Polyphasic and Genomic Characterisation of Streptomyces zimensis sp. nov., a Halotolerant Actinomycetota from the Saline Lake Zima in Morocco

Polyphasic and Genomic Characterisation of Streptomyces zimensis sp. nov., a Halotolerant Actinomycetota from the Saline Lake Zima in Morocco | 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 Polyphasic and Genomic Characterisation of Streptomyces zimensis sp. nov., a Halotolerant Actinomycetota from the Saline Lake Zima in Morocco Ez-Zahra Oubassou, Soukaina Oudchaira, Valérie Cognat, Abdelmalek Alioua, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9413337/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Members of the phylum Actinomycetota are widely distributed across diverse environments and are well known for their metabolic versatility and capacity to produce bioactive compounds. In this study, strain ZE1316R2Aᵀ was isolated from the saline water collected from Lake Zima (Morocco) and subjected to comprehensive polyphasic taxonomic characterisation. Phylogenetic analysis based on the 16S rRNA gene placed strain ZE1316R2Aᵀ within the genus Streptomyces , showing highest sequence similarity with S. albidoflavus DSM 40455 T (99.71%). However, genome-based indices, including average nucleotide identity (ANIb = 94.84%, ANIm = 96.09%) and digital DNA-DNA hybridization (dDDH = 64.9%), supported its distinction as a separate species. The draft genome (7.41 Mb; G + C = 73.26 mol%) comprises 6,464 coding sequences and reveals the presence of strain-specific genomic regions and biosynthetic gene clusters. Comparative analyses highlighted both a conserved core genome and a substantial accessory genome component, reflecting genomic differentiation relative to closely related taxa. Phenotypic and chemotaxonomic characteristics were consistent with assignment to the genus Streptomyces, while supporting its differentiation at the species level. Based on the combined genomic, phenotypic, and chemotaxonomic evidence, strain ZE1316R2Aᵀ represents a novel species of the genus Streptomyces , for which the name Streptomyces zimensis sp. nov., is proposed. This study expands current knowledge of Streptomyces diversity associated with saline environments and highlights the genomic diversity present within closely related taxa. The type strain is ZE1316R2Aᵀ (= CCMM B1331 T = DSM 120541 T ). Comparative analysis Novel species Polyphasic taxonomy Streptomyces Whole-genome Zima Lake Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Extreme saline ecosystems such as salt lakes, salterns, and hypersaline soils represent unique and underexplored habitats characterised by strong physicochemical constraints, including high salinity, temperature fluctuations, and limited nutrient availability. Despite these harsh conditions, such environments harbor diverse and specialised microbial communities, notably within the phylum Actinomycetota (Van der Meij et al., 2017 ; Qin et al., 2019 ; Ngamcharungchit et al., 2023 ). Many Actinomycetota thriving in saline habitats exhibit halotolerant or halophilic lifestyles, enabling them to colonize salt lakes, hypersaline soils, and other hypersaline biotopes (Hozzein, 2015 ). Several genera of Actinomycetota have been reported from these environments, including Nocardiopsis , Actinopolyspora , Actinomadura, Micromonospora , among others (Menasria et al., 2022 ; Carreón-Gaxiola et al., 2025 ). Notably, species of the genus Streptomyces are frequently reported as dominant members of actinomycetal communities in saline and hypersaline ecosystems, owing to their remarkable physiological adaptability, genetic diversity, and metabolism versatility (Ballav et al., 2015 ; Zhao et al., 2016 ; Clavo et al., 2021 ; Gohel, Majithiya, & Singh, 2023 ). The genus Streptomyces , the type genus of the family Streptomycetaceae , represents the largest taxon within the phylum Actinomycetota (Gopalakrishnan et al., 2020 ). Members of this genus are filamentous, Gram-positive bacteria characterised by a complex life cycle involving the formation of vegetative and aerial mycelia and spore chains (Nazari et al., 2023 ). Although nearly 95% of described Streptomyces species have been isolated from soils, where they play a central ecological role as decomposers and nutrient recyclers (De Moura., 2021), representatives of the genus are also widespread in diverse environments, including marine habitats, extreme ecosystems and symbiotic associations with plants and animals (Komaki, 2023 ). To date, approximately 793 Streptomyces species have been validly published (accessed on 05 November 2025; https://lpsn.dsmz.de/search?word=streptomyces ), reflecting the extensive genetic variability and ecological plasticity of this genus. Streptomyces species are widely recognised as the most prolific producers of bioactive secondary metabolites in the bacterial kingdom, with numerous compounds exploited for medical, agricole, and industrial applications (El-Naggar, 2021 ; Alam et al., 2022 ). Their exceptional biosynthetic capacity is largely driven by the presence of numerous biosynthetic gene clusters (BGCs), which encode pathways for the production of antibiotics and other specialised metabolites (Du et al., 2024 ). This metabolic richness contributes to their ability to adapt to a broad range of ecological niches, including saline and hypersaline environments, and highlights their importance as a resource for natural-product discovery, particularly in the context of genomic-guided approaches. Recent advances in Streptomyces genomics have revealed extensive intra-generic diversity and have led to the reclassification of several taxa (Mispelaere et al., 2024 ; Kiepas et al., 2024 ). In Morocco, Streptomyces species have been isolated from different habitats, highlighting the country’s rich microbial diversity. Representative examples include S. marokkonensis from argan-tree rhizospheres (Bouizgarne et al., 2009 ), S. beta-vulgaris from sugar-beet rhizospheric soil (Lebrihi et al., 2009 ), S. thinghirensis from grapevine roots (Loqman et al., 2009 )d youssoufiensis from phosphate mines (Hamdali et al., 2011 ), as well as numerous other strains from agricultural, urban, and endophytic environments (Rammali et al., 2024a ; Rammali et al., 2024b ; Aallam et al., 2021 ; Buchmann et al., 2019 ; Oubaha et al., 2018 ; Katif et al., 2021 ). In contrast, Streptomyces from extreme or saline environments in Morocco remain poorly explored, despite the fact that such habitats often select for microorganisms with unique metabolic traits and adaptive genomic features. The climate and geochemical diversities of arid and semi-arid regions in Morocco provide a wide range of extreme environments, including salt flats, sebkhas, and evaporitic lakes that remain largely unexplored from a microbiological perspective. Among these, Lake Zima, a Ramsar-listed natural wetland located in the Bahira Plain of central Morocco, represents a hypersaline ecosystem shaped by intense evaporation, Triassic evaporite deposits (gypsum and halite), and groundwater inputs enriched in sodium, chloride, and sulfate ions (Karroum et al., 2017 ). The high salinity, seasonal variability, and limited organic input, characteristic of this environment imposes strong selective pressures that may favor the emergence of halophilic and halotolerant microorganisms with distinctive metabolic capacities, including the potential to produce bioactive secondary metabolites. In this context, the present study focuses on strain ZE1316R2Aᵀ, isolated from saline water collected in Lake Zima, Morocco, during an investigation of the biodiversity and biotechnology potential of Actinomycetota inhabiting extreme saline ecosystems. A polyphasic approach combining phylogenomic, phenotypic, and chemotaxonomic analyses was employed to determine its taxonomic position relative to closely related members of the genus Streptomyces . Whole-genome sequencing and comparative analyses revealed that strain ZE1316R2Aᵀ represents a novel species, for which the name Streptomyces zimensis sp. nov., is proposed. Materials and methods Sampling, isolation and culture conditions Saline water samples were collected, under aseptic conditions, from Lake Zima (3,500 km 2 ; 35 km north of Marrakesh; 32°4'48" N, 8°39'36" W), a Ramsar-listed saline wetland in central Morocco. Samples were kept at 4°C until use. Filtration was performed with a sterile filtration cell under reduced pressure, using a 0.45 µm nitrocellulose membrane to retain bacteria. The membranes were then placed on R2A medium supplemented with 3.5% (w/v) NaCl to reflect the lake's natural salinity (Liu et al., 2019 ), as well as cycloheximide (40 µg. mL − 1 ) and nalidixic acid (20 µg. mL − 1 ) to inhibit respectively fungal and Gram-negative bacterial growth (Barakate et al., 2002 ). Incubation occurred at 30°C for 7–10 days, with daily monitoring for colony formation. Colonies exhibiting typical Actinomycetota morphology were selected and subcultured on ISP2 medium to ensure purity. Spore suspensions of purified isolates were preserved at -80°C in sterile 25% (v/v) glycerol. Genomic DNA extraction and 16S rRNA gene sequencing For DNA extraction, strain ZE1316R2Aᵀ was cultivated for 5 days at 30°C in ISP2 medium (4 g.L − 1 Yeast extract; 10 g.L − 1 Malt extract; 4 g.L − 1 Glucose and 20 g.L − 1 Agar) under agitation. Bacterial biomass was collected from 2 mL of culture by centrifugation (16,000 × g for 2 min) and bacterial genomic DNA was extracted using the Genomic-tip 100/G kit (Qiagen, Cat. No. 10243) following the manufacturer’s recommendations. DNA was quantified using the Qubit™ dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific), and purity was verified through A260/280 and A260/230 absorbance measures using the NanoDrop spectrophotometer 2000. The nearly full-length 16S rRNA gene was amplified with Phusion High-Fidelity DNA Polymerase using the universal bacterial primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’ ) (Frank et al., 2008 ). PCR amplification yielded a product of approximately 1.5 kb, as verified by electrophoresis on a 1% (w/v) agarose gel. The corresponding DNA band was excised and purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL). The purified amplicon was subsequently sequenced by the Sanger method using primers 8F and 1492R. The final assembled sequence had a length of 1,417 bp. The 16S rRNA gene sequence of Streptomyces sp. ZE1316R2A (1,417 bp) has been deposited in NCBI GenBank under accession number PZ007821. Whole-genome sequencing, data quality assessment, and genome assembly Whole-genome sequencing was performed using the same genomic DNA preparation reported in the previous section. Libraries were prepared with the Native Barcoding Kit 24 V14 (SQK-NBD114.24, Oxford Nanopore Technologies) using ~ 1 µg of DNA, and sequencing was performed on R10.4.1 PromethION flow cells (FLO-PRO114M) operated with MinKNOW v22.07.9. Raw signal data (fast5 files) were basecalled and demultiplexed with Guppy v6.5.7 (Wick et al., 2019 ) using the super-accuracy model (dna_r10.4.1_e8.2_400bps_hac_prom) and a minimum Q-score cutoff of 7. Read quality was assessed with NanoPlot v0.32.1 (De Coster and Rademakers, 2023 ), and adapter sequences were removed with Porechop v0.2.4 ( https://github.com/rrwick/Porechop ). To screen for contamination, reads were classified against the standard Kraken database using Kraken2 v2.0.9-beta (Wood et al., 2019 ). De novo genome assembly was carried out with Flye v2.9.3-b1797 (Kolmogorov et al., 2019 ), followed by consensus polishing with NextPolish v1.4.1 (Hu et al., 2020 ). Assembly quality and completeness were assessed with QUAST v5.0.2 (Gurevich et al., 2013 ), BUSCO v5.4.3 using the actinobacteria_odb10 lineage dataset, and CheckM v1.2.2 (Parks et al., 2015 ). Phylogenomic analysis The similarity of the 16S rRNA gene sequence of strain ZE1316R2Aᵀ and identification of its closest phylogenetic relatives were evaluated using the EzBioCloud database ( https://www.ezbiocloud.net/ ; accessed September 24, 2025). Accession numbers of 16S rRNA gene sequences from phylogenetically related type and reference strains retrieved from EzBioCloud are provided in Supplementary Table S1 (Chalita et al., 2024 ). Representative Streptomyces 16S rRNA gene sequences were aligned using MAFFT implemented in the NGPhylogeny.fr online platform advanced workflow (Lemoine et al., 2019 ). A maximum-likelihood phylogenetic tree was constructed using the PhyML/OneClick module, with Kitasatospora setae NBRC 14216ᵀ as the outgroup. Branch support was assessed using 1,000 bootstrap replicates, and the resulting tree was visualised using iTOL v7.4.2 (Letunic & Bork, 2024 ). To further confirm the taxonomic placement of Streptomyces sp. ZE1316R2Aᵀ, a whole-genome phylogenetic tree was reconstructed based on a Genome BLAST Distance Phylogeny (GBDP) distance matrix using the Type (Strain) Genome Server (TYGS; https://tygs.dsmz.de ) (Meier-Kolthoff and Göker, 2019 ). Kitasatospora setae NBRC 14216ᵀ was used as an outgroup to root the phylogenetic tree. Accession numbers of the whole-genome assemblies of reference strains included in the analysis are listed in Supplementary Table S2 . The resulting phylogenomic tree was visualised using iTOL version 7.4.2 (Letunic and Bork, 2024 ). Species delineation of ZE1316R2Aᵀ was assessed using genomic similarity metrics. The Average Nucleotide Identity (ANI) values, ANI-BLAST (ANIb) and ANI-MUMmer ultra-rapid aligning tool (ANIm) of the whole genome of strain ZE1316R2Aᵀ with closely related type strain DSM 40455 T were calculated using the JSpeciesWS web server v5.0.2 (Richter et al., 2015 ), and digital DNA-DNA hybridization (dDDH) was calculated with the Genome-to-Genome Distance Calculator (GGDC2.1) with the BLAST+ algorithm, applying Formula 2 (d4) as implemented in the TYGS platform. (Meier-Kolthoff et al., 2021 ). Heat maps were generated to visualize ANI and dDDH-based relationships between strain ZE1316R2A T and multiple closely related Streptomyces species. Whole-genome comparative analyses Whole-genome comparative analyses were performed between strain ZE1316R2Aᵀ and S. albidoflavus DSM 40455 T using the assembled genome sequences deposited under the accession numbers listed in Supplementary Table S2 . Genomic synteny was performed using the D-GENIES web tool (Cabanettes and Klopp, 2018 ) with default parameters and minimap2-based alignments, enabling visualisation of conserved regions and structural rearrangements. Whole-genome pairwise comparisons and alignment visualisations were additionally performed using the Proksee platform (Grant et al., 2023 ). Regions of genomic plasticity (RGPs) were identified using RGP_Finder implemented in the MicroScope platform (Vallenet et al., 2019 ), and secondary metabolite biosynthetic gene clusters (BGCs) were predicted using antiSMASH v7.1.0.1 with default settings. Pan-genome analyses were conducted by computing gene families (MICFAM) using SiLiX with an amino acid identity threshold of 80% and 80% alignment coverage, allowing discrimination of core, variable, and strain-specific genes. Functional annotation and comparative analyses were performed using Clusters of Orthologous Groups (COG) classifications and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways assignments through the MicroScope platform (MicroScope v3.18.1). Carbohydrate-active enzyme (CAZyme) genes were annotated using the dbCAN3 web server (Zheng et al., 2023 ). Predictions were retained when supported by at least two of the three integrated tools (HMMER v3.4, DIAMOND v2.1.21, and dbCAN_sub), ensuring robust identification of CAZyme families. Phenotypic, cultural, and chemotaxonomic characteristics Seven-day-old cultures of Streptomyces sp. ZE1316R2Aᵀ were grown on ISP2 agar at 30°C for 14 days using the inclined coverslip technique as described by Williams and Cross (1971) to allow proper development of aerial and substrate mycelia. The resulting cultures were subsequently analysed by scanning electron microscopy (Quanta FEG 450) following the methodology of Kurtböke ( 2022 ). Cultural characteristics were assessed by cultivating ZE1316R2Aᵀ and its closest relative, S. albidoflavus DSM 40455 T (obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany), on International Streptomyces Project (ISP) media for 14 days at 30°C. Tested media included ISP1 (Tryptone-Yeast Extract), ISP2 (Yeast Extract-Malt Extract Agar), ISP3 (Oatmeal Agar), ISP4 (Inorganic Salts-Starch Agar), ISP5 (Glycerol-Asparagine Agar), ISP6 (Peptone-yeast extract Agar), and ISP7 (Tyrosine Agar) (Shirling and Gottlieb, 1966 ). The growth and the coloration of aerial and substrate mycelia, as well as the production of diffusible pigments, were recorded using the ISCC–NBS color charts, as previously used in the International Streptomyces Project (Shirling and Gottlieb, 1966 ). Tests evaluating growth range of strain ZE1316R2Aᵀ under variable conditions of NaCl concentration, temperature, and pH were conducted using Mannitol Soya Flour agar medium (20 g Mannitol, 20 g Soya Flour, 20 g Agar and 1 L tap water). These tests evaluated NaCl concentration tolerance (0–20%; w/v) in 5% increments, temperature tolerance at 5, 16, 28, 30, 37, and 50°C, and pH range (5.5 to 8.5), in increments of 1 unit. In addition, utilisation of carbon sources was evaluated using a standardised carbohydrate assimilation assay under aerobic conditions. The carbon utilisation profile of strain ZE1316R2Aᵀ was determined and compared with that of S. albidoflavus DSM 40455ᵀ following incubation under identical conditions. All tests were performed in triplicate, and growth was visually evaluated over a 14-day period of incubation at 30°C under aerobic conditions. For the chemotaxonomic analyses, the biomass of ZE1316R2Aᵀ and its closest relative S. albidoflavus DSM 40455 T , was obtained from a 7-day culture in ISP2 broth at 30°C and 250 rpm on a rotary shaker. Cells were harvested by centrifugation, washed three times with sterile distilled water, and preserved in isopropanol. Chemotaxonomic analyses of respiratory quinones, polar lipids, and cellular fatty acids (FAME), commonly used as taxonomic markers in Actinomycetota (Ramasamy & Sudalaimuthu, 2022 ), were performed by DSMZ Services (Leibniz-Institut DSMZ, Braunschweig, Germany) following standard protocols. Results Isolation and 16S rRNA gene analysis of strain ZE1316R2Aᵀ During our investigations of microbial diversity and valorisation in extreme Moroccan saline ecosystems, Actinomycetota were specifically targeted in saline water samples collected from multiple points within Lake Zima, Morocco, as this phylum is well known to include salt-tolerant members and prolific producers of secondary metabolites (Alam et al., 2022 ). More than 100 actinomycetal colonies displaying diverse morphologies were recovered on selective R2A medium supplemented with 3.5% (w/v) NaCl. Among these isolates, strain ZE1316R2Aᵀ was selected for further study as it represented a dominant and recurrent morphotype recovered across sampling points and exhibited stable and reproducible growth under saline conditions. These features made ZE1316R2Aᵀ a suitable representative strain for detailed taxonomic, genomic, and phenotypic characterisation. The nearly full-length 16S rRNA gene (1,417 bp) was sequenced and queried against the EzBioCloud database, which indicated that strain ZE1316R2Aᵀ belongs to the genus Streptomyces within the family Streptomycetaceae . The closest relatives were S. albidoflavus DSM 40455 T (99.71%), S. daghestanicus NRRL B-5418 T (99.64%), and S. violascens ISP 5183 T (99.63%), S. koyangensis VK-A60 T (99.28%), S. cadmiisoli ZFG47 T , while identities with sequence similarities with other phylogenetically related Streptomyces species ranged from 98.92% to 98.28%. The maximum-likelihood phylogenetic tree based on 16S rRNA sequences positioned ZE1316R2Aᵀ within the Streptomyces clade and in close proximity to S. albidoflavus DSM 40455 T , while forming a slightly earlier-branching lineage within the same cluster (Fig. 1 ; accession lists in Table S1 ). This topology supports the affiliation of strain ZE1316R2Aᵀ with the family Streptomycetaceae and reveals a distinct phylogenetic positioning relative to its closest relatives. Beyond its phylogenetic placement, the assignment of strain ZE1316R2Aᵀ to the genus Streptomyces highlighted its potential biotechnological relevance and provided the rationale for further genomic and phenotypic analyses. Genome features and assembly quality The genus Streptomyces displays extensive genetic diversity, making difficult its taxonomic delineation when based solely on 16S rRNA gene sequences, which lack the resolution required to distinguish between closely related species (Kiepas et al., 2024 ; Zakalyukina et al., 2024 ). Therefore, whole-genome sequencing was performed to clarify the taxonomic position of strain ZE1316R2Aᵀ. Sequencing produced 3 contigs, with a total length of 7,407,648 bp. The three contigs measured 370,759 bp, 6,87,3245 bp, and 163,444 bp in length, with average coverages of 169×, 126×, and 124×, respectively. The assembled genome displayed a G + C content of 73.2 mol%, with no detectable contamination and an estimated completeness of 99.89%. To better visualize the chromosomal organisation, the three contigs were aligned against the nearest reference genome of S. albidoflavus DSM 40455ᵀ, allowing their relative positioning along the chromosome and revealing extensive synteny and structural conservation. No additional small contigs or plasmid-like elements were detected, which is consistent with these contigs representing segments of a single linear chromosome typical of the genus. A total of 6,464 protein-coding DNA sequences (CDS), 7 rRNA operons (16S-23S-5S), and 66 tRNA genes were predicted, representing 87.9% of the genome with an average CDS length of 1,018 bp. These assembly and annotation metrics are summarised in Table 1. A linear representation of the 7.4 Mb chromosome of Streptomyces sp. ZE1316R2Aᵀ is shown in Fig. 2 A, illustrating the distribution of coding sequences, rRNA and tRNA genes, GC content, and GC skew. The genome architecture displays the typical large linear organisation characteristic of Streptomyces species, with balanced GC skew and well-defined replication origin and terminus regions. Phylogenomic resolution and species delineation The taxonomic position of Streptomyces sp. ZE1316R2Aᵀ was further evaluated using a whole genome-based phylogenomic approach. The whole-genome tree generated by TYGS, based on the Genome BLAST Distance Phylogeny (GBDP) method, placed strain ZE1316R2Aᵀ within the Streptomyces cluster in a distinct lineage most closely related to S. albidoflavus DSM 40455 T (Fig. 3 ). Notably, strain ZE1316R2Aᵀ branches slightly earlier than S. albidoflavus DSM 40455 T within the clade that also contains S. sampsonii NBRC 13083ᵀ and S. limosus NBRC 12790ᵀ, indicating an independent phylogenomic trajectory consistent with species-level differentiation. This topology was consistent with that obtained from 16S rRNA gene analysis, confirming the close but distinct evolutionary relationship between strain ZE1316R2Aᵀ and S. albidoflavus DSM 40455 T . Pairwise genomic similarity metrics supported the separation of strain ZE1316R2Aᵀ from its closest Streptomyces relatives (Fig. 4 ). The average nucleotide identity values were 94.84% (ANIb) and 96.09% (ANIm), both below the commonly accepted species boundary thresholds of 95–96% and 96,70%, respectively (Richter & Rosselló-Móra, 2009 ; Hu et al., 2022 ). The digital DDH value was 64.9% (confidence interval 61.9–67.7%), also below the 70% delineation threshold. These results confirm that strain ZE1316R2Aᵀ represents a genomically distinct lineage within the genus Streptomyces , and support its recognition as a separate species. Comparative genomics and genome architecture To investigate genomic conservation and structural variation, whole-genome comparisons were performed between Streptomyces sp. ZE1316R2Aᵀ and its closest phylogenetic relative, S. albidoflavus DSM 40455 T . Genome-wide alignment revealed extensive sequence conservation across large chromosomal segments (Fig. 5 A). This high degree of conservation is consistent with their close phylogenomic relationship. Dot-plot analysis further confirmed strong global collinearity between the two chromosomes, as evidenced by the prominent main diagonal (Fig. 5 B). A large chromosomal inversion was observed, together with several local discontinuities and off-diagonal signals reflecting smaller-scale rearrangements and non-syntenic regions. These features illustrate structural diversification between the two genomes while maintaining an overall conserved genomic backbone. A comparative analysis of the pan-, core-, and strain-specific genomes was then conducted to examine the genomic divergence between the two Actinomycetota (Table 2). The pangenome analysis identified 6,464 orthologous gene families. Of these, 5,123 gene families formed the core genome shared by both strains, representing 79.25% of the total coding sequences (CDSs) in strain ZE1316R2Aᵀ and 82.06% in strain DSM 40455 T . In contrast, 1,341 CDSs were specific to strain ZE1316R2Aᵀ and 1,117 CDSs were unique to strain DSM 40455ᵀ, reflecting a substantial accessory genome component contributing to strain-level differentiation. In strain ZE1316R2Aᵀ, 29 distinct Regions of Genomic Plasticity (RGPs) were identified and mapped along the chromosome (Fig. 2 B). These RGPs largely overlapped with strain-specific regions (yellow segments in Fig. 2 B) and were mainly distributed in terminal and subterminal chromosomal compartments. When visualised together with GC content and GC skew profiles (Fig. 2 B), many RGPs coincided with local deviations in base composition and polarity shifts, further supporting their origin as variable genomic compartments. Functional annotation revealed that these RGPs encode diverse adaptive functions, including hydrolases, dioxygenases, aldolases, regulatory STAND NTPases, and trypsin-type proteases. Consistently, strain ZE1316R2Aᵀ harbors a higher number of predicted transposase-encoding genes (51 copies) compared with strain DSM 40455 T (21 copies), suggesting enhanced genomic mobility and recombination potential. Together, these structural and genetic variations delineate strain-specific genomic regions that contribute to the overall divergence between the two genomes. Functional and metabolic potential The biosynthetic potential of Streptomyces sp. ZE1316R2Aᵀ was investigated by genome mining for secondary metabolite biosynthetic gene clusters (BGCs). A total of 20 BGCs were identified in the genome, including two clusters absent in S. albidoflavus DSM 40455 T (Tables S3-S6). One of these ZE1316R2Aᵀ-specific clusters showed no significant similarity to any entry in the MIBiG reference database, suggesting the presence of a potentially novel biosynthetic pathway. Comparative functional annotation based on KEGG pathway mapping revealed broadly similar metabolic profiles between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ (Figure S1 ). In both genomes, genes were predominantly assigned to carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, energy metabolism, and secondary metabolite biosynthesis categories. A focused comparison of lipid metabolic pathways showed that both strains encode the full complement of enzymes required for fatty acid biosynthesis and degradation, together with those involved in glycerophospholipid and glycerolipid metabolisms, indicating a conserved lipid metabolic framework (Table S7). In addition, strain ZE1316R2Aᵀ encoded accessory reactions not identified in strain DSM 40455ᵀ, including genes encoding enzymes involved in the conversion of farnesyl-diphosphate to presqualene-diphosphate and squalene (annotated in KEGG under “steroid biosynthesis”). MicroCyc annotation further revealed partial enzymatic steps associated with the conversion of acetone to acetoacetate and subsequently to acetyl-CoA in strain ZE1316R2Aᵀ, which were absent or incomplete in strain DSM 40455ᵀ. To further investigate the relationship between genomic plasticity and functional specialisation, the 29 previously identified RGPs were cross-referenced with KEGG pathway annotations (Fig. 5 C). This analysis showed that several RGPs contained genes assigned to diverse metabolic categories. Among them, RGP2 displayed the highest number and diversity of KEGG-annotated genes, including genes associated with secondary metabolite biosynthesis (terpenoid and polyketide), amino acid and carbohydrate metabolism, cofactor and vitamin metabolism, energy metabolism, and xenobiotic degradation. Other RGPs contained genes associated with narrower range of metabolic functions. Carbohydrate-active enzyme (CAZymes) analysis revealed that both strains encode diverse repertoires of glycoside hydrolases (GH), glycosyltransferases (GT), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), polysaccharide lyases (PL), and auxiliary activities (AA). Although strain DSM 40455 T exhibited a broader diversity of CAZyme families, particularly in the AA, CBM, GE, GT and PL classes, strain ZE1316R2Aᵀ displayed slight enrichment in several GH families (Table S8). Taken together, these results demonstrate that strain ZE1316R2Aᵀ and strain DSM 40455ᵀ maintain a shared core metabolic architecture but exhibit measurable differences in biosynthetic capacity, lipid-related reactions, genomic plasticity-associated functions, and carbohydrate-active enzyme repertoires. Phenotypic and cultural characteristics To complete