Genome Sequencing and Comparative Analysis of Nocardiopsis moroccensis nov., a Halotolerant Actinomycetota from Moroccan Hypersaline Ecosystem

Genome Sequencing and Comparative Analysis of Nocardiopsis moroccensis nov., a Halotolerant Actinomycetota from Moroccan Hypersaline Ecosystem | 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 Genome Sequencing and Comparative Analysis of Nocardiopsis moroccensis nov., a Halotolerant Actinomycetota from Moroccan Hypersaline Ecosystem Ez-Zahra Oubassou, Valérie Cognat, Abdelmalek Alioua, Florence Arsène-Ploetze, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7605207/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Saline and arid ecosystems are recognized as promising reservoirs of novel actinomycetes with unique adaptations and secondary metabolism potential. In this study, a halotolerant actinomycetota strain, designated ZS3416R2A, was isolated from saline soil in the hypersaline wetland of Lake Zima, Morocco. Phylogenetic analysis using the 16S rRNA gene sequence showed 99.03% similarity to Nocardiopsis terrae DSM 45157, indicating a close affiliation with the genus Nocardiopsis . Whole-genome sequencing revealed a 6.1 Mb circular chromosome with a G + C content of 70.48 mol% and 5,855 coding sequences. Genome-based metrics (ANI 95.07%, dDDH 66.4%) supported its classification as a novel species distinct from N. terrae , while AAI (95.87%) and POCP (82.4%) confirmed its placement within the genus. Comparative analyses revealed unique gene families, biosynthetic gene clusters, and regions of genomic plasticity in ZS3416R2A. Phenotypically, ZS3416R2A grew over a broad range of salinity (0–15%), temperature (16–37°C), and pH (7.0–8.0), with denser aerial mycelium formation than DSM 45157, reflecting adaptation to the arid saline environment of Lake Zima. Chemotaxonomic characterization identified MK-10(H 6 ), MK-10(H 4 ) and MK-9(H 4 ) as predominant menaquinones, iso-C16:0 and 10-methyl-C18:0 as major fatty acids, and diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol as major polar lipids. Based on these polyphasic evidences, strain ZS3416R2A represents a novel species within the genus Nocardiopsis , for which the name Nocardiopsis moroccensis sp. nov. is proposed. The type strain is ZS3416R2A T (= CCMM B1332 T = DSM 120542 T ). Nocardiopsis Novel species Halotolerant Zima Lake Polyphasic taxonomy Comparative analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The phylum Actinomycetota (formerly Actinobacteria) comprises a highly diverse group of Gram-positive bacteria, characterized by their filamentous morphology and major ecological importance. These microorganisms display a high level of biodiversity across a wide range of ecological niches, including soils, marine sediments, and symbiotic relationships (Ait Barka et al., 2015 ). Within this phylum, the genus Nocardiopsis has attracted increasing scientific interest owning to its broad ecological distribution and its ability to produce a broad spectrum of bioactive secondary metabolites with biotechnological and pharmaceutical relevance (Shi et al., 2022 ; Ouchene et al., 2024 ). Most species of Nocardiopsis are halophilic or halotolerant and are frequently found in extreme environments such as saline soils, dry salt lakes, and other hypersaline ecosystems, where their metabolic versatility supports unique adaptive strategies (Parihar et al., 2021 ; Chantavorakit et al., 2023 ). Saline habitats in particular represent extreme and selective environments, often yielding halophilic and halotolerant actinomycetes with distinctive biosynthetic repertoires and secondary metabolites not found in other ecosystems (Ventura et al., 2007 ; Genilloud, 2017 ). In recent years, the diversity of Nocardiopsis has been extensively expanded through the description of numerous novel species, isolated from diverse regions, particularly in arid and saline habitats. Examples include N. changdeensis , isolated from the roots of Eucommia ulmoides (Mo et al., 2023 ), as well as N. oceani and N. nanhaiensis from marine sediments (Pan et al., 2015 ). In North Africa, several species have also been described, such as N. aegyptia SNG49 T from Egyptian marine sediments (Sabry et al., 2004 ), N. alkaliphila YIM 80379 T from Egyptian desert soil (Hozzein et al., 2004 )d algeriensis B32 T from Saharan soil in Algeria (Bouras et al., 2015 ). Despite progress in characterizing Nocardiopsis diversity worldwide, Moroccan habitats remain largely unexplored. Their saline and arid ecosystems, located at the intersection of Mediterranean, Atlantic, and Saharan biomes, create a unique ecological mosaic that likely harbors untapped reservoirs of actinomycetota with distinctive adaptations and metabolic capacities. These environments therefore represent promising hotspots for the discovery of novel microbial taxa. In this study, we present the first description of a Nocardiopsis species from Morocco, a halotolerant strain isolated from saline soil at Lake Zima in central Morocco. approach that integrates genomic, phenotypic, and chemotaxonomic analyses, we demonstrate that this isolate represents a novel species, for which we propose the name Nocardiopsis moroccensis sp. nov. Materials and methods Sample collection and bacterial isolation Soil samples were collected from Lake Zima (Sebkha of Zima), a Ramsar-listed continental saline wetland in central Morocco (3,500 km 2 ; 35 km north of Marrakesh; 32°4'48" N et 8°39'36" W). Samples were aseptically transferred into sterile polyethylene bags, transported in a cooler, and stored at 4°C until use. Isolation of halophilic and halotolerant actinomycetota was performed using the dilution-suspension plating method in physiological saline (0.9% NaCl) (Anwar et al., 2016 ). Serial dilutions were plated on R2A medium supplemented with 3.5% (w/v) NaCl to mimic natural salinity (Liu et al., 2019 ), and with cycloheximide (40 µg.mL − 1 ) and nalidixic acid (20 µg.mL − 1 ) to inhibit fungal and Gram-negative bacterial growth (Barakate et al., 2002 ). Plates were incubated at 30°C for 7 to 10 days, and colony development was monitored daily. Colonies with typical actinomycetota morphology (chalky to leathery appearance) were selected and subcultured on ISP2 medium to ensure purity. Spores suspensions of the purified isolate were preserved in sterile 25% (w/v) glycerol at -80°C. For comparative analyses, Nocardiopsis terrae DSM45157 was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). Genomic DNA extraction and 16S rRNA gene sequencing Bacterial biomass was prepared by culturing the strain in Luria-Bertani (LB) liquid medium (10 g.L − 1 tryptone, 5 g.L − 1 yeast extract, 10 g.L − 1 NaCl, pH 7.0) at 30°C for 5 days on a rotary shaker. Cells (2mL) were harvested by centrifugation at 16,000 × g for 2 min. Genomic DNA was extracted using the Genomic-tip 100/G kit (Qiagen, Cat. No. 10243) according to the manufacturer’s instructions. DNA concentrations were measured with the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific), and purity was assessed spectrophotometrically (A260/280 ~ 1.8; A260/230 > 2.0). The 16S rRNA gene was PCR-amplified with Phusion High-Fidelity DNA Polymerase using universal primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) (JoVE Science Education Database., 2023). The expected ~ 1.5Kb amplicon was verified by agarose gel electrophoresis (1% w/v). PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) and sequenced using the same primers 8F and 1492R. Whole genome sequencing, quality control, and assembly The same genomic DNA preparation was used for whole-genome sequencing. 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, and CheckM v1.2.2 (Parks et al., 2015 ). Final assemblies were reoriented to start at the dnaA gene using Circlator fixstart v1.5.5 (Hunt et al., 2015 ). Phylogenomic analysis Reference genomes of relevant Nocardiopsis species were retrieved from the EzBioCloud database ( https://www.ezbiocloud.net/ ) (accessed May 21, 2025) (Chalita et al., 2024 ) and the NCBI GenBank database ( www.ncbi.nlm.nih.gov ) (accessed May 21, 2025) using BLASTn (Sayers et al., 2021 ). Accession numbers are provided in Supplementary Tables S8 and S9. Genome annotation was performed using Prokka v1.14.6 (Seemann, 2014 ), and pangenome analyses were conducted with Roary v3.13.0 (Page et al., 2015 ) to identify core, accessory, and strain-specific genes. Core gene alignments were generated using PRANK v170427 (Löytynoja, 2014 ). A phylogenomic tree was reconstructed from the concatenated core gene alignment using the maximum likelihood method in FastTree v2.1.11 (Price et al., 2010 ), and visualized with FigTree v1.4.4 (Rambaut, 2018 ). Genome relatedness indices were calculated to assess taxonomic placement. Average Nucleotide Identity (ANIb) was determined using JSpeciesWS v5.0.2 (Richter et al., 2015 ), and digital DNA-DNA hybridization (dDDH) was calculated with the Genome-to-Genome Distance Calculator (GGDC2.1) using the BLAST + method and Formula 2 (Meier-Kolthoff et al., 2021 ). Average Amino Acid Identity (AAI) was computed with CompareM v0.1.2 ( https://github.com/dparks1134/CompareM ) and the Percentage of Conserved