernatant of the polar actinomycete \u003cem\u003eN. antarcticus\u003c/em\u003e CCTCC AB 2014053T, which contains the RpfB protein, can resuscitate VBNC bacteria, promote the growth of normal bacterial cells, and alter soil bacterial diversity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenome annotation and protein purification confirmed the presence of the rpfB gene in \u003cem\u003eN. antarcticus\u003c/em\u003e and the secretion of RpfB into the culture supernatant. The RpfB protein shared low sequence similarity (36.3%) with characterized Rpf proteins from other actinomycetes, suggesting it may represent a novel Rpf homolog with unique functions. The high-molecular-weight fraction of the supernatant contained higher levels of protein and polysaccharide, including RpfB, while the low-molecular-weight fraction lacked detectable proteins, consistent with the size of RpfB (~47 kDa). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree VBNC bacterial species—\u003cem\u003eB. paraconglomeratum\u003c/em\u003e, \u003cem\u003eS. edaphicus\u003c/em\u003e, and \u003cem\u003eP. psychrotolerans\u003c/em\u003e—were successfully resuscitated from the limit dilution (10⁻¹⁰) of Antarctic soil treated with the culture supernatant. This is consistent with previous studies showing that Rpf can resuscitate VBNC cells from various environments [6, 16, 26]. \u003cem\u003eP. psychrotolerans\u003c/em\u003e was isolated by both conventional and supernatant-treated methods, indicating it exists in both active and VBNC states, which may be an adaptive strategy for surviving extreme polar conditions. The potential novel taxa (\u003cem\u003eB. paraconglomeratum\u003c/em\u003e and \u003cem\u003eD. naphthalenivorans\u003c/em\u003e) isolated in this study highlight the utility of Rpf-containing supernatants for discovering new microbial resources. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe high-molecular-weight fraction of the supernatant significantly promoted the growth of \u003cem\u003eB. paraconglomeratum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. psychrotolerans\u003c/em\u003e, likely due to the presence of RpfB and other growth-promoting factors (e.g., polysaccharides, amino acids). In contrast, the low-molecular-weight fraction inhibited the growth of \u003cem\u003eS. edaphicus\u003c/em\u003e, which may be attributed to the presence of antibiotics (e.g., vancomycin) or toxins secreted by \u003cem\u003eN. antarcticus\u003c/em\u003e [35]. The resistance of \u003cem\u003eB. paraconglomeratum\u003c/em\u003e and \u003cem\u003eP. psychrotolerans\u003c/em\u003e to these inhibitors suggests they have evolved adaptive mechanisms to coexist with \u003cem\u003eN. antarcticus\u003c/em\u003e in the polar environment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreatment with the high-molecular-weight fraction increased soil bacterial diversity and richness, while the low-molecular-weight fraction had the opposite effect. This is likely because the high-molecular-weight fraction contains RpfB and other functional molecules that resuscitate VBNC cells and promote bacterial growth, leading to the expansion of the bacterial community. In contrast, the low-molecular-weight fraction contains inhibitory compounds that reduce the abundance of sensitive taxa (e.g., \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eCyanobacteria\u003c/em\u003e). The increased relative abundances of \u003cem\u003eAcidobacteriota\u003c/em\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e, and \u003cem\u003eGemmatimonadota\u003c/em\u003e in both fractions may be due to their resistance to inhibitory compounds and ability to utilize nutrients released by other bacteria. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study is the first to report the resuscitative and growth-promoting effects of the culture supernatant from a polar actinomycete (\u003cem\u003eN. antarcticus\u003c/em\u003e) on VBNC and normal bacterial cells in Antarctic soil. The results demonstrate that the supernatant, particularly its high-molecular-weight fraction containing RpfB, can resuscitate VBNC bacteria, promote bacterial growth, and enhance soil bacterial diversity. These findings provide a novel approach for isolating novel microbial taxa from extreme environments and highlight the potential of polar actinomycetes and their secreted factors for biotechnological applications. Future studies should focus on identifying additional growth-promoting/inhibitory factors in the supernatant, investigating the molecular mechanism of RpfB-mediated resuscitation, and exploring the application of \u003cem\u003eN. antarcticus\u003c/em\u003e culture supernatant in environmental remediation and microbial resource exploitation. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the China Center for Type Culture Collection (CCTCC) for providing the *N. antarcticus* strain and Antarctic soil samples. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChol Song Kim and Jin Hyok Kim designed the study, analyzed the data, and wrote the manuscript. Chan Hyon Han, Tong Ryul Kim, Hye Gyong Mun, Ju Hyok Kim, Il Hyang Pang, and Ju Hyang Hyon performed the experiments and collected data. All authors reviewed and approved the final manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no funding sources. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDong K, Pan H, Yang D, et al. Induction, detection, formation, and resuscitation of viable but non-culturable state microorganisms. Compr Rev Food Sci Food Saf. 2020;19:149\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eRozema J, Boelen P, Blokker P, et al. 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Front Microbiol. 2019;10:1067. https://doi.org/10.3389/fmicb.2019.01067. \u003c/li\u003e\n\u003cli\u003eMukamolova GV, Kaprelyants AS, Young DI, et al. A bacterial cytokine. Proc Natl Acad Sci U S A. 1998;95:8916\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eDhillon J, Lowrie DB, Mitchison DA. \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e from chronic murine infections that grows in liquid but not on solid medium. BMC Infect Dis. 2004;4:51. \u003c/li\u003e\n\u003cli\u003eRomano M, Aryan E, Korf H, et al. Potential of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines. Microbes Infect. 2012;14:86\u0026ndash;95. \u003c/li\u003e\n\u003cli\u003eArroyo L, Mar\u0026iacute;n D, Franken KLM, et al. 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UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10:996\u0026ndash;8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nocardioides antarcticus, Culture supernatant, Antarctic soil, Resuscitation-promoting factor (Rpf), Viable but non-culturable (VBNC) bacteria, Bacterial diversity ","lastPublishedDoi":"10.21203/rs.3.rs-8668372/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8668372/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Resuscitation-promoting factor (Rpf) is a secreted protein of some Gram-positive bacteria that exerts significant resuscitative effects on viable but non-culturable (VBNC) microorganisms. Culture supernatants of Rpf-secreting strains, like Rpf itself, can recruit VBNC bacteria and modify soil bacterial diversity. This study investigated the resuscitative and growth-promoting effects of the culture supernatant from the polar actinomycete Nocardioides antarcticus CCTCC AB 2014053T (harboring the rpf gene) on VBNC and normal bacterial cells in Antarctic soil, as well as its impact on soil bacterial diversity. Three VBNC bacterial species—Brachybacterium paraconglomeratum, Staphylococcus edaphicus, and Pseudarthrobacter psychrotolerans—were successfully isolated from 10⁻¹⁰-fold serially diluted Antarctic soil treated with the culture supernatant. The culture supernatant, particularly its high-molecular-weight fraction, significantly promoted the growth of B. paraconglomeratum and P. psychrotolerans (growth rates: 125.0–191.7% relative to the untreated control). In contrast, the low-molecular-weight fraction inhibited the growth of S. edaphicus (growth rates: 52.9–91.7% relative to the control). High-throughput sequencing revealed that treatment with the high-molecular-weight fraction resulted in the classification of 782 soil bacterial genera, an 8.8% increase compared to the control, while the low-molecular-weight fraction reduced the number of genera to 90.9% of the control. Additionally, the culture supernatant altered the soil bacterial community composition, with distinct differences between the high- and low-molecular-weight fractions. These findings demonstrate that the N. antarcticus culture supernatant, containing Rpf, holds potential for isolating novel bacterial taxa by resuscitating VBNC cells and promoting bacterial growth in extreme environments.","manuscriptTitle":"Culture Supernatant of the Polar Actinomycete Nocardioides antarcticus CCTCC AB 2014053T Resuscitates VBNC Cells, Promotes Bacterial Growth, and Alters Bacterial Diversity in Antarctic Soil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 14:27:57","doi":"10.21203/rs.3.rs-8668372/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"331596af-4755-4a16-8be1-16cce25a9a78","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T14:27:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 14:27:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8668372","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8668372","identity":"rs-8668372","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}