the taxonomic characterisation of strain ZE1316R2Aᵀ, its morphological, cultural, and physiological properties were examined and compared with those of S. albidoflavus DSM 40455ᵀ. On Mannitol Soy Flour (MSF) agar after 14 days of incubation at 30°C, both strains produced creamy-white, folded colonies typical of the genus Streptomyces (Fig. 6 B). However, strain ZE1316R2Aᵀ formed smaller and more compact colonies than strain DSM 40455ᵀ. Scanning electron microscopy (SEM) of strain ZE1316R2Aᵀ grown on ISP2 agar at 30°C for 14 days revealed a well-developed, branched aerial mycelia forming long chains of smooth-surfaced, globose spores (Fig. 6 A). When cultivated on the International Streptomyces Project (ISP) media, strain ZE1316R2Aᵀ exhibited growth ranging from poor to good after 14 days of incubation at 30°C (Table 3). The strain produced both substrate and aerial mycelia, with coloration varying depending on the medium. On ISP6, strain ZE1316R2Aᵀ displayed a distinct orange pigmentation. Overall, its growth profile was comparable to that of S. albidoflavus DSM 40455ᵀ, although the latter generally formed more abundant aerial mycelia on certain media ( e.g . ISP3 and ISP7). Representative colony morphologies on ISP1-ISP7 media are shown in Figure S2 . Growth responses under varying salinity, temperature, and pH conditions are summarised in Table S9 and illustrated in Figures S3-S5. Both strains ZE1316R2Aᵀ and DSM 40455ᵀ exhibited similar growth characteristics across all tested conditions. Optimal growth occurred in the absence of NaCl (0%; w/v), and both strains tolerated NaCl concentration up to 5% (w/v). Growth was observed between 16 and 37°C. Both strains grew over a pH range of 5.5 and 8.5, with optimal growth within this interval (Figures S3-S5). Carbohydrate utilisation patterns revealed phenotypic differences between strain ZE1316R2Aᵀ and S. albidoflavus DSM 40455ᵀ.Strain ZE1316R2Aᵀ utilised D-mannose but not the other tested carbohydrates, clearly distinguishing it from S. albidoflavus DSM 40455ᵀ (Table S10). Chemotaxonomic characteristics Chemotaxonomic analyses were performed to determine the polar lipid, menaquinone, and cellular fatty acid composition of Streptomyces sp. ZE1316R2Aᵀ and its closest relative S. albidoflavus DSM 40455ᵀ (Table 4). Two-dimensional thin-layer chromatography (TLC) revealed broadly similar polar lipid profiles in both strains (Figure S6). The major detected lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Additional components, including phospholipids (PL), aminophospholipids (APL), glycolipids (GL), glycophospholipids (GPL), and unidentified lipids (L), were also detected (Table 4). The predominant menaquinones in strain ZE1316R2Aᵀ were MK-9(H 2 ) (15.2%), MK-9(H 4 ) (31%) and MK-9(H 6 ) (33%). A similar menaquinone profile was detected in strain DSM 40455ᵀ, although the relative proportions differed (Table 4). Fatty acid methyl ester (FAME) analysis of strain ZE1316R2Aᵀ revealed that the dominant components (> 10%) were anteiso-C 15:0 (23.3%), iso-C 16:0 (22.0%), and anteiso-C 17:0 (18.4%; Table 4). In DSM 40455 T , the same fatty acids predominated, with slightly higher proportions of iso-C 16:0 (27.7%) and lower proportions of anteiso-C 15:0 (17.6%) and anteiso-C 17:0 (15.6%). Polyphasic characterisation and taxonomic conclusion The combined genomic, phenotypic, and chemotaxonomic data support the differentiation of strain ZE1316R2Aᵀ from its closest phylogenetic relative, S. albidoflavus DSM 40455ᵀ. Strain ZE1316R2Aᵀ is a Gram-positive, aerobic, non-motile Actinomycetota forming branched substrate and aerial mycelia that differentiates into long chains of smooth, globose spores. Growth occurs between 16 and 37°C, at pH 5.5–8.5, and in the presence of 0–5% (w/v) NaCl, with optimal growth in the absence of NaCl. Good growth was observed on nutrient agar (NA), ISP5, and MSF media. The predominant menaquinones are MK-9(H 2 ), MK-9(H 4 ), and MK-9(H 6 ). The polar lipid profile includes DPG, PE, PI, APL, GPL, GL, PL and L. The major cellular fatty acids are anteiso-C 15:0 , iso-C 16:0 , and anteiso-C 17:0 . The draft genome of strain ZE1316R2Aᵀ comprises 3 contigs totaling 7.4 Mb with a G + C content of 73.2 mol%. It contains 6,464 predicted coding sequences, 7 rRNA operons, and 66 tRNA genes. Whole-genome comparative analyses yielded ANIm and dDDH values of 96.09% and 64.9%, respectively, when compared with strain S. albidoflavus DSM 40455 T , values below the accepted thresholds for species delineation. Phylogenomic analysis based on whole-genome sequences further placed strain ZE1316R2Aᵀ as a distinct lineage within the genus Streptomyces . Based on the polyphasic evidence presented, strain ZE1316R2Aᵀ represents a novel species of the genus Streptomyces , for which the name Streptomyces zimensis sp. nov. is proposed (zi.men’sis. L. masc./fem. adj. zimensis , referring to the Zima ecosystem). The type strain is ZE1316R2Aᵀ (= CCMM B1331ᵀ = DSM 120541ᵀ), isolated from saline water collected from Lake Zima, Morocco. Discussion Members of the genus Streptomyces represent one of the most taxonomically and functionally diverse lineages within the phylum Actinomycetota , and their extensive genomic and phenotypic variability can complicate species delineation when relying solely on classical markers (Chandra & Chater, 2014 ; Alam et al., 2022 ; Nikolaidis et al., 2023 ). In the present study, a polyphasic approach combining phenotypic, chemotaxonomic, and whole-genome analyses was applied to determine the taxonomic status of strain ZE1316R2Aᵀ, isolated from the hypersaline Lake Zima in Morocco, a habitat that remains underexplored microbiologically. Phylogenetic analyses based on the 16S rRNA gene placed strain ZE1316R2Aᵀ in close proximity to S. albidoflavus DSM 40455ᵀ, S. daghestanicus NRRL B-5418ᵀ, and S. violascens ISP 5183ᵀ, with sequence similarities exceeding 99%. These values are well above the 98.7% threshold historically proposed for species delineation, underscoring the limited resolving power of the 16S rRNA gene within Streptomyces , where this marker is highly conserved and frequently fails to discriminate among genomically distinct taxa (Chun et al., 2018 ; Kiepas et al., 2024 ). Genome-scale comparisons were therefore essential to achieve reliable classification. In this context, strain ZE1316R2Aᵀ displayed ANIm (96.09%) and dDDH (64.9%) values below the accepted species thresholds when compared with strain DSM 40455ᵀ (Hu et al., 2022 ). These results underscore the necessity of genome-based criteria for accurate species circumscription in Streptomyces , where high 16S rRNA gene similarity alone may mask substantial genomic divergence (Zakalyukina et al., 2024 ). Beyond species-level delineation, comparative genome structure provides insight into the evolutionary processes underlying this divergence. Syntenic analysis revealed extensive collinearity between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, indicating a strongly conserved chromosomal backbone, while a large inversion and additional local discontinuities point to structural remodeling over evolutionary time. Such patterns are consistent with the dynamic organisation of linear Streptomyces chromosomes, in which recombination and rearrangements contribute to genome evolution (Bury-Moné et al., 2023 ; Nikolaidis et al., 2023 ). The identification of 29 Regions of Genomic Plasticity (RGPs) in strain ZE1316R2Aᵀ, several overlapping with strain-specific segments, aligns with the bipartite organisational model described for Streptomyces , where a conserved central chromosomal core is flanked by more variable peripheral compartments enriched in accessory and horizontally acquired genes (Bury-Moné et al., 2023 ; Nikolaidis et al., 2023 ). Thus, despite high global synteny, localised plastic regions can account for meaningful genomic divergence between closely related taxa. Consistent with this model, pan-genome comparison revealed that approximately 80% of genes are shared between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, whereas ~ 20% are strain-specific. Such proportions fall within the range of intra-species variability reported for bacteria (Martiny et al., 2006 ; Ventura et al., 2007 ) and underscore the evolutionary importance of the accessory genome in shaping strain-level diversity. In strain ZE1316R2Aᵀ, many strain-specific genes were located within Regions of Genomic Plasticity (RGPs), reinforcing the view that these regions represent dynamic compartments contributing to genomic diversification in Streptomyces (Ogier et al., 2010 ). From a functional perspective, both strain ZE1316R2Aᵀ and strain DSM 40455ᵀ share a broadly conserved metabolic backbone, including enrichment in genes associated with carbohydrate and amino acid metabolism. This is consistent with previously described core metabolic features of the genus Streptomyces (Bentley et al., 2002 ), reflecting its saprophytic lifestyle and capacity to exploit complex organic substrates. At the same time, strain ZE1316R2Aᵀ exhibits signatures of functional diversification within its accessory genome. Within strain ZE1316R2Aᵀ, several RGPs ( i.e. notably RGP2) contained a broader diversity of annotated metabolic functions compared to other plastic regions. These included genes associated with carbohydrate and amino acid metabolism, cofactor-related pathways, and secondary metabolite biosynthesis. In Streptomyces , biosynthetic gene clusters are frequently located within recombination-prone chromosomal regions that facilitate horizontal acquisition and metabolic innovation (Van der Meij et al., 2017 ). The enrichment of both primary and specialised metabolic functions within RGPs in strain ZE1316R2Aᵀ suggests that genome plasticity contributes to strain-specific functional configurations beyond the conserved core genome. Comparative analysis of lipid-related pathways further illustrates this functional divergence. In addition to the conserved enzymatic repertoire required for fatty acid, glycerolipid, and glycerophospholipid metabolism, strain ZE1316R2Aᵀ encodes accessory reactions, including early triterpenoid biosynthesis steps converting farnesyl-diphosphate to presqualene-diphosphate and squalene. MicroCyc annotation also identified partial enzymatic steps associated with acetone-to-acetoacetate-to-acetyl-CoA conversion that were absent or incompletely represented in strain DSM 40455ᵀ. Although these do not constitute complete metabolic modules, their strain-specific presence expands the enzymatic repertoire of strain ZE1316R2Aᵀ relative to its closest phylogenetic neighbor. Lipid remodeling and isoprenoid-derived compounds have been implicated in stress adaptation and membrane stability in halophilic and halotolerant microorganisms (Ventosa et al., 1998). While experimental validation is required to determine the physiological relevance of these accessory reactions, their presence in a strain isolated from a hypersaline ecosystem is consistent with potential ecological differentiation. Likewise, enrichment of amino acid metabolic functions may relate to osmoadaptive mechanisms such as compatible solute synthesis, although this hypothesis remains to be tested experimentally (Niu et al., 2021 ). Genome mining further emphasised the biosynthetic potential of strain ZE1316R2Aᵀ. The genome encodes 20 predicted BGCs, including two not detected in strain DSM 40455ᵀ. Notably, one of these BGCs showed no match to known entries in the MIBiG database, suggesting the capacity to synthesize previously uncharacterised metabolites. Such strain-specific BGC content reflects the contribution of the accessory genome to specialised metabolism and reinforces the genomic differentiation between the two closely related taxa. Streptomyces species are widely recognised as prolific producers of bioactive natural compounds with agricultural and medical relevance (Alam et al., 2022 ). In this context, the diversity and partial uniqueness of BGCs identified in strain ZE1316R2Aᵀ are consistent with the metabolic plasticity characteristic of the genus. However, experimental validation will be necessary to determine the chemical products and biological activities associated with these predicted clusters. In addition to BGC diversity, CAZymes revealed differences in family composition between the two strains. While strain DSM 40455ᵀ exhibited broader representation in certain AA, CBM, GT, and PL families, strain ZE1316R2Aᵀ showed enrichment in several GH families. Such genetic diversity is a hallmark of Streptomyces evolution and underlies the genus’s capacity to generate structurally diverse natural products. In this context, the characterisation of new species contributes to expanding the documented genomic and biosynthetic diversity within the genus (Du et al., 2024 ). The phenotypic characterisation revealed broadly similar morphological and cultural features between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, including comparable colony morphology, growth ranges with respect to salinity, temperature, and pH. Such similarities are common among closely related Streptomyces taxa, as the genetic determinants of morphological development are largely conserved across the genus (Otani et al., 2022 ). Consequently, phenotypic traits alone provide limited resolution for species delineation and must be interpreted in conjunction with genomic data. Chemotaxonomic analyses confirmed the genus affiliation of strain ZE1316R2Aᵀ. The strain displayed characteristics polar lipid and menaquinone profiles, and a fatty acid composition dominated by branched-chain iso- and anteiso-fatty acids, a hallmark of the genus (Shi et al., 2022 ; Pansomsuay et al., 2025 ; Zhang et al., 2025 ). Although the overall chemotaxonomic patterns were consistent with those of strain DSM 40455ᵀ, minor differences in the relative proportions of major components provide additional phenotypic support for differentiation at the species level. Together with whole-genome evidence, these data complete the polyphasic framework supporting the recognition of strain ZE1316R2Aᵀ as a distinct species. In conclusion, the polyphasic evidence presented here supports the recognition of strain ZE1316R2Aᵀ as a novel species of the genus Streptomyces , for which the name Streptomyces zimensis sp. nov. is proposed (type strain ZE1316R2Aᵀ = CCMM B1331ᵀ = DSM 120541ᵀ). Genome-based analyses, combined with phenotypic and chemotaxonomic data, demonstrate that this strain constitutes a distinct genomic lineage despite its high 16S rRNA gene similarity to closely related taxa. This work contributes to expanding the taxonomic and genomic diversity of Streptomyces associated with underexplored Moroccan saline ecosystems and provides a foundation for future studies exploring its metabolic potential and potential biotechnological applications. Nucleotide sequence accession numbers The complete genome sequence of strain ZE1316R2Aᵀ (= CCMM B1331ᵀ = DSM 120541ᵀ) has been deposited in the NCBI GenBank under the accession number JBSXQD000000000 and the BioProject number PRJNA1345039. The 16S rRNA gene sequence of strain ZE1316R2Aᵀ has been deposited in GenBank under accession number PZ007821. Declarations Nucleotide sequence accession numbers The complete genome sequence of strain ZE1316R2Aᵀ (= CCMM B1331ᵀ = DSM 120541ᵀ) has been deposited in the NCBI GenBank under the accession number JBSXQD000000000 and the BioProject number PRJNA1345039. The 16S rRNA gene sequence of strain ZE1316R2Aᵀ has been deposited in GenBank under accession number PZ007821. Acknowledgements The authors are grateful to all colleagues for their valuable discussions and technical assistance during this study. Funding This work was financially supported by the Partnership Hubert Curien (PHC) Maghreb (PHC 23MAG09) program and by the CNRST Labeled Research Unit N°4 grant. Author contribution A. B. and M. B. initiated and coordinated the project. E-Z. O., A. B. and M. B. designed the experiments. E-Z. O., V. C., A. A., M. E., D. P., A. B. and M. B. performed experiments. E-Z. O, S.O, A. B. and M. B. made figures and wrote the manuscript. V. C., A. A., M. E., D. P. and F. A.P. revised the manuscript. All authors approved the final version of the manuscript. Declaration of interest The authors declare no competing interests. 