Proteins (POCP) was determined following the method described by Qin et al. ( 2014 ). Comparative genome analyses Comparative genomic analyses were performed between strain ZS3416R2A and N. terrae DSM 45157. Synteny was assessed with the D-GENIES web tool (Cabanettes and Klopp, 2018 ). Integrative and conjugative elements were detected using ICEfinder (Liu et al., 2018 ), and regions of genomic plasticity were identified with RGP_Finder. Secondary metabolite biosynthetic gene clusters (BGCs) were predicted using AntiSMASH v7.1.0.1 integrated in the MicroScope annotation platform (Vallenet et al., 2019 ). Pan-genome analyses were conducted in MicroScope using gene families (MICFAM) computed with SiLiX, allowing the identification of core, variable, and strain-specific genes. Functional annotation and comparative analyses of gene distributions across Clusters of Orthologous Groups (COG) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were also performed in MicroScope. Phenotypic, cultural, and chemotaxonomic characteristics Morphological features of strain ZS3416R2A were examined after 7 days of incubation on nutrient agar at 30°C using scanning electron microscopy (Quanta FEG 450) following the methodology of Kurtböke ( 2022 ). The inclined coverslip technique described by Williams and Cross (1971) was employed to observe aerial mycelium and spore morphology. Cultural properties of strain ZS3416R2A and its closest relative, N. terrae DSM 45157, were assessed on International Streptomyces Project (ISP) media supplemented with 4% NaCl after 14 days of incubation 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 colors of aerial mycelium, substrate mycelium, and diffusible pigments were recorded using the ISCC-NBS color system, as previously used in the International Streptomyces Project (Shirling and Gottlieb, 1966 ). Physiological traits were evaluated under different temperature, pH, and salinity conditions. Temperature tolerance was determined on LB agar at 5, 16, 28, 30, 37, and 50°C for 7 days. pH tolerance was tested in media adjusted from pH 5.0 to 10.0 (increments of 1 unit) using acetate buffer (pH 5.0), phosphate buffer (pH 6.0–8.0), and Tris buffer (pH 9.0–10.0) (Girão et al., 2024 ). Salt tolerance was tested on LB agar supplemented with 0 to 20% NaCl (w/v) in 5% increments. All tests were performed in triplicate, and growth was evaluated visually after 7 days of incubation at 30°C under aerobic conditions. Chemotaxonomic analyses of respiratory quinones, polar lipids, and cellular fatty acids (FAME), all widely used as taxonomic markers in actinomycetota (Ramasamy & Sudalaimuthu, 2022 ), were carried out by DSMZ Services (Leibniz-Institut DSMZ, Braunschweig, Germany) using standard protocols. Results Isolation of strain ZS3416R2A and 16S rRNA gene analysis While the ecological and geochemical features of Lake Zima have been documented (El Mokhtar et al., 2012 ), no microbiological studies have yet addressed this unique hypersaline environment. Soil samples yielded several colonies with typical actinomycetota morphology when plated on selective R2A medium containing 3.5% NaCl. Among the isolates obtained, one strain displaying the typical chalky appearance of the genus Nocardiopsis was selected for further study and designated ZS3416R2A. The isolate was purified on ISP2 medium and preserved at -80°C in glycerol suspensions. To determine the taxonomic position of ZS3416R2A, the nearly full-length 16S rRNA gene was amplified with primers 8F and 1492R, yielding a fragment of about 1,500 bp. The PCR product was sequenced, and BLAST analysis against the GenBank and EzBioCloud databases confirmed affiliation with the genus Nocardiopsis . A maximum-likelihood phylogenetic tree was constructed from a 1,457 bp alignment of 16S rRNA gene sequences from multiple Nocardiopsis strains. The tree was rooted using Streptomonospora arabica S186 and Strm. halophila YIM 91355 as outgroups. This analysis placed ZS3416R2A in a clade with N. terrae DSM 45157, originally isolated from saline soil in China (Chen et al., 2010 ), showing 99.03% similarity (Fig. 1). This clade also includes N. oceani 1010A04, recovered from marine sediment in the South China Sea (Pan et al., 2015 ). Whole genome sequencing and comparative genomic analysis 16S rRNA gene similarity values above ~ 98.7% are often insufficient to reliably delineate bacterial species (Chun et al., 2018 ) and Several Nocardiopsis taxa are known to share > 99% similarity (Chantavorakit et al., 2023 ; Mo et al., 2023 ; Girão et al., 2024 ). Therefore, whole-genome sequencing using Oxford Nanopore technology was carried out to clarify the taxonomic position of ZS3416R2A. The assembly produced a single circular contig of 6.1 Mb. Genome completeness was estimated at 99.45%, with no detectable contamination, and sequencing coverage reached 132×. The calculated G + C content was 70.48 mol%. Using the MicroScope platform (Vallenet et al., 2019 ) for functional annotation, a total of 5,855 coding DNA sequences (CDS) were predicted, together with 5 rRNA operons and 60 tRNA genes. Key assembly and annotation metrics for ZS3416R2A, alongside values for N. terrae DSM 45157, are summarized in Table 1. A circular map generated with Bakta (Beyvers et al., 2025 ) is shown in Fig. 3a, highlighting annotated coding sequences, tRNA and rRNA genes, GC content, GC skew, and Regions of Genomic Plasticity (RGPs). The high-quality assembly was therefore suitable for comparative genomic analyses. Only Nocardiopsis strains for which complete genomes closely related to N. terrae DSM 45157 were included in the phylogenomic analysis. The resulting core-genome tree displayed a topology consistent with the 16S rRNA gene phylogeny (Fig. 2), confirming the close relationship between ZS3416R2A and N. terrae DSM 45157. Interestingly, ZS3416R2A did not cluster with other North African strains such as N. aegyptia DSM 44442 (Sabry et al., 2004 )d algeriensis DSM 45462 (Bouras et al., 2015 ). Instead, it grouped with additional Chinese Nocardiopsis species recovered from saline or marine environments (Pan et al., 2015 ). Comparative analyses further supported species-level distinction. The Average Nucleotide Identity (ANIb) between ZS3416R2A and N. terrae DSM 45157 was 95.07%, while the digital DNA-DNA hybridization (dDDH) value was estimated at 66.4% (63.4–69.2%). Although the ANI value approached the typical cutoff of 95–96%, the dDDH value fell below the conventional 70% threshold, indicating that the two strains represent separate species within the genus Nocardiopsis . Additional genomic metrics reinforced this conclusion: the Average Amino Acid Identity (AAI) was 95.87%, consistent with genus-level affiliation, and the Percentage Of Conserved Proteins (POCP) was 82.4%, exceeding the 50% threshold proposed for genus boundaries (Konstantinidis & Tiedje, 2005 ; Qin et al., 2014 ). To evaluate genomic conservation between strain ZS3416R2A and its closest relative, N. terrae DSM 45157, a synteny analysis was conducted. The resulting syntenic dot plot (Fig. 3b) revealed a prominent continuous diagonal line, indicating extensive collinearity and conserved gene order between the two genomes. However, several minor interruptions and shifts are visible along the diagonal, reflecting localized genomic rearrangements, such as small insertions, deletions, or sequence inversions. In addition, scattered points located away from the main diagonal suggest the presence of non-syntenic regions, which may represent strain-specific genomic islands, mobile genetic elements, or horizontally acquired sequences absent in one of the genomes (Okuno, 2025). To further assess the genomic divergence between strain ZS3416R2A and its closest relative, a comparative analysis of the pan-, core-, and strain-specific genomes was conducted. This analysis revealed a pan-genome of 6,037 orthologous gene families, of which 4,338 formed the core genome shared between the two strains. In terms of individual CDS, this core corresponds to 4,357 CDS in N. terrae DSM 45157 (79.20% of its genome) and 4,373 in ZS3416R2A (74.68% of its genome), with slight differences reflecting gene duplications (Table 2). The analysis also revealed genomic differences outside this core. Strain-specific fractions accounted for 1,464 strain-specific gene families (1,482 CDS) in ZS3416R2A and 1,135 gene families (1,144 CDS) in DSM 45157, representing approximately one-quarter of each genome. These variable regions included 38 distinct regions of genomic plasticity (RGPs) in ZS3416R2A, all of which overlapped with strain-specific genomic regions (Fig. 3a). These RGPs were enriched in genes encoding membrane transporters ( e.g .: permease, phosphate ABC transporter membrane proteins), secondary-metabolism enzymes, and antibiotic-resistance determinants such as capreomycidine synthase and enediyne polyketide synthase. Two Actinomycete Integrative and Conjugative Elements (AICEs) and three prophages (two Siphoviridae and one unclassified) were detected in ZS3416R2A, compared to four AICEs and a single Podoviridae prophage in DSM 45157. The Moroccan strain also carried a higher number of transposase sites (41 vs. 27). Given the genomic