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Zhao, F., Qin, Y.-H., Zheng, X., Zhao, H.-W., Chai, D.-Y., Li, W., Pu, M.-X., Zuo, X.-S., Qian, W., Ni, P., Zhang, Y., Mei, H. and He, S.-T. (2016). Biogeography and Adaptive evolution of Streptomyces Strains from saline environments. Scientific Reports, 6. doi:https://doi.org/10.1038/srep32718. Zheng, J., Ge, Q., Yan, Y., Zhang, X., Huang, L. and Yin, Y. (2023). dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Research, 51, p.Pages W115–W121. doi:https://doi.org/10.1093/nar/gkad328. Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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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-9413337","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632236443,"identity":"403de940-20e4-43a1-8625-874975228ab1","order_by":0,"name":"Ez-Zahra Oubassou","email":"","orcid":"","institution":"Cadi Ayyad University (UCA), Faculty of Sciences-Semlalia","correspondingAuthor":false,"prefix":"","firstName":"Ez-Zahra","middleName":"","lastName":"Oubassou","suffix":""},{"id":632236444,"identity":"575d9de0-7846-48fa-a931-0272026d079e","order_by":1,"name":"Soukaina Oudchaira","email":"","orcid":"","institution":"Cadi Ayyad University","correspondingAuthor":false,"prefix":"","firstName":"Soukaina","middleName":"","lastName":"Oudchaira","suffix":""},{"id":632236445,"identity":"f4cf68f4-6707-4863-ad7a-c7198865e7b3","order_by":2,"name":"Valérie Cognat","email":"","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":false,"prefix":"","firstName":"Valérie","middleName":"","lastName":"Cognat","suffix":""},{"id":632236446,"identity":"96bea1f9-2cec-4a7f-b0ec-3d8a7167fcc6","order_by":3,"name":"Abdelmalek Alioua","email":"","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":false,"prefix":"","firstName":"Abdelmalek","middleName":"","lastName":"Alioua","suffix":""},{"id":632236448,"identity":"988e013c-73fa-4316-9450-68ee1db6043c","order_by":4,"name":"Florence Arsène-Ploetze","email":"","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":false,"prefix":"","firstName":"Florence","middleName":"","lastName":"Arsène-Ploetze","suffix":""},{"id":632236450,"identity":"ff0d2dee-6186-4ad1-a168-20d71680d084","order_by":5,"name":"Mathieu Erhardt","email":"","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":false,"prefix":"","firstName":"Mathieu","middleName":"","lastName":"Erhardt","suffix":""},{"id":632236451,"identity":"1057faa9-cf0c-495f-844b-98b626f8fdab","order_by":6,"name":"David Pflieger","email":"","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Pflieger","suffix":""},{"id":632236452,"identity":"7d7816e0-466b-4b27-97f1-ed7baefeaae5","order_by":7,"name":"Alexandre Berr","email":"data:image/png;base64,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","orcid":"","institution":"Institut de Biologie Moléculaire des Plantes","correspondingAuthor":true,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Berr","suffix":""},{"id":632236453,"identity":"a20e674a-8ba6-415b-b9e4-12160e073d67","order_by":8,"name":"Mustapha Barakate","email":"","orcid":"","institution":"Cadi Ayyad University (UCA), Faculty of Sciences-Semlalia","correspondingAuthor":false,"prefix":"","firstName":"Mustapha","middleName":"","lastName":"Barakate","suffix":""}],"badges":[],"createdAt":"2026-04-14 09:25:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9413337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9413337/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108804519,"identity":"1afbf9d9-60e0-4a0c-9e06-ffc97fb7f1f7","added_by":"auto","created_at":"2026-05-08 15:21:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":325226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showing the position of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. ZE1316R2A\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e within the genus \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Sequence alignment was performed using MAFFT and phylogenetic inference using PhyML under the GTR+G+I substitution model via the NGPhylogeny.fr platform. Bootstrap support values were calculated from 1,000 replicates; only values ≥50% are shown. The tree was rooted with \u003cem\u003eKitasatospora setae\u003c/em\u003e NBRC 14216ᵀ as the outgroup. Bar, 0.01 substitutions per nucleotide position.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/41fe55146f2c9eabb1f0ccb6.png"},{"id":108591242,"identity":"a004927f-372c-4909-8227-75d8d5e24748","added_by":"auto","created_at":"2026-05-06 09:48:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":180855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinear chromosome representation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. ZE1316R2Aᵀ. A)\u003c/strong\u003e Organisation of the 7.4 Mb chromosome showing GC content (black), coding sequences (CDSs, red), RNA genes (tRNA green, rRNA blue), and GC skew (positive green, negative purple). \u003cstrong\u003eB)\u003c/strong\u003eDistribution of Regions of Genomic Plasticity (RGPs, red) and strain-specific regions (yellow) along the linear chromosome. GC content and GC skew variations highlight the localisation of variable genomic compartments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/0e8f3ca58da5218b34977851.png"},{"id":108805509,"identity":"233f0634-1526-4867-ab2c-4d2f70dd552a","added_by":"auto","created_at":"2026-05-08 15:26:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree highlighting the position of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. ZE1316R2Aᵀ relative to phylogenetically close type strains within the genus \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces.\u003c/strong\u003e\u003c/em\u003e The maximum-likelihood phylogenomic tree based on whole-genome sequences generated using the Genome BLAST Distance Phylogeny (GBDP) method. The tree shows the relationship of strain ZE1316R2Aᵀ (highlighted in red) to type and reference strains of the genus. Bootstrap support values are indicated by blue circles. \u003cem\u003eKitasatospora setae\u003c/em\u003e NBRC 14216ᵀ was used as the outgroup. The scale bar represents 0.01 nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/424edef21990eb3dfc6afbe7.png"},{"id":108591247,"identity":"49c0bd8d-4301-4ed4-8662-67b3311a48c7","added_by":"auto","created_at":"2026-05-06 09:48:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":399812,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomic heat maps showing pairwise genome relatedness between \u003cem\u003eStreptomyces\u003c/em\u003e sp. strain ZE1316R2Aᵀ and 16 closely related \u003cem\u003eStreptomyces\u003c/em\u003etype strains. Heat maps are based on average nucleotide identity calculated using MUMmer (ANIm) or BLAST (ANIb) in comparison with digital DNA–DNA hybridization (dDDH) values. Color intensity reflects the degree of genomic similarity. Species delineation thresholds corresponding to 95–96% for ANIb, 96.7% for ANIm as proposed for the genus \u003cem\u003eStreptomyces\u003c/em\u003e, and 70% for dDDH.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/f6ed7e443ab14c40255cd73d.png"},{"id":108804620,"identity":"f58a6737-757c-42ef-9307-c6565deb6662","added_by":"auto","created_at":"2026-05-08 15:22:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":334585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genome alignment, synteny, and functional mapping of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. ZE1316R2Aᵀ and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces albidoflavus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DSM 40455ᵀ.\u003c/strong\u003e \u003cstrong\u003eA) \u003c/strong\u003eLinear genome alignment between DSM 40455ᵀ (top) and ZE1316R2Aᵀ (bottom). Homologous genomic regions are connected by ribbons, illustrating overall synteny and structural rearrangements between the two chromosomes. Color intensity of the ribbons reflects nucleotide identity (74–100%). Colored segments along the genomes are displayed to facilitate visualisation of large-scale rearrangements. \u003cstrong\u003eB)\u003c/strong\u003e Dot-plot comparison showing pairwise genome alignment between the two strains. The prominent diagonal indicates strong collinearity and conserved gene order, whereas a large inversion and several minor discontinuities reveal structural rearrangements between the genomes. Scattered off-diagonal points correspond to non-syntenic regions, likely representing strain-specific genes, genomic islands, or horizontally acquired elements. \u003cstrong\u003eC)\u003c/strong\u003e Functional categorisation of the 29 Regions of Genomic Plasticity (RGPs) identified in \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ, based on KEGG pathway annotation. Bars represent the number of genes within each RGP assigned to distinct metabolic categories. RGP2 contains the highest number and diversity of KEGG-annotated functions, including genes associated with secondary metabolite biosynthesis, amino acid and carbohydrate metabolism, terpenoid/polyketide biosynthesis, energy metabolism, metabolism of cofactors and vitamins, and xenobiotic degradation. The multifunctional nature of RGP2 and several other RGPs highlights the contribution of variable genomic regions to the adaptive metabolic potential of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/5dbd2415640ced7ff55c66c9.png"},{"id":108804621,"identity":"5e4db2b5-4d42-44e9-a18c-fea1197bdb05","added_by":"auto","created_at":"2026-05-08 15:22:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":301927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological features of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. ZE1316R2Aᵀ and comparison with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. albidoflavus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DSM 40455ᵀ.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Scanning electron micrograph of strain ZE1316R2Aᵀ grown ISP2 medium at 30 °C for 14 days, showing well-developed aerial mycelia with branched hyphae and long chains of smooth-surfaced spores (arrow). Scale bar = 5 µm. \u003cstrong\u003eB)\u003c/strong\u003e Colony morphology of \u003cem\u003eS. zimensis\u003c/em\u003e sp. nov. (right) and \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ (left) grown on Mannitol Soy Flour (MSF) agar for 7 days at 30 °C, showing creamy-white, folded colonies typical of the genus \u003cem\u003eStreptomyces.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/13505f55596a6380fb76061b.png"},{"id":108977474,"identity":"3ef4c351-3c94-4d13-b93d-fd92afbe5cd3","added_by":"auto","created_at":"2026-05-11 11:31:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1969891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/f1582655-603c-41d2-8d1d-38b8a8da0ada.pdf"},{"id":108591241,"identity":"de515cb3-4ff4-4627-8ed3-eebeaeba0e42","added_by":"auto","created_at":"2026-05-06 09:48:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":255628,"visible":true,"origin":"","legend":"","description":"","filename":"TablesV22032026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/54891d79115a2bf98872108c.pdf"},{"id":108804521,"identity":"b702342e-ddca-4d3e-95c0-57ad34249390","added_by":"auto","created_at":"2026-05-08 15:21:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":413941,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTablesV22032026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/e0a8f78e03afd6c995c953ad.pdf"},{"id":108591244,"identity":"7bec6679-64f9-49d5-bc8c-6e92a5d95bf6","added_by":"auto","created_at":"2026-05-06 09:48:33","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":797774,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFiguresV22032026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9413337/v1/81624fd7ef212197a555599c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polyphasic and Genomic Characterisation of Streptomyces zimensis sp. nov., a Halotolerant Actinomycetota from the Saline Lake Zima in Morocco","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtreme saline ecosystems such as salt lakes, salterns, and hypersaline soils represent unique and underexplored habitats characterised by strong physicochemical constraints, including high salinity, temperature fluctuations, and limited nutrient availability. Despite these harsh conditions, such environments harbor diverse and specialised microbial communities, notably within the phylum \u003cem\u003eActinomycetota\u003c/em\u003e (Van der Meij et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ngamcharungchit et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Many Actinomycetota thriving in saline habitats exhibit halotolerant or halophilic lifestyles, enabling them to colonize salt lakes, hypersaline soils, and other hypersaline biotopes (Hozzein, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several genera of Actinomycetota have been reported from these environments, including \u003cem\u003eNocardiopsis\u003c/em\u003e, \u003cem\u003eActinopolyspora\u003c/em\u003e, \u003cem\u003eActinomadura, Micromonospora\u003c/em\u003e, among others (Menasria et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Carre\u0026oacute;n-Gaxiola et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notably, species of the genus \u003cem\u003eStreptomyces\u003c/em\u003e are frequently reported as dominant members of actinomycetal communities in saline and hypersaline ecosystems, owing to their remarkable physiological adaptability, genetic diversity, and metabolism versatility (Ballav et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Clavo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gohel, Majithiya, \u0026amp; Singh, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eStreptomyces\u003c/em\u003e, the type genus of the family \u003cem\u003eStreptomycetaceae\u003c/em\u003e, represents the largest taxon within the phylum \u003cem\u003eActinomycetota\u003c/em\u003e (Gopalakrishnan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Members of this genus are filamentous, Gram-positive bacteria characterised by a complex life cycle involving the formation of vegetative and aerial mycelia and spore chains (Nazari et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although nearly 95% of described \u003cem\u003eStreptomyces\u003c/em\u003e species have been isolated from soils, where they play a central ecological role as decomposers and nutrient recyclers (De Moura., 2021), representatives of the genus are also widespread in diverse environments, including marine habitats, extreme ecosystems and symbiotic associations with plants and animals (Komaki, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To date, approximately 793 \u003cem\u003eStreptomyces\u003c/em\u003e species have been validly published (accessed on 05 November 2025; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lpsn.dsmz.de/search?word=streptomyces\u003c/span\u003e\u003cspan address=\"https://lpsn.dsmz.de/search?word=streptomyces\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), reflecting the extensive genetic variability and ecological plasticity of this genus.