variability between the two strains, their biosynthetic potential was compared. Secondary-metabolism potential differed substantially between the two strains. ZS3416R2A harbored 19 biosynthesis gene clusters (BGCs), including 15 not detected in DSM 45157 (Table S1 -S4). Six of these unique clusters showed no significant similarity to known entries in the MIBiG database. Beyond specialized metabolism, KEGG-based annotation indicated that ZS3416R2A encodes a broader repertoire of metabolic pathways compared to DSM 45157 (Figure S1 ). In addition, CAZyme analysis revealed enrichment of the PL11 family, associated with pectate lyase activity, which was absent in DSM 45157 (Table S5). Together, these genomic features indicate that ZS3416R2A shares a close evolutionary relationship with N. terrae DSM 45157 but also possesses substantial strain-specific content, supporting its recognition as a distinct Nocardiopsis species. Phenotypic and cultural characteristics The morphological traits of strain ZS3416R2A, assigned to the genus Nocardiopsis based on 16S rRNA and whole-genome analyses, were examined by scanning electron microscopy (SEM). The strain produced well-developed aerial mycelia composed of long branched hyphae that fragmented similarly to fungal arthrospores (Fig. 4). Spores were typically arranged in short chains and exhibited surfaces ranging from smooth to slightly wrinkled, consistent with description for the genus Nocardiopsis (Goodfellow et al., 2012). The substrate mycelium appeared compact and extensively branched, providing structural support to the aerial hyphae and anchoring the colony to the agar surface. These morphological features were used to compare ZS3416R2A with its closest relative N. terrae DSM 45157. Despite being closely related, the two Nocardiopsis strains display morphological differences. ZS3416R2A formed abundant aerial mycelia with a powdery colony surface, while DSM 45157 produced a more rugose and elevated colony morphology with limited aerial mycelium development on ISP2 medium. In DSM 45157, fine hyphal projections were visible at the colony periphery, indicating initial stage of aerial mycelium formation. However, these structures were sparser than in ZS3416R2A. The aerial mycelium of DSM 45157 was yellowish-white and less extensive than that of ZS3416R2A. Cultural characteristics were assessed after 14 days of incubation at 30°C on various International Streptomyces Project (ISP) media supplemented with 4% NaCl, the optimal concentration for DSM 45157 (Chen et al., 2010 ). Detailed cultural characteristics for both strains across these media are summarized in Table S6, and representative colony morphologies are shown in Figure S2 . Strain ZS3416R2A exhibited moderate to abundant growth on most media, producing both substrate and aerial mycelia. Colors of aerial and substrate mycelia were documented using the ISCC-NBS Color System. Melanin production was tested on ISP6, where ZS3416R2A displayed a markedly stronger pigmentation response compared to DSM 45157. Colonies of ZS3416R2A had a powdery texture, filamentous margins, and a pronounced earthy odor, consistent with traits commonly observed in actinomycetota (Shirling and Gottlieb, 1966 ; Williams et al., 1983 ). In contrast, DSM 45157 colonies were more rugose and displayed reduced aerial mycelium. Physiological profiling revealed that ZS3416R2A grew at temperatures ranging from 16°C to 37°C, with optimal growth between 28°C and 37°C, consistent with a mesophilic profile. This strain tolerated pH values from pH 7.0 to 8.0, with optimal growth at pH 8.0, indicating preference for neutral to slightly alkaline conditions. In term of salinity, ZS3416R2A grew in media containing up to 10% (w/v) NaCl, with optimal growth between 0% and 10%, confirming a moderately halotolerant phenotype. Compared with DSM 45157, ZS3416R2A exhibited faster growth rates and increased sporulation across the tested ranges of NaCl, temperature, and pH (Figures S3-S8). A detailed summary of growth responses under the tested conditions is provided in Table S7. Chemotaxonomic characteristics The chemotaxonomic profile of ZS3416R2A was consistent with its assignment to the genus Nocardiopsis . The predominant respiratory quinones of strain ZS3416R2A were MK-10(H 6 ) (42.1%), MK-10(H 4 ) (29.3%) and MK-9(H4) (10.6%). Minor components include MK-9(H 6 ) (6.4%), MK-10(H 2 ) (3%), MK-10(H 8 ) (1.6%), MK-10 (1.3%), MK-9 (0.7%) and MK-8(H 4 ) (0.5%). The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). The major cellular fatty acids were iso-C16:0 (44.1%), 10-methyl-C18:0 (14.5%), anteiso-C17:0 (9.7%), C18:0 (7.1%) and iso-C18:0 (7%). Several additional fatty acids were detected in lower proportions, including 10-methyl-C17:0 (4.3%), iso-C14:0 (3.1%), anteiso-C15:0 (2.5%), C18:1 ω9c (1.5%), 10-methyl-C16:0 (1.4%), iso-C17:0 (1.1%), and C16:0 (1.0%). Comparison with N. terrae DSM 45157 revealed both shared chemotaxonomic features, typical of the genus Nocardiopsis , and clear differences. In particular, ZS3416R2A exhibited a higher proportion of iso-C16:0 and anteiso-C17:0, a distinct distribution of minor menaquinones, and a stronger representation of phosphatidylcholine compared to DSM 45157 (Table 3). Description of Nocardiopsis moroccensis sp. nov Strain ZS3416R2A is an aerobic, Gram-positive, spore-forming, and non-motile actinomycete that produces short spore chains and develops branched substrate mycelium and aerial hyphae, with the mycelium fragmenting as it ages. On ISP2 medium, colonies are powdery, with pale yellow aerial mycelium and strong brown substrate mycelium. Diffusible pigments are produced on ISP6 medium. Growth occurs at temperatures between 16–50°C (optimum 28–37°C), at pH values from 5.0 to 10.0 (optimum 7.0–8.0), and in the presence of up to 10% (w/v) NaCl (optimum 5%), indicating moderate halotolerance. Phylogenetic analysis based on the 16S rRNA gene sequence places strain ZS3416R2A within the genus Nocardiopsis , showing highest sequence similarity (99.03%) to Nocardiopsis terrae DSM 45157. A whole-genome phylogenomic tree based on core gene alignments confirmed this close relationship. The draft genome of ZS3416R2A assembles into a single circular contig of 6.1 Mb in size with a G + C content of 70.48 mol%, comprising 5,855 coding sequences, 5 rRNA operons, and 59 tRNA genes. Whole-genome comparisons further supported its distinctiveness: the ANI and dDDH values between ZS3416R2A and DSM 45157 are 95.07% and 66.4%, respectively, while the AAI and POCP are 95.87% and 82.4%, respectively. The predominant respiratory quinones are MK-10(H 6 ), MK-10(H 4 ) and MK-9(H 4 ), accompanied by minor components including MK-9(H 6 ), MK-10(H 2 ), MK-10(H 8 ), MK-10, MK-9, and MK-8(H 4 ). The polar lipid profile consists of DPG, PC, PG, PE and PI. The major fatty acids are iso-C16:0, 10-methyl-C18:0, anteiso-C17:0, C18:0 and iso-C18:0 with additional components present in smaller amounts including 10-methyl-C17:0, iso-C14:0, anteiso-C15:0, C18:1 ω9c, 10-methyl-C16:0, iso-C17:0, and C16:0. The type strain is ZS3416R2A T (= CCMM B1332 T = DSM 120542 T ), isolated from saline soil collected from Lake Zima’s ecosystem, Morocco. The type strain is deposited in two distinct collections, CCMM and DSMZ under accession numbers [CCMM B1332ᵀ = DSM 120542ᵀ]. The species epithet moroccensis (mo.roc.cen’sis. L. masc./fem. adj. moroccensis, pertaining to Morocco) is proposed. Discussion The purpose of this study was to explore the bacterial diversity of Moroccan saline ecosystems, with a focus on Lake Zima (Sebkha of Zima), a Ramsar-listed hypersaline wetland that has remained poorly explored from a microbiological perspective. Among the isolates obtained, strain ZS3416R2A was selected for detailed characterization due to its distinctive morphology and halotolerant growth profile. Phylogenetic, genomic, phenotypic, and chemotaxonomic analyses were undertaken to clarify its taxonomic position within the genus Nocardiopsis and to assess its potential ecological and biotechnological significance. Phylogenetic analysis of the 16S rRNA gene placed strain ZS3416R2A in the genus Nocardiopsis , with 99.03% sequence similarity to N. terrae DSM 45157, isolated from saline soil in China (Chen et al., 2010 ). However, due to the high 16S rRNA gene similarity among Nocardiopsis species, this marker alone is insufficient for reliable species delineation, necessitating additional analyses (Chun et al., 2018 ; Chantavorakit et al., 2023 ; Mo et al., 2023 ; Girão et al., 2024 ). Whole-genome analyses therefore provided a more robust framework: the ANI between ZS3416R2A and DSM 45157 was 95.07%, and the dDDH value was 66.4%, both falling below species thresholds. In contrast, the AAI (95.87%) and POCP (82.4%) confirmed their placement within the same genus. The genomic comparisons also revealed notable differences between both strains. Approximately 25% of each genome consisted of strain-specific genes, consistent with earlier estimates that 20–35% of bacterial genes are variable within a species (Martiny et al., 2006 ; Ventura et al., 2007 ). These unique fractions underscore the high genomic plasticity of actinomycetes and suggest that