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStreptomyces\u003c/em\u003e species are widely recognised as the most prolific producers of bioactive secondary metabolites in the bacterial kingdom, with numerous compounds exploited for medical, agricole, and industrial applications (El-Naggar, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alam et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Their exceptional biosynthetic capacity is largely driven by the presence of numerous biosynthetic gene clusters (BGCs), which encode pathways for the production of antibiotics and other specialised metabolites (Du et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This metabolic richness contributes to their ability to adapt to a broad range of ecological niches, including saline and hypersaline environments, and highlights their importance as a resource for natural-product discovery, particularly in the context of genomic-guided approaches.\u003c/p\u003e \u003cp\u003eRecent advances in \u003cem\u003eStreptomyces\u003c/em\u003e genomics have revealed extensive intra-generic diversity and have led to the reclassification of several taxa (Mispelaere et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kiepas et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In Morocco, \u003cem\u003eStreptomyces\u003c/em\u003e species have been isolated from different habitats, highlighting the country\u0026rsquo;s rich microbial diversity. Representative examples include \u003cem\u003eS. marokkonensis\u003c/em\u003e from argan-tree rhizospheres (Bouizgarne et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), S. \u003cem\u003ebeta-vulgaris\u003c/em\u003e from sugar-beet rhizospheric soil (Lebrihi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), S. \u003cem\u003ethinghirensis\u003c/em\u003e from grapevine roots (Loqman et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e)d \u003cem\u003eyoussoufiensis\u003c/em\u003e from phosphate mines (Hamdali et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), as well as numerous other strains from agricultural, urban, and endophytic environments (Rammali et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Rammali et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Aallam et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Buchmann et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oubaha et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Katif et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, \u003cem\u003eStreptomyces\u003c/em\u003e from extreme or saline environments in Morocco remain poorly explored, despite the fact that such habitats often select for microorganisms with unique metabolic traits and adaptive genomic features. The climate and geochemical diversities of arid and semi-arid regions in Morocco provide a wide range of extreme environments, including salt flats, sebkhas, and evaporitic lakes that remain largely unexplored from a microbiological perspective. Among these, Lake Zima, a Ramsar-listed natural wetland located in the Bahira Plain of central Morocco, represents a hypersaline ecosystem shaped by intense evaporation, Triassic evaporite deposits (gypsum and halite), and groundwater inputs enriched in sodium, chloride, and sulfate ions (Karroum et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The high salinity, seasonal variability, and limited organic input, characteristic of this environment imposes strong selective pressures that may favor the emergence of halophilic and halotolerant microorganisms with distinctive metabolic capacities, including the potential to produce bioactive secondary metabolites.\u003c/p\u003e \u003cp\u003eIn this context, the present study focuses on strain ZE1316R2Aᵀ, isolated from saline water collected in Lake Zima, Morocco, during an investigation of the biodiversity and biotechnology potential of Actinomycetota inhabiting extreme saline ecosystems. A polyphasic approach combining phylogenomic, phenotypic, and chemotaxonomic analyses was employed to determine its taxonomic position relative to closely related members of the genus \u003cem\u003eStreptomyces\u003c/em\u003e. Whole-genome sequencing and comparative analyses revealed that strain ZE1316R2Aᵀ represents a novel species, for which the name \u003cem\u003eStreptomyces zimensis\u003c/em\u003e sp. nov., is proposed.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling, isolation and culture conditions\u003c/h2\u003e \u003cp\u003eSaline water samples were collected, under aseptic conditions, from Lake Zima (3,500 km\u003csup\u003e2\u003c/sup\u003e; 35 km north of Marrakesh; 32\u0026deg;4'48\" N, 8\u0026deg;39'36\" W), a Ramsar-listed saline wetland in central Morocco. Samples were kept at 4\u0026deg;C until use. Filtration was performed with a sterile filtration cell under reduced pressure, using a 0.45 \u0026micro;m nitrocellulose membrane to retain bacteria. The membranes were then placed on R2A medium supplemented with 3.5% (w/v) NaCl to reflect the lake's natural salinity (Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), as well as cycloheximide (40 \u0026micro;g. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and nalidixic acid (20 \u0026micro;g. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to inhibit respectively fungal and Gram-negative bacterial growth (Barakate et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Incubation occurred at 30\u0026deg;C for 7\u0026ndash;10 days, with daily monitoring for colony formation. Colonies exhibiting typical Actinomycetota morphology were selected and subcultured on ISP2 medium to ensure purity. Spore suspensions of purified isolates were preserved at -80\u0026deg;C in sterile 25% (v/v) glycerol.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenomic DNA extraction and 16S rRNA gene sequencing\u003c/h3\u003e\n\u003cp\u003eFor DNA extraction, strain ZE1316R2Aᵀ was cultivated for 5 days at 30\u0026deg;C in ISP2 medium (4 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Yeast extract; 10 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Malt extract; 4 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Glucose and 20 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Agar) under agitation. Bacterial biomass was collected from 2 mL of culture by centrifugation (16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 min) and bacterial genomic DNA was extracted using the Genomic-tip 100/G kit (Qiagen, Cat. No. 10243) following the manufacturer\u0026rsquo;s recommendations. DNA was quantified using the Qubit\u0026trade; dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific), and purity was verified through A260/280 and A260/230 absorbance measures using the NanoDrop spectrophotometer 2000. The nearly full-length 16S rRNA gene was amplified with Phusion High-Fidelity DNA Polymerase using the universal bacterial primers 8F (5\u0026rsquo;-AGAGTTTGATCCTGGCTCAG-3\u0026rsquo;) and 1492R (5\u0026rsquo;-GGTTACCTTGTTACGACTT-3\u0026rsquo;\u003cb\u003e)\u003c/b\u003e (Frank et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). PCR amplification yielded a product of approximately 1.5 kb, as verified by electrophoresis on a 1% (w/v) agarose gel. The corresponding DNA band was excised and purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL). The purified amplicon was subsequently sequenced by the Sanger method using primers 8F and 1492R. The final assembled sequence had a length of 1,417 bp. The 16S rRNA gene sequence of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2A (1,417 bp) has been deposited in NCBI GenBank under accession number PZ007821.\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing, data quality assessment, and genome assembly\u003c/h3\u003e\n\u003cp\u003eWhole-genome sequencing was performed using the same genomic DNA preparation reported in the previous section. Libraries were prepared with the Native Barcoding Kit 24 V14 (SQK-NBD114.24, Oxford Nanopore Technologies) using\u0026thinsp;~\u0026thinsp;1 \u0026micro;g of DNA, and sequencing was performed on R10.4.1 PromethION flow cells (FLO-PRO114M) operated with MinKNOW v22.07.9. Raw signal data (fast5 files) were basecalled and demultiplexed with Guppy v6.5.7 (Wick et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) using the super-accuracy model (dna_r10.4.1_e8.2_400bps_hac_prom) and a minimum Q-score cutoff of 7. Read quality was assessed with NanoPlot v0.32.1 (De Coster and Rademakers, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and adapter sequences were removed with Porechop v0.2.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Porechop\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Porechop\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e To screen for contamination, reads were classified against the standard Kraken database using Kraken2 v2.0.9-beta (Wood et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). De novo genome assembly was carried out with Flye v2.9.3-b1797 (Kolmogorov et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), followed by consensus polishing with NextPolish v1.4.1 (Hu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Assembly quality and completeness were assessed with QUAST v5.0.2 (Gurevich et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), BUSCO v5.4.3 using the actinobacteria_odb10 lineage dataset, and CheckM v1.2.2 (Parks et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhylogenomic analysis\u003c/h3\u003e\n\u003cp\u003eThe similarity of the 16S rRNA gene sequence of strain ZE1316R2Aᵀ and identification of its closest phylogenetic relatives were evaluated using the EzBioCloud database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed September 24, 2025). Accession numbers of 16S rRNA gene sequences from phylogenetically related type and reference strains retrieved from EzBioCloud are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Chalita et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Representative \u003cem\u003eStreptomyces\u003c/em\u003e 16S rRNA gene sequences were aligned using MAFFT implemented in the NGPhylogeny.fr online platform advanced workflow (Lemoine et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A maximum-likelihood phylogenetic tree was constructed using the PhyML/OneClick module, with \u003cem\u003eKitasatospora setae\u003c/em\u003e NBRC 14216ᵀ as the outgroup. Branch support was assessed using 1,000 bootstrap replicates, and the resulting tree was visualised using iTOL v7.4.2 (Letunic \u0026amp; Bork, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To further confirm the taxonomic placement of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ, a whole-genome phylogenetic tree was reconstructed based on a Genome BLAST Distance Phylogeny (GBDP) distance matrix using the Type (Strain) Genome Server (TYGS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Meier-Kolthoff and G\u0026ouml;ker, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eKitasatospora setae\u003c/em\u003e NBRC 14216ᵀ was used as an outgroup to root the phylogenetic tree. Accession numbers of the whole-genome assemblies of reference strains included in the analysis are listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The resulting phylogenomic tree was visualised using iTOL version 7.4.2 (Letunic and Bork, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecies delineation of ZE1316R2Aᵀ was assessed using genomic similarity metrics. The Average Nucleotide Identity (ANI) values, ANI-BLAST (ANIb) and ANI-MUMmer ultra-rapid aligning tool (ANIm) of the whole genome of strain ZE1316R2Aᵀ with closely related type strain DSM 40455\u003csup\u003eT\u003c/sup\u003e were calculated using the JSpeciesWS web server v5.0.2 (Richter et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and digital DNA-DNA hybridization (dDDH) was calculated with the Genome-to-Genome Distance Calculator (GGDC2.1) with the BLAST+ algorithm, applying Formula 2 (d4) as implemented in the TYGS platform. (Meier-Kolthoff et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Heat maps were generated to visualize ANI and dDDH-based relationships between strain ZE1316R2A\u003csup\u003eT\u003c/sup\u003e and multiple closely related \u003cem\u003eStreptomyces\u003c/em\u003e species.