both local adaptation and horizontal gene transfer may have shaped the divergence of ZS3416R2A and DSM 45157. Indeed, RGPs in ZS3416R2A were enriched in genes encoding transport systems and secondary metabolism enzymes, with lower GC content suggestive of horizontal acquisition, a mechanism long recognized as a driver of actinomycete diversification (Ochman et al., 2000 ). Mobile genetic elements further highlighted the potential for differential adaptation. ZS3416R2A contained two AICEs, compared to four in DSM 45157. AICEs are known to mediate horizontal transfer and often carry accessory genes for antibiotic resistance, stress tolerance, or specialized metabolism (te Poele et al., 2008 ). Additionally, three prophages (two Siphoviridae and one unclassified) and a higher number of transposases (41 vs. 27) were detected in ZS3416R2A, again pointing to an active role of horizontal gene flux in its evolutionary history. Such elements may have contributed to niche-specific adaptations, despite both species being recovered from saline soils on different continents. This observation reinforces the notion that ecological similarity alone does not explain phylogenetic relationships in Nocardiopsis , other factors such as micro-environmental heterogeneity, historical biogeography, and lateral gene transfer may all play important roles (Martiny et al., 2006 ; Martiny et al., 2011 ). Supporting this evidence of genomic differentiation, ZS3416R2A displayed an expanded secondary metabolism potential relative to DSM 45157. Indeed, it harbored 19 BGCs, including 15 totally absent from its closest relative. Six of these showed no similarity to known entries in the MIBiG database, suggesting the capacity to produce novel metabolites. These findings are consistent with the view that actinomycetes remain a prolific source of new natural products, with nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and terpene pathways frequently linked to antibiotics, anticancer agents, and other valuable bioactive molecules (Ait Barka et al., 2015 ; Genilloud, 2017 ). KEGG-based annotation further indicated that ZS3416R2A encodes a broader repertoire of metabolic pathways than DSM 45157, further suggesting the potential for biotechnological exploitation as a reservoir of novel enzymes or stress-adaptive functions possibly relevant to industrial processes (Alanzi, 2024 ). CAZyme profiling also revealed an enrichment in the PL11 family (pectate lyases), which was absent in DSM 45157. These enzymes are of particular interest in agriculture and industry because of their role in degrading pectin-rich plant biomass and possible involvement in plant-microbe interactions and stress alleviation (Jayani et al., 2005 ; Ventura et al., 2007 ). Such metabolic distinctiveness suggests that ZS3416R2A has adapted to its specific ecological niche, potentially shaped by the selective pressures of the Moroccan saline and arid environment. Phenotypic and cultural characteristics provided additional evidence for differentiation. Compared to DSM 45157, strain ZS3416R2A exhibited denser aerial mycelium formation, more rapid growth, and stronger sporulation across different ranges of temperature, salinity, and pH. These traits may reflect ecological specialization, as previously observed for actinomycetes from extreme or marginal environments (Ventura et al., 2007 ). In particular, the robust growth of ZS3416R2A under saline conditions suggests a high degree of halotolerance that may be advantageous in the fluctuating salt and arid regimes of Lake Zima. Chemotaxonomic markers further reinforced its distinctiveness. Although the overall profiles of quinones, polar lipids, and fatty acids were consistent with the genus Nocardiopsis , quantitative differences were evident. For instance, ZS3416R2A exhibited higher proportions of iso-C16:0 and anteiso-C17:0, a distinct distribution of minor menaquinones, and stronger representation of phosphatidylcholine compared to DSM 45157. Such differences, when combined with genomic and phenotypic traits, provide reliable chemotaxonomic signatures for species delineation within the genus. In conclusion, we isolated and characterized strain ZS3416R2A from saline soil in the salt ecosystem of Lake Zima, Morocco. Comprehensive polyphasic and comparative genomic analyses, including phylogenomic, phenotypic, and chemotaxonomic approaches, demonstrated that ZS3416R2A represents a novel species within the genus Nocardiopsis and, to our knowledge, the first one described in Morocco. This discovery expands our understanding of actinomycetota diversity in saline environments and underscores the potential of Moroccan ecosystems as sources of unique microbial taxa with possible biotechnological applications. We propose the name Nocardiopsis moroccensis sp. nov. for this new species, with strain ZS3416R2Aᵀ (= CCMM B1332ᵀ = DSM 120542 ᵀ) designated as the type strain. Declarations Nucleotide sequence accession numbers The complete genome sequence of strain ZS3416R2Aᵀ (= CCMM B1332ᵀ = DSM 120542ᵀ) has been deposited in the NCBI GenBank under the accession number CP199747 and the BioProject number PRJNA1321125. Acknowledgements 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., 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. References Ait Barka, E., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H.-P., … van Wezel, G. P. (2015). Taxonomy, Physiology, and Natural Products of Actinobacteria. Microbiology and Molecular Biology Reviews, 80(1), 1–43. https://doi.org/10.1128/mmbr.00019-15 Alanzi, A. R. (2024). 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2","display":"","copyAsset":false,"role":"figure","size":171352,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/1f445dc3eda248f3a5df43d2.jpg"},{"id":94596442,"identity":"1ac17a86-cc68-486d-8a44-c2adcb64a12c","added_by":"auto","created_at":"2025-10-28 18:42:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":572199,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/7d31a38911fe4654728ab9b4.jpg"},{"id":94583548,"identity":"8b1a6fdb-5a2b-4396-92e6-4b9bd6cff47f","added_by":"auto","created_at":"2025-10-28 18:14:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":204438,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/ad2072d3e4272fae9b002e38.jpg"},{"id":94582606,"identity":"42e88e4e-5eef-4b45-b39e-3d223a44c053","added_by":"auto","created_at":"2025-10-28 18:13:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":258974,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/e719d2f10c99ef705ca98fbb.jpg"},{"id":94600140,"identity":"607e4538-683f-4ffe-ad69-933c18b693d6","added_by":"auto","created_at":"2025-10-28 19:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2356632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/19f39d95-2a5d-423e-9974-ed0fe42179df.pdf"},{"id":94583351,"identity":"95bcc9c9-8c4e-41c6-8135-79d74c341da6","added_by":"auto","created_at":"2025-10-28 18:14:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":326394,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/f6daedfea6abcf46664cbb8d.pdf"},{"id":94582720,"identity":"e228b68a-0335-48bb-9587-367c0902a4fa","added_by":"auto","created_at":"2025-10-28 18:13:25","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1392571,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/e1d32dd1452f5f95cd7d7045.pdf"},{"id":94583403,"identity":"311a3b90-52b9-40da-a993-0a76cd8599b4","added_by":"auto","created_at":"2025-10-28 18:14:01","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":213088,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7605207/v1/40f149f3b35fbba85cf6a216.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome Sequencing and Comparative Analysis of Nocardiopsis moroccensis nov., a Halotolerant Actinomycetota from Moroccan Hypersaline Ecosystem","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe phylum Actinomycetota (formerly Actinobacteria) comprises a highly diverse group of Gram-positive bacteria, characterized by their filamentous morphology and major ecological importance. These microorganisms display a high level of biodiversity across a wide range of ecological niches, including soils, marine sediments, and symbiotic relationships (Ait Barka et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Within this phylum, the genus \u003cem\u003eNocardiopsis\u003c/em\u003e has attracted increasing scientific interest owning to its broad ecological distribution and its ability to produce a broad spectrum of bioactive secondary metabolites with biotechnological and pharmaceutical relevance (Shi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ouchene et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Most species of \u003cem\u003eNocardiopsis\u003c/em\u003e are halophilic or halotolerant and are frequently found in extreme environments such as saline soils, dry salt lakes, and other hypersaline ecosystems, where their metabolic versatility supports unique adaptive strategies (Parihar et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chantavorakit et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Saline habitats in particular represent extreme and selective environments, often yielding halophilic and halotolerant