\u003c/p\u003e\n\u003ch3\u003eWhole-genome comparative analyses\u003c/h3\u003e\n\u003cp\u003eWhole-genome comparative analyses were performed between strain ZE1316R2Aᵀ and \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e using the assembled genome sequences deposited under the accession numbers listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Genomic synteny was performed using the D-GENIES web tool (Cabanettes and Klopp, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with default parameters and minimap2-based alignments, enabling visualisation of conserved regions and structural rearrangements. Whole-genome pairwise comparisons and alignment visualisations were additionally performed using the Proksee platform (Grant et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Regions of genomic plasticity (RGPs) were identified using RGP_Finder implemented in the MicroScope platform (Vallenet et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and secondary metabolite biosynthetic gene clusters (BGCs) were predicted using antiSMASH v7.1.0.1 with default settings. Pan-genome analyses were conducted by computing gene families (MICFAM) using SiLiX with an amino acid identity threshold of 80% and 80% alignment coverage, allowing discrimination of core, variable, and strain-specific genes. Functional annotation and comparative analyses were performed using Clusters of Orthologous Groups (COG) classifications and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways assignments through the MicroScope platform (MicroScope v3.18.1). Carbohydrate-active enzyme (CAZyme) genes were annotated using the dbCAN3 web server (Zheng et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Predictions were retained when supported by at least two of the three integrated tools (HMMER v3.4, DIAMOND v2.1.21, and dbCAN_sub), ensuring robust identification of CAZyme families.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic, cultural, and chemotaxonomic characteristics\u003c/h2\u003e \u003cp\u003eSeven-day-old cultures of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ were grown on ISP2 agar at 30\u0026deg;C for 14 days using the inclined coverslip technique as described by Williams and Cross (1971) to allow proper development of aerial and substrate mycelia. The resulting cultures were subsequently analysed by scanning electron microscopy (Quanta FEG 450) following the methodology of Kurtb\u0026ouml;ke (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCultural characteristics were assessed by cultivating ZE1316R2Aᵀ and its closest relative, \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e (obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany), on International \u003cem\u003eStreptomyces\u003c/em\u003e Project (ISP) media for 14 days at 30\u0026deg;C. Tested media included ISP1 (Tryptone-Yeast Extract), ISP2 (Yeast Extract-Malt Extract Agar), ISP3 (Oatmeal Agar), ISP4 (Inorganic Salts-Starch Agar), ISP5 (Glycerol-Asparagine Agar), ISP6 (Peptone-yeast extract Agar), and ISP7 (Tyrosine Agar) (Shirling and Gottlieb, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). The growth and the coloration of aerial and substrate mycelia, as well as the production of diffusible pigments, were recorded using the ISCC\u0026ndash;NBS color charts, as previously used in the International \u003cem\u003eStreptomyces\u003c/em\u003e Project (Shirling and Gottlieb, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1966\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTests evaluating growth range of strain ZE1316R2Aᵀ under variable conditions of NaCl concentration, temperature, and pH were conducted using Mannitol Soya Flour agar medium (20 g Mannitol, 20 g Soya Flour, 20 g Agar and 1 L tap water). These tests evaluated NaCl concentration tolerance (0\u0026ndash;20%; w/v) in 5% increments, temperature tolerance at 5, 16, 28, 30, 37, and 50\u0026deg;C, and pH range (5.5 to 8.5), in increments of 1 unit. In addition, utilisation of carbon sources was evaluated using a standardised carbohydrate assimilation assay under aerobic conditions. The carbon utilisation profile of strain ZE1316R2Aᵀ was determined and compared with that of \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ following incubation under identical conditions. All tests were performed in triplicate, and growth was visually evaluated over a 14-day period of incubation at 30\u0026deg;C under aerobic conditions.\u003c/p\u003e \u003cp\u003eFor the chemotaxonomic analyses, the biomass of ZE1316R2Aᵀ and its closest relative \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e, was obtained from a 7-day culture in ISP2 broth at 30\u0026deg;C and 250 rpm on a rotary shaker. Cells were harvested by centrifugation, washed three times with sterile distilled water, and preserved in isopropanol. Chemotaxonomic analyses of respiratory quinones, polar lipids, and cellular fatty acids (FAME), commonly used as taxonomic markers in Actinomycetota (Ramasamy \u0026amp; Sudalaimuthu, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), were performed by DSMZ Services (Leibniz-Institut DSMZ, Braunschweig, Germany) following standard protocols.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and 16S rRNA gene analysis of strain ZE1316R2Aᵀ\u003c/h2\u003e \u003cp\u003eDuring our investigations of microbial diversity and valorisation in extreme Moroccan saline ecosystems, Actinomycetota were specifically targeted in saline water samples collected from multiple points within Lake Zima, Morocco, as this phylum is well known to include salt-tolerant members and prolific producers of secondary metabolites (Alam et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). More than 100 actinomycetal colonies displaying diverse morphologies were recovered on selective R2A medium supplemented with 3.5% (w/v) NaCl. Among these isolates, strain ZE1316R2Aᵀ was selected for further study as it represented a dominant and recurrent morphotype recovered across sampling points and exhibited stable and reproducible growth under saline conditions. These features made ZE1316R2Aᵀ a suitable representative strain for detailed taxonomic, genomic, and phenotypic characterisation.\u003c/p\u003e \u003cp\u003eThe nearly full-length 16S rRNA gene (1,417 bp) was sequenced and queried against the EzBioCloud database, which indicated that strain ZE1316R2Aᵀ belongs to the genus \u003cem\u003eStreptomyces\u003c/em\u003e within the family \u003cem\u003eStreptomycetaceae\u003c/em\u003e. The closest relatives were \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e (99.71%), \u003cem\u003eS. daghestanicus\u003c/em\u003e NRRL B-5418\u003csup\u003eT\u003c/sup\u003e (99.64%), and \u003cem\u003eS. violascens\u003c/em\u003e ISP 5183\u003csup\u003eT\u003c/sup\u003e (99.63%), \u003cem\u003eS. koyangensis\u003c/em\u003e VK-A60\u003csup\u003eT\u003c/sup\u003e (99.28%), \u003cem\u003eS. cadmiisoli\u003c/em\u003e ZFG47\u003csup\u003eT\u003c/sup\u003e, while identities with sequence similarities with other phylogenetically related \u003cem\u003eStreptomyces\u003c/em\u003e species ranged from 98.92% to 98.28%. The maximum-likelihood phylogenetic tree based on 16S rRNA sequences positioned ZE1316R2Aᵀ within the \u003cem\u003eStreptomyces\u003c/em\u003e clade and in close proximity to \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e, while forming a slightly earlier-branching lineage within the same cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; accession lists in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This topology supports the affiliation of strain ZE1316R2Aᵀ with the family \u003cem\u003eStreptomycetaceae\u003c/em\u003e and reveals a distinct phylogenetic positioning relative to its closest relatives. Beyond its phylogenetic placement, the assignment of strain ZE1316R2Aᵀ to the genus \u003cem\u003eStreptomyces\u003c/em\u003e highlighted its potential biotechnological relevance and provided the rationale for further genomic and phenotypic analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenome features and assembly quality\u003c/h2\u003e \u003cp\u003eThe genus \u003cem\u003eStreptomyces\u003c/em\u003e displays extensive genetic diversity, making difficult its taxonomic delineation when based solely on 16S rRNA gene sequences, which lack the resolution required to distinguish between closely related species (Kiepas et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zakalyukina et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, whole-genome sequencing was performed to clarify the taxonomic position of strain ZE1316R2Aᵀ. Sequencing produced 3 contigs, with a total length of 7,407,648 bp. The three contigs measured 370,759 bp, 6,87,3245 bp, and 163,444 bp in length, with average coverages of 169\u0026times;, 126\u0026times;, and 124\u0026times;, respectively. The assembled genome displayed a G\u0026thinsp;+\u0026thinsp;C content of 73.2 mol%, with no detectable contamination and an estimated completeness of 99.89%. To better visualize the chromosomal organisation, the three contigs were aligned against the nearest reference genome of \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ, allowing their relative positioning along the chromosome and revealing extensive synteny and structural conservation. No additional small contigs or plasmid-like elements were detected, which is consistent with these contigs representing segments of a single linear chromosome typical of the genus. A total of 6,464 protein-coding DNA sequences (CDS), 7 rRNA operons (16S-23S-5S), and 66 tRNA genes were predicted, representing 87.9% of the genome with an average CDS length of 1,018 bp. These assembly and annotation metrics are summarised in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eA linear representation of the 7.4 Mb chromosome of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, illustrating the distribution of coding sequences, rRNA and tRNA genes, GC content, and GC skew. The genome architecture displays the typical large linear organisation characteristic of \u003cem\u003eStreptomyces\u003c/em\u003e species, with balanced GC skew and well-defined replication origin and terminus regions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenomic resolution and species delineation\u003c/h2\u003e \u003cp\u003eThe taxonomic position of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ was further evaluated using a whole genome-based phylogenomic approach. The whole-genome tree generated by TYGS, based on the Genome BLAST Distance Phylogeny (GBDP) method, placed strain ZE1316R2Aᵀ within the \u003cem\u003eStreptomyces\u003c/em\u003e cluster in a distinct lineage most closely related to \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, strain ZE1316R2Aᵀ branches slightly earlier than \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e within the clade that also contains \u003cem\u003eS. sampsonii\u003c/em\u003e NBRC 13083ᵀ and \u003cem\u003eS. limosus\u003c/em\u003e NBRC 12790ᵀ, indicating an independent phylogenomic trajectory consistent with species-level differentiation. This topology was consistent with that obtained from 16S rRNA gene analysis, confirming the close but distinct evolutionary relationship between strain ZE1316R2Aᵀ and \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePairwise genomic similarity metrics supported the separation of strain ZE1316R2Aᵀ from its closest \u003cem\u003eStreptomyces\u003c/em\u003e relatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The average nucleotide identity values were 94.84% (ANIb) and 96.09% (ANIm), both below the commonly accepted species boundary thresholds of 95\u0026ndash;96% and 96,70%, respectively (Richter \u0026amp; Rossell\u0026oacute;-M\u0026oacute;ra, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The digital DDH value was 64.9% (confidence interval 61.9\u0026ndash;67.7%), also below the 70% delineation threshold. These results confirm that strain ZE1316R2Aᵀ represents a genomically distinct lineage within the genus \u003cem\u003eStreptomyces\u003c/em\u003e, and support its recognition as a separate species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparative genomics and genome architecture\u003c/h2\u003e \u003cp\u003eTo investigate genomic conservation and structural variation, whole-genome comparisons were performed between \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ and its closest phylogenetic relative, \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e. Genome-wide alignment revealed extensive sequence conservation across large chromosomal segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This high degree of conservation is consistent with their close phylogenomic relationship.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDot-plot analysis further confirmed strong global collinearity between the two chromosomes, as evidenced by the prominent main diagonal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A large chromosomal inversion was observed, together with several local discontinuities and off-diagonal signals reflecting smaller-scale rearrangements and non-syntenic regions. These features illustrate structural diversification between the two genomes while maintaining an overall conserved genomic backbone.\u003c/p\u003e \u003cp\u003eA comparative analysis of the pan-, core-, and strain-specific genomes was then conducted to examine the genomic divergence between the two Actinomycetota (Table\u0026nbsp;2). The pangenome analysis identified 6,464 orthologous gene families. Of these, 5,123 gene families formed the core genome shared by both strains, representing 79.25% of the total coding sequences (CDSs) in strain ZE1316R2Aᵀ and 82.06% in strain DSM 40455\u003csup\u003eT\u003c/sup\u003e. In contrast, 1,341 CDSs were specific to strain ZE1316R2Aᵀ and 1,117 CDSs were unique to strain DSM 40455ᵀ, reflecting a substantial accessory genome component contributing to strain-level differentiation. In strain ZE1316R2Aᵀ, 29 distinct Regions of Genomic Plasticity (RGPs) were identified and mapped along the chromosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These RGPs largely overlapped with strain-specific regions (yellow segments in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and were mainly distributed in terminal and subterminal chromosomal compartments. When visualised together with GC content and GC skew profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), many RGPs coincided with local deviations in base composition and polarity shifts, further supporting their origin as variable genomic compartments. Functional annotation revealed that these RGPs encode diverse adaptive functions, including hydrolases, dioxygenases, aldolases, regulatory STAND NTPases, and trypsin-type proteases. Consistently, strain ZE1316R2Aᵀ harbors a higher number of predicted transposase-encoding genes (51 copies) compared with strain DSM 40455\u003csup\u003eT\u003c/sup\u003e (21 copies), suggesting enhanced genomic mobility and recombination potential.\u003c/p\u003e \u003cp\u003eTogether, these structural and genetic variations delineate strain-specific genomic regions that contribute to the overall divergence between the two genomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFunctional and metabolic potential\u003c/h2\u003e \u003cp\u003eThe biosynthetic potential of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ was investigated by genome mining for secondary metabolite biosynthetic gene clusters (BGCs). A total of 20 BGCs were identified in the genome, including two clusters absent in \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e (Tables S3-S6). One of these ZE1316R2Aᵀ-specific clusters showed no significant similarity to any entry in the MIBiG reference database, suggesting the presence of a potentially novel biosynthetic pathway.\u003c/p\u003e \u003cp\u003eComparative functional annotation based on KEGG pathway mapping revealed broadly similar metabolic profiles between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In both genomes, genes were predominantly assigned to carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, energy metabolism, and secondary metabolite biosynthesis categories.\u003c/p\u003e \u003cp\u003eA focused comparison of lipid metabolic pathways showed that both strains encode the full complement of enzymes required for fatty acid biosynthesis and degradation, together with those involved in glycerophospholipid and glycerolipid metabolisms, indicating a conserved lipid metabolic framework (Table S7). In addition, strain ZE1316R2Aᵀ encoded accessory reactions not identified in strain DSM 40455ᵀ, including genes encoding enzymes involved in the conversion of farnesyl-diphosphate to presqualene-diphosphate and squalene (annotated in KEGG under \u0026ldquo;steroid biosynthesis\u0026rdquo;). MicroCyc annotation further revealed partial enzymatic steps associated with the conversion of acetone to acetoacetate and subsequently to acetyl-CoA in strain ZE1316R2Aᵀ, which were absent or incomplete in strain DSM 40455ᵀ.\u003c/p\u003e \u003cp\u003eTo further investigate the relationship between genomic plasticity and functional specialisation, the 29 previously identified RGPs were cross-referenced with KEGG pathway annotations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This analysis showed that several RGPs contained genes assigned to diverse metabolic categories. Among them, RGP2 displayed the highest number and diversity of KEGG-annotated genes, including genes associated with secondary metabolite biosynthesis (terpenoid and polyketide), amino acid and carbohydrate metabolism, cofactor and vitamin metabolism, energy metabolism, and xenobiotic degradation. Other RGPs contained genes associated with narrower range of metabolic functions.\u003c/p\u003e \u003cp\u003eCarbohydrate-active enzyme (CAZymes) analysis revealed that both strains encode diverse repertoires of glycoside hydrolases (GH), glycosyltransferases (GT), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), polysaccharide lyases (PL), and auxiliary activities (AA). Although strain DSM 40455\u003csup\u003eT\u003c/sup\u003e exhibited a broader diversity of CAZyme families, particularly in the AA, CBM, GE, GT and PL classes, strain ZE1316R2Aᵀ displayed slight enrichment in several GH families (Table S8).\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that strain ZE1316R2Aᵀ and strain DSM 40455ᵀ maintain a shared core metabolic architecture but exhibit measurable differences in biosynthetic capacity, lipid-related reactions, genomic plasticity-associated functions, and carbohydrate-active enzyme repertoires.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic and cultural characteristics\u003c/h2\u003e \u003cp\u003eTo complete the taxonomic characterisation of strain ZE1316R2Aᵀ, its morphological, cultural, and physiological properties were examined and compared with those of \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ. On Mannitol Soy Flour (MSF) agar after 14 days of incubation at 30\u0026deg;C, both strains produced creamy-white, folded colonies typical of the genus \u003cem\u003eStreptomyces\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, strain ZE1316R2Aᵀ formed smaller and more compact colonies than strain DSM 40455ᵀ. Scanning electron microscopy (SEM) of strain ZE1316R2Aᵀ grown on ISP2 agar at 30\u0026deg;C for 14 days revealed a well-developed, branched aerial mycelia forming long chains of smooth-surfaced, globose spores (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen cultivated on the International \u003cem\u003eStreptomyces\u003c/em\u003e Project (ISP) media, strain ZE1316R2Aᵀ exhibited growth ranging from poor to good after 14 days of incubation at 30\u0026deg;C (Table\u0026nbsp;3). The strain produced both substrate and aerial mycelia, with coloration varying depending on the medium. On ISP6, strain ZE1316R2Aᵀ displayed a distinct orange pigmentation. Overall, its growth profile was comparable to that of \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ, although the latter generally formed more abundant aerial mycelia on certain media (\u003cem\u003ee.g\u003c/em\u003e. ISP3 and ISP7). Representative colony morphologies on ISP1-ISP7 media are shown in Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eGrowth responses under varying salinity, temperature, and pH conditions are summarised in Table S9 and illustrated in Figures S3-S5. Both strains ZE1316R2Aᵀ and DSM 40455ᵀ exhibited similar growth characteristics across all tested conditions. Optimal growth occurred in the absence of NaCl (0%; w/v), and both strains tolerated NaCl concentration up to 5% (w/v). Growth was observed between 16 and 37\u0026deg;C. Both strains grew over a pH range of 5.5 and 8.5, with optimal growth within this interval (Figures S3-S5). Carbohydrate utilisation patterns revealed phenotypic differences between strain ZE1316R2Aᵀ and \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ.Strain ZE1316R2Aᵀ utilised D-mannose but not the other tested carbohydrates, clearly distinguishing it from \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ (Table S10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChemotaxonomic characteristics\u003c/h2\u003e \u003cp\u003eChemotaxonomic analyses were performed to determine the polar lipid, menaquinone, and cellular fatty acid composition of \u003cem\u003eStreptomyces\u003c/em\u003e sp. ZE1316R2Aᵀ and its closest relative \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ (Table\u0026nbsp;4). Two-dimensional thin-layer chromatography (TLC) revealed broadly similar polar lipid profiles in both strains (Figure S6). The major detected lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Additional components, including phospholipids (PL), aminophospholipids (APL), glycolipids (GL), glycophospholipids (GPL), and unidentified lipids (L), were also detected (Table\u0026nbsp;4). The predominant menaquinones in strain ZE1316R2Aᵀ were MK-9(H\u003csub\u003e2\u003c/sub\u003e) (15.2%), MK-9(H\u003csub\u003e4\u003c/sub\u003e) (31%) and MK-9(H\u003csub\u003e6\u003c/sub\u003e) (33%). A similar menaquinone profile was detected in strain DSM 40455ᵀ, although the relative proportions differed (Table\u0026nbsp;4). Fatty acid methyl ester (FAME) analysis of strain ZE1316R2Aᵀ revealed that the dominant components (\u0026gt;\u0026thinsp;10%) were anteiso-C\u003csub\u003e15:0\u003c/sub\u003e (23.3%), iso-C\u003csub\u003e16:0\u003c/sub\u003e (22.0%), and anteiso-C\u003csub\u003e17:0\u003c/sub\u003e (18.4%; Table\u0026nbsp;4). In DSM 40455\u003csup\u003eT\u003c/sup\u003e, the same fatty acids predominated, with slightly higher proportions of iso-C\u003csub\u003e16:0\u003c/sub\u003e (27.7%) and lower proportions of anteiso-C\u003csub\u003e15:0\u003c/sub\u003e (17.6%) and anteiso-C\u003csub\u003e17:0\u003c/sub\u003e (15.6%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePolyphasic characterisation and taxonomic conclusion\u003c/h2\u003e \u003cp\u003eThe combined genomic, phenotypic, and chemotaxonomic data support the differentiation of strain ZE1316R2Aᵀ from its closest phylogenetic relative, \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ. Strain ZE1316R2Aᵀ is a Gram-positive, aerobic, non-motile Actinomycetota forming branched substrate and aerial mycelia that differentiates into long chains of smooth, globose spores. Growth occurs between 16 and 37\u0026deg;C, at pH 5.5\u0026ndash;8.5, and in the presence of 0\u0026ndash;5% (w/v) NaCl, with optimal growth in the absence of NaCl. Good growth was observed on nutrient agar (NA), ISP5, and MSF media.\u003c/p\u003e \u003cp\u003eThe predominant menaquinones are MK-9(H\u003csub\u003e2\u003c/sub\u003e), MK-9(H\u003csub\u003e4\u003c/sub\u003e), and MK-9(H\u003csub\u003e6\u003c/sub\u003e). The polar lipid profile includes DPG, PE, PI, APL, GPL, GL, PL and L. The major cellular fatty acids are anteiso-C\u003csub\u003e15:0\u003c/sub\u003e, iso-C\u003csub\u003e16:0\u003c/sub\u003e, and anteiso-C\u003csub\u003e17:0\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe draft genome of strain ZE1316R2Aᵀ comprises 3 contigs totaling 7.4 Mb with a G\u0026thinsp;+\u0026thinsp;C content of 73.2 mol%. It contains 6,464 predicted coding sequences, 7 rRNA operons, and 66 tRNA genes. Whole-genome comparative analyses yielded ANIm and dDDH values of 96.09% and 64.9%, respectively, when compared with strain \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e, values below the accepted thresholds for species delineation. Phylogenomic analysis based on whole-genome sequences further placed strain ZE1316R2Aᵀ as a distinct lineage within the genus \u003cem\u003eStreptomyces\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBased on the polyphasic evidence presented, strain ZE1316R2Aᵀ represents a novel species of the genus \u003cem\u003eStreptomyces\u003c/em\u003e, for which the name \u003cem\u003eStreptomyces zimensis\u003c/em\u003e sp. nov. is proposed (zi.men\u0026rsquo;sis. L. masc./fem. adj. \u003cem\u003ezimensis\u003c/em\u003e, referring to the Zima ecosystem). The type strain is ZE1316R2Aᵀ (=\u0026thinsp;CCMM B1331ᵀ = DSM 120541ᵀ), isolated from saline water collected from Lake Zima, Morocco.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMembers of the genus \u003cem\u003eStreptomyces\u003c/em\u003e represent one of the most taxonomically and functionally diverse lineages within the phylum \u003cem\u003eActinomycetota\u003c/em\u003e, and their extensive genomic and phenotypic variability can complicate species delineation when relying solely on classical markers (Chandra \u0026amp; Chater, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Alam et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nikolaidis et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, a polyphasic approach combining phenotypic, chemotaxonomic, and whole-genome analyses was applied to determine the taxonomic status of strain ZE1316R2Aᵀ, isolated from the hypersaline Lake Zima in Morocco, a habitat that remains underexplored microbiologically.\u003c/p\u003e \u003cp\u003ePhylogenetic analyses based on the 16S rRNA gene placed strain ZE1316R2Aᵀ in close proximity to \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455ᵀ, \u003cem\u003eS. daghestanicus\u003c/em\u003e NRRL B-5418ᵀ, and \u003cem\u003eS. violascens\u003c/em\u003e ISP 5183ᵀ, with sequence similarities exceeding 99%. These values are well above the 98.7% threshold historically proposed for species delineation, underscoring the limited resolving power of the 16S rRNA gene within \u003cem\u003eStreptomyces\u003c/em\u003e, where this marker is highly conserved and frequently fails to discriminate among genomically distinct taxa (Chun et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kiepas et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Genome-scale comparisons were therefore essential to achieve reliable classification. In this context, strain ZE1316R2Aᵀ displayed ANIm (96.09%) and dDDH (64.9%) values below the accepted species thresholds when compared with strain DSM 40455ᵀ (Hu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results underscore the necessity of genome-based criteria for accurate species circumscription in \u003cem\u003eStreptomyces\u003c/em\u003e, where high 16S rRNA gene similarity alone may mask substantial genomic divergence (Zakalyukina et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond species-level delineation, comparative genome structure provides insight into the evolutionary processes underlying this divergence. Syntenic analysis revealed extensive collinearity between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, indicating a strongly conserved chromosomal backbone, while a large inversion and additional local discontinuities point to structural remodeling over evolutionary time. Such patterns are consistent with the dynamic organisation of linear \u003cem\u003eStreptomyces\u003c/em\u003e chromosomes, in which recombination and rearrangements contribute to genome evolution (Bury-Mon\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nikolaidis et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The identification of 29 Regions of Genomic Plasticity (RGPs) in strain ZE1316R2Aᵀ, several overlapping with strain-specific segments, aligns with the bipartite organisational model described for \u003cem\u003eStreptomyces\u003c/em\u003e, where a conserved central chromosomal core is flanked by more variable peripheral compartments enriched in accessory and horizontally acquired genes (Bury-Mon\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nikolaidis et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, despite high global synteny, localised plastic regions can account for meaningful genomic divergence between closely related taxa.\u003c/p\u003e \u003cp\u003eConsistent with this model, pan-genome comparison revealed that approximately 80% of genes are shared between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, whereas ~\u0026thinsp;20% are strain-specific. Such proportions fall within the range of intra-species variability reported for bacteria (Martiny et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ventura et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and underscore the evolutionary importance of the accessory genome in shaping strain-level diversity. In strain ZE1316R2Aᵀ, many strain-specific genes were located within Regions of Genomic Plasticity (RGPs), reinforcing the view that these regions represent dynamic compartments contributing to genomic diversification in \u003cem\u003eStreptomyces\u003c/em\u003e (Ogier et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom a functional perspective, both strain ZE1316R2Aᵀ and strain DSM 40455ᵀ share a broadly conserved metabolic backbone, including enrichment in genes associated with carbohydrate and amino acid metabolism. This is consistent with previously described core metabolic features of the genus \u003cem\u003eStreptomyces\u003c/em\u003e (Bentley et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), reflecting its saprophytic lifestyle and capacity to exploit complex organic substrates. At the same time, strain ZE1316R2Aᵀ exhibits signatures of functional diversification within its accessory genome. Within strain ZE1316R2Aᵀ, several RGPs (\u003cem\u003ei.e.