actinomycetes with distinctive biosynthetic repertoires and secondary metabolites not found in other ecosystems (Ventura et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Genilloud, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, the diversity of \u003cem\u003eNocardiopsis\u003c/em\u003e has been extensively expanded through the description of numerous novel species, isolated from diverse regions, particularly in arid and saline habitats. Examples include \u003cem\u003eN. changdeensis\u003c/em\u003e, isolated from the roots of \u003cem\u003eEucommia ulmoides\u003c/em\u003e (Mo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), as well as \u003cem\u003eN. oceani\u003c/em\u003e and \u003cem\u003eN. nanhaiensis\u003c/em\u003e from marine sediments (Pan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In North Africa, several species have also been described, such as \u003cem\u003eN. aegyptia\u003c/em\u003e SNG49\u003csup\u003eT\u003c/sup\u003e from Egyptian marine sediments (Sabry et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), N. \u003cem\u003ealkaliphila\u003c/em\u003e YIM 80379\u003csup\u003eT\u003c/sup\u003e from Egyptian desert soil (Hozzein et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)d \u003cem\u003ealgeriensis B32\u003c/em\u003e\u003csup\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sup\u003e from Saharan soil in Algeria (Bouras et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite progress in characterizing \u003cem\u003eNocardiopsis\u003c/em\u003e diversity worldwide, Moroccan habitats remain largely unexplored. Their saline and arid ecosystems, located at the intersection of Mediterranean, Atlantic, and Saharan biomes, create a unique ecological mosaic that likely harbors untapped reservoirs of actinomycetota with distinctive adaptations and metabolic capacities. These environments therefore represent promising hotspots for the discovery of novel microbial taxa.\u003c/p\u003e\u003cp\u003eIn this study, we present the first description of a Nocardiopsis species from Morocco, a halotolerant strain isolated from saline soil at Lake Zima in central Morocco. approach that integrates genomic, phenotypic, and chemotaxonomic analyses, we demonstrate that this isolate represents a novel species, for which we propose the name \u003cem\u003eNocardiopsis moroccensis sp. nov.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample collection and bacterial isolation\u003c/h2\u003e\u003cp\u003eSoil samples were collected from Lake Zima (Sebkha of Zima), a Ramsar-listed continental saline wetland in central Morocco (3,500 km\u003csup\u003e2\u003c/sup\u003e; 35 km north of Marrakesh; 32\u0026deg;4'48\" N et 8\u0026deg;39'36\" W). Samples were aseptically transferred into sterile polyethylene bags, transported in a cooler, and stored at 4\u0026deg;C until use. Isolation of halophilic and halotolerant actinomycetota was performed using the dilution-suspension plating method in physiological saline (0.9% NaCl) (Anwar et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Serial dilutions were plated on R2A medium supplemented with 3.5% (w/v) NaCl to mimic natural salinity (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and with 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 fungal and Gram-negative bacterial growth (Barakate et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Plates were incubated at 30\u0026deg;C for 7 to 10 days, and colony development was monitored daily. Colonies with typical actinomycetota morphology (chalky to leathery appearance) were selected and subcultured on ISP2 medium to ensure purity. Spores suspensions of the purified isolate were preserved in sterile 25% (w/v) glycerol at -80\u0026deg;C. For comparative analyses, \u003cem\u003eNocardiopsis terrae\u003c/em\u003e DSM45157 was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenomic DNA extraction and 16S rRNA gene sequencing\u003c/h3\u003e\n\u003cp\u003eBacterial biomass was prepared by culturing the strain in Luria-Bertani (LB) liquid medium (10 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tryptone, 5 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yeast extract, 10 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, pH 7.0) at 30\u0026deg;C for 5 days on a rotary shaker. Cells (2mL) were harvested by centrifugation at 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 min. Genomic DNA was extracted using the Genomic-tip 100/G kit (Qiagen, Cat. No. 10243) according to the manufacturer\u0026rsquo;s instructions. DNA concentrations were measured with the Qubit\u0026trade; dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific), and purity was assessed spectrophotometrically (A260/280\u0026thinsp;~\u0026thinsp;1.8; A260/230\u0026thinsp;\u0026gt;\u0026thinsp;2.0). The 16S rRNA gene was PCR-amplified with Phusion High-Fidelity DNA Polymerase using universal primers 8F (5\u0026rsquo;-AGAGTTTGATCCTGGCTCAG-3\u0026rsquo;) and 1492R (5\u0026rsquo;-GGTTACCTTGTTACGACTT-3\u0026rsquo;) (JoVE Science Education Database., 2023). The expected\u0026thinsp;~\u0026thinsp;1.5Kb amplicon was verified by agarose gel electrophoresis (1% w/v). PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) and sequenced using the same primers 8F and 1492R.\u003c/p\u003e\n\u003ch3\u003eWhole genome sequencing, quality control, and assembly\u003c/h3\u003e\n\u003cp\u003eThe same genomic DNA preparation was used for whole-genome sequencing. 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=\"CR53\" 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=\"CR12\" 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). To screen for contamination, reads were classified against the standard Kraken database using Kraken2 v2.0.9-beta (Wood et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). De novo genome assembly was carried out with Flye v2.9.3-b1797 (Kolmogorov et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), followed by consensus polishing with NextPolish v1.4.1 (Hu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Assembly quality and completeness were assessed with QUAST v5.0.2 (Gurevich et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), BUSCO, and CheckM v1.2.2 (Parks et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Final assemblies were reoriented to start at the \u003cem\u003ednaA\u003c/em\u003e gene using Circlator fixstart v1.5.5 (Hunt et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhylogenomic analysis\u003c/h3\u003e\n\u003cp\u003eReference genomes of relevant \u003cem\u003eNocardiopsis\u003c/em\u003e species were retrieved from the EzBioCloud database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003c/span\u003e) (accessed May 21, 2025) (Chalita et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) and the NCBI GenBank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e) (accessed May 21, 2025) using BLASTn (Sayers et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Accession numbers are provided in Supplementary Tables S8 and S9. Genome annotation was performed using Prokka v1.14.6 (Seemann, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), and pangenome analyses were conducted with Roary v3.13.0 (Page et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) to identify core, accessory, and strain-specific genes. Core gene alignments were generated using PRANK v170427 (L\u0026ouml;ytynoja, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). A phylogenomic tree was reconstructed from the concatenated core gene alignment using the maximum likelihood method in FastTree v2.1.11 (Price et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), and visualized with FigTree v1.4.4 (Rambaut, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Genome relatedness indices were calculated to assess taxonomic placement. Average Nucleotide Identity (ANIb) was determined using JSpeciesWS v5.0.2 (Richter et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e), and digital DNA-DNA hybridization (dDDH) was calculated with the Genome-to-Genome Distance Calculator (GGDC2.1) using the BLAST\u0026thinsp;+\u0026thinsp;method and Formula 2 (Meier-Kolthoff et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Average Amino Acid Identity (AAI) was computed with CompareM v0.1.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/dparks1134/CompareM\u003c/span\u003e\u003c/span\u003e) and the Percentage of Conserved Proteins (POCP) was determined following the method described by Qin et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eComparative genome analyses\u003c/h3\u003e\n\u003cp\u003eComparative genomic analyses were performed between strain ZS3416R2A and \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157. Synteny was assessed with the D-GENIES web tool (Cabanettes and Klopp, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Integrative and conjugative elements were detected using ICEfinder (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and regions of genomic plasticity were identified with RGP_Finder. Secondary metabolite biosynthetic gene clusters (BGCs) were predicted using AntiSMASH v7.1.0.1 integrated in the MicroScope annotation platform (Vallenet et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Pan-genome analyses were conducted in MicroScope using gene families (MICFAM) computed with SiLiX, allowing the identification of core, variable, and strain-specific genes. Functional annotation and comparative analyses of gene distributions across Clusters of Orthologous Groups (COG) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were also performed in MicroScope.