\u003c/em\u003e notably RGP2) contained a broader diversity of annotated metabolic functions compared to other plastic regions. These included genes associated with carbohydrate and amino acid metabolism, cofactor-related pathways, and secondary metabolite biosynthesis. In \u003cem\u003eStreptomyces\u003c/em\u003e, biosynthetic gene clusters are frequently located within recombination-prone chromosomal regions that facilitate horizontal acquisition and metabolic innovation (Van der Meij et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The enrichment of both primary and specialised metabolic functions within RGPs in strain ZE1316R2Aᵀ suggests that genome plasticity contributes to strain-specific functional configurations beyond the conserved core genome.\u003c/p\u003e \u003cp\u003eComparative analysis of lipid-related pathways further illustrates this functional divergence. In addition to the conserved enzymatic repertoire required for fatty acid, glycerolipid, and glycerophospholipid metabolism, strain ZE1316R2Aᵀ encodes accessory reactions, including early triterpenoid biosynthesis steps converting farnesyl-diphosphate to presqualene-diphosphate and squalene. MicroCyc annotation also identified partial enzymatic steps associated with acetone-to-acetoacetate-to-acetyl-CoA conversion that were absent or incompletely represented in strain DSM 40455ᵀ. Although these do not constitute complete metabolic modules, their strain-specific presence expands the enzymatic repertoire of strain ZE1316R2Aᵀ relative to its closest phylogenetic neighbor. Lipid remodeling and isoprenoid-derived compounds have been implicated in stress adaptation and membrane stability in halophilic and halotolerant microorganisms (Ventosa et al., 1998). While experimental validation is required to determine the physiological relevance of these accessory reactions, their presence in a strain isolated from a hypersaline ecosystem is consistent with potential ecological differentiation. Likewise, enrichment of amino acid metabolic functions may relate to osmoadaptive mechanisms such as compatible solute synthesis, although this hypothesis remains to be tested experimentally (Niu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenome mining further emphasised the biosynthetic potential of strain ZE1316R2Aᵀ. The genome encodes 20 predicted BGCs, including two not detected in strain DSM 40455ᵀ. Notably, one of these BGCs showed no match to known entries in the MIBiG database, suggesting the capacity to synthesize previously uncharacterised metabolites. Such strain-specific BGC content reflects the contribution of the accessory genome to specialised metabolism and reinforces the genomic differentiation between the two closely related taxa. \u003cem\u003eStreptomyces\u003c/em\u003e species are widely recognised as prolific producers of bioactive natural compounds with agricultural and medical relevance (Alam et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this context, the diversity and partial uniqueness of BGCs identified in strain ZE1316R2Aᵀ are consistent with the metabolic plasticity characteristic of the genus. However, experimental validation will be necessary to determine the chemical products and biological activities associated with these predicted clusters. In addition to BGC diversity, CAZymes revealed differences in family composition between the two strains. While strain DSM 40455ᵀ exhibited broader representation in certain AA, CBM, GT, and PL families, strain ZE1316R2Aᵀ showed enrichment in several GH families. Such genetic diversity is a hallmark of \u003cem\u003eStreptomyces\u003c/em\u003e evolution and underlies the genus\u0026rsquo;s capacity to generate structurally diverse natural products. In this context, the characterisation of new species contributes to expanding the documented genomic and biosynthetic diversity within the genus (Du et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phenotypic characterisation revealed broadly similar morphological and cultural features between strain ZE1316R2Aᵀ and strain DSM 40455ᵀ, including comparable colony morphology, growth ranges with respect to salinity, temperature, and pH. Such similarities are common among closely related \u003cem\u003eStreptomyces\u003c/em\u003e taxa, as the genetic determinants of morphological development are largely conserved across the genus (Otani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, phenotypic traits alone provide limited resolution for species delineation and must be interpreted in conjunction with genomic data. Chemotaxonomic analyses confirmed the genus affiliation of strain ZE1316R2Aᵀ. The strain displayed characteristics polar lipid and menaquinone profiles, and a fatty acid composition dominated by branched-chain iso- and anteiso-fatty acids, a hallmark of the genus (Shi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pansomsuay et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although the overall chemotaxonomic patterns were consistent with those of strain DSM 40455ᵀ, minor differences in the relative proportions of major components provide additional phenotypic support for differentiation at the species level. Together with whole-genome evidence, these data complete the polyphasic framework supporting the recognition of strain ZE1316R2Aᵀ as a distinct species.\u003c/p\u003e \u003cp\u003eIn conclusion, the polyphasic evidence presented here supports the recognition of strain ZE1316R2Aᵀ as a novel species of the genus \u003cem\u003eStreptomyces\u003c/em\u003e, for which the name \u003cem\u003eStreptomyces zimensis\u003c/em\u003e sp. nov. is proposed (type strain ZE1316R2Aᵀ = CCMM B1331ᵀ = DSM 120541ᵀ). Genome-based analyses, combined with phenotypic and chemotaxonomic data, demonstrate that this strain constitutes a distinct genomic lineage despite its high 16S rRNA gene similarity to closely related taxa. This work contributes to expanding the taxonomic and genomic diversity of \u003cem\u003eStreptomyces\u003c/em\u003e associated with underexplored Moroccan saline ecosystems and provides a foundation for future studies exploring its metabolic potential and potential biotechnological applications.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNucleotide sequence accession numbers\u003c/h2\u003e \u003cp\u003eThe complete genome sequence of strain ZE1316R2Aᵀ (=\u0026thinsp;CCMM B1331ᵀ = DSM 120541ᵀ) has been deposited in the NCBI GenBank under the accession number JBSXQD000000000 and the BioProject number PRJNA1345039. The 16S rRNA gene sequence of strain ZE1316R2Aᵀ has been deposited in GenBank under accession number PZ007821.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eNucleotide sequence accession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequence of strain ZE1316R2Aᵀ (= CCMM B1331ᵀ = DSM 120541ᵀ) has been deposited in the NCBI GenBank under the accession number JBSXQD000000000 and the BioProject number PRJNA1345039. The 16S rRNA gene sequence of strain ZE1316R2Aᵀ has been deposited in GenBank under accession number PZ007821.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to all colleagues for their valuable discussions and technical assistance during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Partnership Hubert Curien (PHC) Maghreb (PHC 23MAG09) program and by the CNRST Labeled Research Unit N\u0026deg;4 grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. B. and M. B. initiated and coordinated the project. E-Z. O., A. B. and M. B. designed the experiments. E-Z. O., V. C., A. A., M. E., D. P., A. B. and M. B. performed experiments. E-Z. O, S.O, A. B. and M. B. made figures and wrote the manuscript. V. C., A. A., M. E., D. P. and F. A.P. revised the manuscript. All authors approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eNucleotide sequence accession numbersThe complete genome sequence of strain ZE1316R2Aᵀ (= CCMM B1331ᵀ = DSM 120541ᵀ) has been deposited in the NCBI GenBank under the accession number JBSXQD000000000 and the BioProject number PRJNA1345039. The 16S rRNA gene sequence of strain ZE1316R2Aᵀ has been deposited in GenBank under accession number PZ007821.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAallam, Y., Dhiba, D., Lemriss, S., Souiri, A., Karray, F., Rasafi, T.E., Sa\u0026iuml;di, N., Haddioui, A., El Kabbaj, S., Virolle, M.J. and Hamdali, H. (2021). Isolation and Characterization of Phosphate Solubilizing \u003cem\u003eStreptomyces\u003c/em\u003e sp. Endemic from Sugar Beet Fields of the Beni-Mellal Region in Morocco. Microorganisms, 9, p.914. doi:https://doi.org/10.3390/microorganisms9050914.\u003c/li\u003e\n\u003cli\u003eAlam, K., Mazumder, A., Sikdar, S., Zhao, Y.-M., Hao, J., Song, C., Wang, Y., Sarkar, R., Islam, S., Zhang, Y. and Li, A. (2022). \u003cem\u003eStreptomyces\u003c/em\u003e: The biofactory of secondary metabolites. 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Retrieved from \u003cu\u003ehttps://doi.org/10.1016/S0580-9517(09)70016-9\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eWood, D.E., Lu, J. and Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20. doi:https://doi.org/10.1186/s13059-019-1891-0.\u003c/li\u003e\n\u003cli\u003eZakalyukina, Y.V., Alferova, V.A., Nikandrova, A.A., Kiriy, A.R., Chernyshova, A.P., Kabilov, M.R., Baturina, O.A., Biryukov, M.V., Sergiev, P.V. and Lukianov, D.A. (2024). Genomic and Phenotypic Characterization of \u003cem\u003eStreptomyces sirii\u003c/em\u003e sp. nov., Amicetin-Producing Actinobacteria Isolated from Bamboo Rhizospheric Soil. Microorganisms, 12, pp.2628\u0026ndash;2628. doi:https://doi.org/10.3390/microorganisms12122628.\u003c/li\u003e\n\u003cli\u003eZhang, B., Xiang, X., Ma, X., Chen, L., Li, J., Sun, C. and Yin, H. (2025). \u003cem\u003eStreptomyces longbaonensis\u003c/em\u003e sp. nov., a novel actinomycete from an alpine meadow that enhances plant salt tolerance via antioxidative and osmotic regulation. Plant Stress, 18, pp.101018\u0026ndash;101018. doi:https://doi.org/10.1016/j.stress.2025.101018.\u003c/li\u003e\n\u003cli\u003eZhao, F., Qin, Y.-H., Zheng, X., Zhao, H.-W., Chai, D.-Y., Li, W., Pu, M.-X., Zuo, X.-S., Qian, W., Ni, P., Zhang, Y., Mei, H. and He, S.-T. (2016). Biogeography and Adaptive evolution of \u003cem\u003eStreptomyces\u003c/em\u003e Strains from saline environments. Scientific Reports, 6. doi:https://doi.org/10.1038/srep32718.\u003c/li\u003e\n\u003cli\u003eZheng, J., Ge, Q., Yan, Y., Zhang, X., Huang, L. and Yin, Y. (2023). dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Research, 51, p.Pages W115\u0026ndash;W121. doi:https://doi.org/10.1093/nar/gkad328.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"annals-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amoa","sideBox":"Learn more about [Annals of Microbiology](https://www.springer.com/journal/13213)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/amoa/default.aspx","title":"Annals of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Comparative analysis, Novel species, Polyphasic taxonomy, Streptomyces, Whole-genome, Zima Lake","lastPublishedDoi":"10.21203/rs.3.rs-9413337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9413337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembers of the phylum Actinomycetota are widely distributed across diverse environments and are well known for their metabolic versatility and capacity to produce bioactive compounds. In this study, strain ZE1316R2Aᵀ was isolated from the saline water collected from Lake Zima (Morocco) and subjected to comprehensive polyphasic taxonomic characterisation. Phylogenetic analysis based on the 16S rRNA gene placed strain ZE1316R2Aᵀ within the genus \u003cem\u003eStreptomyces\u003c/em\u003e, showing highest sequence similarity with \u003cem\u003eS. albidoflavus\u003c/em\u003e DSM 40455\u003csup\u003eT\u003c/sup\u003e (99.71%). However, genome-based indices, including average nucleotide identity (ANIb\u0026thinsp;=\u0026thinsp;94.84%, ANIm\u0026thinsp;=\u0026thinsp;96.09%) and digital DNA-DNA hybridization (dDDH\u0026thinsp;=\u0026thinsp;64.9%), supported its distinction as a separate species. The draft genome (7.41 Mb; G\u0026thinsp;+\u0026thinsp;C = 73.26 mol%) comprises 6,464 coding sequences and reveals the presence of strain-specific genomic regions and biosynthetic gene clusters. Comparative analyses highlighted both a conserved core genome and a substantial accessory genome component, reflecting genomic differentiation relative to closely related taxa. Phenotypic and chemotaxonomic characteristics were consistent with assignment to the genus Streptomyces, while supporting its differentiation at the species level. Based on the combined genomic, phenotypic, and chemotaxonomic evidence, strain ZE1316R2Aᵀ represents a novel species of the genus \u003cem\u003eStreptomyces\u003c/em\u003e, for which the name \u003cem\u003eStreptomyces zimensis\u003c/em\u003e sp. nov., is proposed. This study expands current knowledge of Streptomyces diversity associated with saline environments and highlights the genomic diversity present within closely related taxa. The type strain is ZE1316R2Aᵀ (=\u0026thinsp;CCMM B1331\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 120541\u003csup\u003eT\u003c/sup\u003e).\u003c/p\u003e","manuscriptTitle":"Polyphasic and Genomic Characterisation of Streptomyces zimensis sp. nov., a Halotolerant Actinomycetota from the Saline Lake Zima in Morocco","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 09:48:28","doi":"10.21203/rs.3.rs-9413337/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-11T09:12:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33869974765134927904533380927048545126","date":"2026-04-30T02:48:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221262759265744229741438987164378934561","date":"2026-04-28T13:48:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T02:42:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T14:41:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T09:40:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Microbiology","date":"2026-04-14T09:18:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amoa","sideBox":"Learn more about [Annals of Microbiology](https://www.springer.com/journal/13213)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/amoa/default.aspx","title":"Annals of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d86947a6-c042-42ad-8ed7-341ad0370dbb","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-11T09:12:36+00:00","index":10,"fulltext":""},{"type":"reviewerAgreed","content":"33869974765134927904533380927048545126","date":"2026-04-30T02:48:58+00:00","index":8,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T09:48:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 09:48:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9413337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9413337","identity":"rs-9413337","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}