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePhenotypic, cultural, and chemotaxonomic characteristics\u003c/h2\u003e\u003cp\u003eMorphological features of strain ZS3416R2A were examined after 7 days of incubation on nutrient agar at 30\u0026deg;C using scanning electron microscopy (Quanta FEG 450) following the methodology of Kurtb\u0026ouml;ke (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The inclined coverslip technique described by Williams and Cross (1971) was employed to observe aerial mycelium and spore morphology. Cultural properties of strain ZS3416R2A and its closest relative, \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157, were assessed on International Streptomyces Project (ISP) media supplemented with 4% NaCl after 14 days of incubation 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=\"CR49\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). The colors of aerial mycelium, substrate mycelium, and diffusible pigments were recorded using the ISCC-NBS color system, as previously used in the International Streptomyces Project (Shirling and Gottlieb, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Physiological traits were evaluated under different temperature, pH, and salinity conditions. Temperature tolerance was determined on LB agar at 5, 16, 28, 30, 37, and 50\u0026deg;C for 7 days. pH tolerance was tested in media adjusted from pH 5.0 to 10.0 (increments of 1 unit) using acetate buffer (pH 5.0), phosphate buffer (pH 6.0\u0026ndash;8.0), and Tris buffer (pH 9.0\u0026ndash;10.0) (Gir\u0026atilde;o et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Salt tolerance was tested on LB agar supplemented with 0 to 20% NaCl (w/v) in 5% increments. All tests were performed in triplicate, and growth was evaluated visually after 7 days of incubation at 30\u0026deg;C under aerobic conditions. Chemotaxonomic analyses of respiratory quinones, polar lipids, and cellular fatty acids (FAME), all widely used as taxonomic markers in \u003cem\u003eactinomycetota\u003c/em\u003e (Ramasamy \u0026amp; Sudalaimuthu, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), were carried out by DSMZ Services (Leibniz-Institut DSMZ, Braunschweig, Germany) using standard protocols.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eIsolation of strain ZS3416R2A and 16S rRNA gene analysis\u003c/h2\u003e\u003cp\u003eWhile the ecological and geochemical features of Lake Zima have been documented (El Mokhtar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), no microbiological studies have yet addressed this unique hypersaline environment. Soil samples yielded several colonies with typical actinomycetota morphology when plated on selective R2A medium containing 3.5% NaCl. Among the isolates obtained, one strain displaying the typical chalky appearance of the genus \u003cem\u003eNocardiopsis\u003c/em\u003e was selected for further study and designated ZS3416R2A. The isolate was purified on ISP2 medium and preserved at -80\u0026deg;C in glycerol suspensions.\u003c/p\u003e\u003cp\u003eTo determine the taxonomic position of ZS3416R2A, the nearly full-length 16S rRNA gene was amplified with primers 8F and 1492R, yielding a fragment of about 1,500 bp. The PCR product was sequenced, and BLAST analysis against the GenBank and EzBioCloud databases confirmed affiliation with the genus \u003cem\u003eNocardiopsis\u003c/em\u003e. A maximum-likelihood phylogenetic tree was constructed from a 1,457 bp alignment of 16S rRNA gene sequences from multiple \u003cem\u003eNocardiopsis\u003c/em\u003e strains. The tree was rooted using \u003cem\u003eStreptomonospora arabica\u003c/em\u003e S186 and \u003cem\u003eStrm. halophila\u003c/em\u003e YIM 91355 as outgroups. This analysis placed ZS3416R2A in a clade with \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157, originally isolated from saline soil in China (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), showing 99.03% similarity (Fig.\u0026nbsp;1). This clade also includes \u003cem\u003eN. oceani\u003c/em\u003e 1010A04, recovered from marine sediment in the South China Sea (Pan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWhole genome sequencing and comparative genomic analysis\u003c/h2\u003e\u003cp\u003e16S rRNA gene similarity values above ~\u0026thinsp;98.7% are often insufficient to reliably delineate bacterial species (Chun et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Several \u003cem\u003eNocardiopsis\u003c/em\u003e taxa are known to share\u0026thinsp;\u0026gt;\u0026thinsp;99% similarity (Chantavorakit et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gir\u0026atilde;o et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, whole-genome sequencing using Oxford Nanopore technology was carried out to clarify the taxonomic position of ZS3416R2A. The assembly produced a single circular contig of 6.1 Mb. Genome completeness was estimated at 99.45%, with no detectable contamination, and sequencing coverage reached 132\u0026times;. The calculated G\u0026thinsp;+\u0026thinsp;C content was 70.48 mol%. Using the MicroScope platform (Vallenet et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) for functional annotation, a total of 5,855 coding DNA sequences (CDS) were predicted, together with 5 rRNA operons and 60 tRNA genes. Key assembly and annotation metrics for ZS3416R2A, alongside values for \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157, are summarized in Table\u0026nbsp;1. A circular map generated with Bakta (Beyvers et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) is shown in Fig.\u0026nbsp;3a, highlighting annotated coding sequences, tRNA and rRNA genes, GC content, GC skew, and Regions of Genomic Plasticity (RGPs). The high-quality assembly was therefore suitable for comparative genomic analyses.\u003c/p\u003e\u003cp\u003eOnly \u003cem\u003eNocardiopsis\u003c/em\u003e strains for which complete genomes closely related to \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157 were included in the phylogenomic analysis. The resulting core-genome tree displayed a topology consistent with the 16S rRNA gene phylogeny (Fig.\u0026nbsp;2), confirming the close relationship between ZS3416R2A and \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157. Interestingly, ZS3416R2A did not cluster with other North African strains such as \u003cem\u003eN. aegyptia\u003c/em\u003e DSM 44442 (Sabry et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)d \u003cem\u003ealgeriensis\u003c/em\u003e DSM 45462 (Bouras et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Instead, it grouped with additional Chinese \u003cem\u003eNocardiopsis\u003c/em\u003e species recovered from saline or marine environments (Pan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eComparative analyses further supported species-level distinction. The Average Nucleotide Identity (ANIb) between ZS3416R2A and \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157 was 95.07%, while the digital DNA-DNA hybridization (dDDH) value was estimated at 66.4% (63.4\u0026ndash;69.2%). Although the ANI value approached the typical cutoff of 95\u0026ndash;96%, the dDDH value fell below the conventional 70% threshold, indicating that the two strains represent separate species within the genus \u003cem\u003eNocardiopsis\u003c/em\u003e. Additional genomic metrics reinforced this conclusion: the Average Amino Acid Identity (AAI) was 95.87%, consistent with genus-level affiliation, and the Percentage Of Conserved Proteins (POCP) was 82.4%, exceeding the 50% threshold proposed for genus boundaries (Konstantinidis \u0026amp; Tiedje, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo evaluate genomic conservation between strain ZS3416R2A and its closest relative, \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157, a synteny analysis was conducted. The resulting syntenic dot plot (Fig.\u0026nbsp;3b) revealed a prominent continuous diagonal line, indicating extensive collinearity and conserved gene order between the two genomes. However, several minor interruptions and shifts are visible along the diagonal, reflecting localized genomic rearrangements, such as small insertions, deletions, or sequence inversions. In addition, scattered points located away from the main diagonal suggest the presence of non-syntenic regions, which may represent strain-specific genomic islands, mobile genetic elements, or horizontally acquired sequences absent in one of the genomes (Okuno, 2025).\u003c/p\u003e\u003cp\u003eTo further assess the genomic divergence between strain ZS3416R2A and its closest relative, a comparative analysis of the pan-, core-, and strain-specific genomes was conducted. This analysis revealed a pan-genome of 6,037 orthologous gene families, of which 4,338 formed the core genome shared between the two strains. In terms of individual CDS, this core corresponds to 4,357 CDS in \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157 (79.20% of its genome) and 4,373 in ZS3416R2A (74.68% of its genome), with slight differences reflecting gene duplications (Table\u0026nbsp;2). The analysis also revealed genomic differences outside this core. Strain-specific fractions accounted for 1,464 strain-specific gene families (1,482 CDS) in ZS3416R2A and 1,135 gene families (1,144 CDS) in DSM 45157, representing approximately one-quarter of each genome. These variable regions included 38 distinct regions of genomic plasticity (RGPs) in ZS3416R2A, all of which overlapped with strain-specific genomic regions (Fig.\u0026nbsp;3a). These RGPs were enriched in genes encoding membrane transporters (\u003cem\u003ee.g\u003c/em\u003e.: permease, phosphate ABC transporter membrane proteins), secondary-metabolism enzymes, and antibiotic-resistance determinants such as capreomycidine synthase and enediyne polyketide synthase. Two Actinomycete Integrative and Conjugative Elements (AICEs) and three prophages (two \u003cem\u003eSiphoviridae\u003c/em\u003e and one unclassified) were detected in ZS3416R2A, compared to four AICEs and a single \u003cem\u003ePodoviridae\u003c/em\u003e prophage in DSM 45157. The Moroccan strain also carried a higher number of transposase sites (41 vs. 27).\u003c/p\u003e\u003cp\u003eGiven the genomic variability between the two strains, their biosynthetic potential was compared. Secondary-metabolism potential differed substantially between the two strains. ZS3416R2A harbored 19 biosynthesis gene clusters (BGCs), including 15 not detected in DSM 45157 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S4). Six of these unique clusters showed no significant similarity to known entries in the MIBiG database. Beyond specialized metabolism, KEGG-based annotation indicated that ZS3416R2A encodes a broader repertoire of metabolic pathways compared to DSM 45157 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In addition, CAZyme analysis revealed enrichment of the PL11 family, associated with pectate lyase activity, which was absent in DSM 45157 (Table S5).\u003c/p\u003e\u003cp\u003eTogether, these genomic features indicate that ZS3416R2A shares a close evolutionary relationship with \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157 but also possesses substantial strain-specific content, supporting its recognition as a distinct \u003cem\u003eNocardiopsis\u003c/em\u003e species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePhenotypic and cultural characteristics\u003c/h2\u003e\u003cp\u003eThe morphological traits of strain ZS3416R2A, assigned to the genus \u003cem\u003eNocardiopsis\u003c/em\u003e based on 16S rRNA and whole-genome analyses, were examined by scanning electron microscopy (SEM). The strain produced well-developed aerial mycelia composed of long branched hyphae that fragmented similarly to fungal arthrospores (Fig.\u0026nbsp;4). Spores were typically arranged in short chains and exhibited surfaces ranging from smooth to slightly wrinkled, consistent with description for the genus \u003cem\u003eNocardiopsis\u003c/em\u003e (Goodfellow et al., 2012). The substrate mycelium appeared compact and extensively branched, providing structural support to the aerial hyphae and anchoring the colony to the agar surface. These morphological features were used to compare ZS3416R2A with its closest relative \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157. Despite being closely related, the two \u003cem\u003eNocardiopsis\u003c/em\u003e strains display morphological differences. ZS3416R2A formed abundant aerial mycelia with a powdery colony surface, while DSM 45157 produced a more rugose and elevated colony morphology with limited aerial mycelium development on ISP2 medium. In DSM 45157, fine hyphal projections were visible at the colony periphery, indicating initial stage of aerial mycelium formation. However, these structures were sparser than in ZS3416R2A. The aerial mycelium of DSM 45157 was yellowish-white and less extensive than that of ZS3416R2A.\u003c/p\u003e\u003cp\u003eCultural characteristics were assessed after 14 days of incubation at 30\u0026deg;C on various \u003cem\u003eInternational Streptomyces Project\u003c/em\u003e (ISP) media supplemented with 4% NaCl, the optimal concentration for DSM 45157 (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Detailed cultural characteristics for both strains across these media are summarized in Table S6, and representative colony morphologies are shown in Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Strain ZS3416R2A exhibited moderate to abundant growth on most media, producing both substrate and aerial mycelia. Colors of aerial and substrate mycelia were documented using the ISCC-NBS Color System. Melanin production was tested on ISP6, where ZS3416R2A displayed a markedly stronger pigmentation response compared to DSM 45157. Colonies of ZS3416R2A had a powdery texture, filamentous margins, and a pronounced earthy odor, consistent with traits commonly observed in actinomycetota (Shirling and Gottlieb, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Williams et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). In contrast, DSM 45157 colonies were more rugose and displayed reduced aerial mycelium.\u003c/p\u003e\u003cp\u003ePhysiological profiling revealed that ZS3416R2A grew at temperatures ranging from 16\u0026deg;C to 37\u0026deg;C, with optimal growth between 28\u0026deg;C and 37\u0026deg;C, consistent with a mesophilic profile. This strain tolerated pH values from pH 7.0 to 8.0, with optimal growth at pH 8.0, indicating preference for neutral to slightly alkaline conditions. In term of salinity, ZS3416R2A grew in media containing up to 10% (w/v) NaCl, with optimal growth between 0% and 10%, confirming a moderately halotolerant phenotype. Compared with DSM 45157, ZS3416R2A exhibited faster growth rates and increased sporulation across the tested ranges of NaCl, temperature, and pH (Figures S3-S8). A detailed summary of growth responses under the tested conditions is provided in Table S7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eChemotaxonomic characteristics\u003c/h2\u003e\u003cp\u003eThe chemotaxonomic profile of ZS3416R2A was consistent with its assignment to the genus \u003cem\u003eNocardiopsis\u003c/em\u003e. The predominant respiratory quinones of strain ZS3416R2A were MK-10(H\u003csub\u003e6\u003c/sub\u003e) (42.1%), MK-10(H\u003csub\u003e4\u003c/sub\u003e) (29.3%) and MK-9(H4) (10.6%). Minor components include MK-9(H\u003csub\u003e6\u003c/sub\u003e) (6.4%), MK-10(H\u003csub\u003e2\u003c/sub\u003e) (3%), MK-10(H\u003csub\u003e8\u003c/sub\u003e) (1.6%), MK-10 (1.3%), MK-9 (0.7%) and MK-8(H\u003csub\u003e4\u003c/sub\u003e) (0.5%). The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). The major cellular fatty acids were iso-C16:0 (44.1%), 10-methyl-C18:0 (14.5%), anteiso-C17:0 (9.7%), C18:0 (7.1%) and iso-C18:0 (7%). Several additional fatty acids were detected in lower proportions, including 10-methyl-C17:0 (4.3%), iso-C14:0 (3.1%), anteiso-C15:0 (2.5%), C18:1 ω9c (1.5%), 10-methyl-C16:0 (1.4%), iso-C17:0 (1.1%), and C16:0 (1.0%). Comparison with \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157 revealed both shared chemotaxonomic features, typical of the genus \u003cem\u003eNocardiopsis\u003c/em\u003e, and clear differences. In particular, ZS3416R2A exhibited a higher proportion of iso-C16:0 and anteiso-C17:0, a distinct distribution of minor menaquinones, and a stronger representation of phosphatidylcholine compared to DSM 45157 (Table\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDescription of Nocardiopsis moroccensis\u003c/b\u003e \u003cb\u003esp. nov\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStrain ZS3416R2A is an aerobic, Gram-positive, spore-forming, and non-motile actinomycete that produces short spore chains and develops branched substrate mycelium and aerial hyphae, with the mycelium fragmenting as it ages. On ISP2 medium, colonies are powdery, with pale yellow aerial mycelium and strong brown substrate mycelium. Diffusible pigments are produced on ISP6 medium. Growth occurs at temperatures between 16\u0026ndash;50\u0026deg;C (optimum 28\u0026ndash;37\u0026deg;C), at pH values from 5.0 to 10.0 (optimum 7.0\u0026ndash;8.0), and in the presence of up to 10% (w/v) NaCl (optimum 5%), indicating moderate halotolerance.\u003c/p\u003e\u003cp\u003ePhylogenetic analysis based on the 16S rRNA gene sequence places strain ZS3416R2A within the genus \u003cem\u003eNocardiopsis\u003c/em\u003e, showing highest sequence similarity (99.03%) to \u003cem\u003eNocardiopsis terrae\u003c/em\u003e DSM 45157. A whole-genome phylogenomic tree based on core gene alignments confirmed this close relationship. The draft genome of ZS3416R2A assembles into a single circular contig of 6.1 Mb in size with a G\u0026thinsp;+\u0026thinsp;C content of 70.48 mol%, comprising 5,855 coding sequences, 5 rRNA operons, and 59 tRNA genes. Whole-genome comparisons further supported its distinctiveness: the ANI and dDDH values between ZS3416R2A and DSM 45157 are 95.07% and 66.4%, respectively, while the AAI and POCP are 95.87% and 82.4%, respectively.\u003c/p\u003e\u003cp\u003eThe predominant respiratory quinones are MK-10(H\u003csub\u003e6\u003c/sub\u003e), MK-10(H\u003csub\u003e4\u003c/sub\u003e) and MK-9(H\u003csub\u003e4\u003c/sub\u003e), accompanied by minor components including MK-9(H\u003csub\u003e6\u003c/sub\u003e), MK-10(H\u003csub\u003e2\u003c/sub\u003e), MK-10(H\u003csub\u003e8\u003c/sub\u003e), MK-10, MK-9, and MK-8(H\u003csub\u003e4\u003c/sub\u003e). The polar lipid profile consists of DPG, PC, PG, PE and PI. The major fatty acids are iso-C16:0, 10-methyl-C18:0, anteiso-C17:0, C18:0 and iso-C18:0 with additional components present in smaller amounts including 10-methyl-C17:0, iso-C14:0, anteiso-C15:0, C18:1 ω9c, 10-methyl-C16:0, iso-C17:0, and C16:0.\u003c/p\u003e\u003cp\u003eThe type strain is ZS3416R2A\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;CCMM B1332\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 120542\u003csup\u003eT\u003c/sup\u003e), isolated from saline soil collected from Lake Zima\u0026rsquo;s ecosystem, Morocco. The type strain is deposited in two distinct collections, CCMM and DSMZ under accession numbers [CCMM B1332ᵀ = DSM 120542ᵀ]. The species epithet \u003cem\u003emoroccensis\u003c/em\u003e (mo.roc.cen\u0026rsquo;sis. L. masc./fem. adj. moroccensis, pertaining to Morocco) is proposed.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe purpose of this study was to explore the bacterial diversity of Moroccan saline ecosystems, with a focus on Lake Zima (Sebkha of Zima), a Ramsar-listed hypersaline wetland that has remained poorly explored from a microbiological perspective. Among the isolates obtained, strain ZS3416R2A was selected for detailed characterization due to its distinctive morphology and halotolerant growth profile. Phylogenetic, genomic, phenotypic, and chemotaxonomic analyses were undertaken to clarify its taxonomic position within the genus \u003cem\u003eNocardiopsis\u003c/em\u003e and to assess its potential ecological and biotechnological significance.\u003c/p\u003e\u003cp\u003ePhylogenetic analysis of the 16S rRNA gene placed strain ZS3416R2A in the genus \u003cem\u003eNocardiopsis\u003c/em\u003e, with 99.03% sequence similarity to \u003cem\u003eN. terrae\u003c/em\u003e DSM 45157, isolated from saline soil in China (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, due to the high 16S rRNA gene similarity among \u003cem\u003eNocardiopsis\u003c/em\u003e species, this marker alone is insufficient for reliable species delineation, necessitating additional analyses (Chun et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chantavorakit et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gir\u0026atilde;o et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Whole-genome analyses therefore provided a more robust framework: the ANI between ZS3416R2A and DSM 45157 was 95.07%, and the dDDH value was 66.4%, both falling below species thresholds. In contrast, the AAI (95.87%) and POCP (82.4%) confirmed their placement within the same genus.\u003c/p\u003e\u003cp\u003eThe genomic comparisons also revealed notable differences between both strains. Approximately 25% of each genome consisted of strain-specific genes, consistent with earlier estimates that 20\u0026ndash;35% of bacterial genes are variable within a species (Martiny et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ventura et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These unique fractions underscore the high genomic plasticity of actinomycetes and suggest that both local adaptation and horizontal gene transfer may have shaped the divergence of ZS3416R2A and DSM 45157. Indeed, RGPs in ZS3416R2A were enriched in genes encoding transport systems and secondary metabolism enzymes, with lower GC content suggestive of horizontal acquisition, a mechanism long recognized as a driver of actinomycete diversification (Ochman et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Mobile genetic elements further highlighted the potential for differential adaptation. ZS3416R2A contained two AICEs, compared to four in DSM 45157. AICEs are known to mediate horizontal transfer and often carry accessory genes for antibiotic resistance, stress tolerance, or specialized metabolism (te Poele et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, three prophages (two \u003cem\u003eSiphoviridae\u003c/em\u003e and one unclassified) and a higher number of transposases (41 vs. 27) were detected in ZS3416R2A, again pointing to an active role of horizontal gene flux in its evolutionary history. Such elements may have contributed to niche-specific adaptations, despite both species being recovered from saline soils on different continents. This observation reinforces the notion that ecological similarity alone does not explain phylogenetic relationships in \u003cem\u003eNocardiopsis\u003c/em\u003e, other factors such as micro-environmental heterogeneity, historical biogeography, and lateral gene transfer may all play important roles (Martiny et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Martiny et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSupporting this evidence of genomic differentiation, ZS3416R2A displayed an expanded secondary metabolism potential relative to DSM 45157. Indeed, it harbored 19 BGCs, including 15 totally absent from its closest relative. Six of these showed no similarity to known entries in the MIBiG database, suggesting the capacity to produce novel metabolites. These findings are consistent with the view that actinomycetes remain a prolific source of new natural products, with nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and terpene pathways frequently linked to antibiotics, anticancer agents, and other valuable bioactive molecules (Ait Barka et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Genilloud, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). KEGG-based annotation further indicated that ZS3416R2A encodes a broader repertoire of metabolic pathways than DSM 45157, further suggesting the potential for biotechnological exploitation as a reservoir of novel enzymes or stress-adaptive functions possibly relevant to industrial processes (Alanzi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). CAZyme profiling also revealed an enrichment in the PL11 family (pectate lyases), which was absent in DSM 45157. These enzymes are of particular interest in agriculture and industry because of their role in degrading pectin-rich plant biomass and possible involvement in plant-microbe interactions and stress alleviation (Jayani et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ventura et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Such metabolic distinctiveness suggests that ZS3416R2A has adapted to its specific ecological niche, potentially shaped by the selective pressures of the Moroccan saline and arid environment.\u003c/p\u003e\u003cp\u003ePhenotypic and cultural characteristics provided additional evidence for differentiation. Compared to DSM 45157, strain ZS3416R2A exhibited denser aerial mycelium formation, more rapid growth, and stronger sporulation across different ranges of temperature, salinity, and pH. These traits may reflect ecological specialization, as previously observed for actinomycetes from extreme or marginal environments (Ventura et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In particular, the robust growth of ZS3416R2A under saline conditions suggests a high degree of halotolerance that may be advantageous in the fluctuating salt and arid regimes of Lake Zima. Chemotaxonomic markers further reinforced its distinctiveness. Although the overall profiles of quinones, polar lipids, and fatty acids were consistent with the genus \u003cem\u003eNocardiopsis\u003c/em\u003e, quantitative differences were evident. For instance, ZS3416R2A exhibited higher proportions of iso-C16:0 and anteiso-C17:0, a distinct distribution of minor menaquinones, and stronger representation of phosphatidylcholine compared to DSM 45157. Such differences, when combined with genomic and phenotypic traits, provide reliable chemotaxonomic signatures for species delineation within the genus.\u003c/p\u003e\u003cp\u003eIn conclusion, we isolated and characterized strain ZS3416R2A from saline soil in the salt ecosystem of Lake Zima, Morocco. Comprehensive polyphasic and comparative genomic analyses, including phylogenomic, phenotypic, and chemotaxonomic approaches, demonstrated that ZS3416R2A represents a novel species within the genus \u003cem\u003eNocardiopsis\u003c/em\u003e and, to our knowledge, the first one described in Morocco. This discovery expands our understanding of actinomycetota diversity in saline environments and underscores the potential of Moroccan ecosystems as sources of unique microbial taxa with possible biotechnological applications. We propose the name \u003cem\u003eNocardiopsis moroccensis sp. nov.\u003c/em\u003e for this new species, with strain ZS3416R2Aᵀ (=\u0026thinsp;CCMM B1332ᵀ = DSM 120542 ᵀ) designated as the type strain.\u003c/p\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 ZS3416R2Aᵀ (= CCMM B1332ᵀ = DSM 120542ᵀ) has been deposited in the NCBI GenBank under the accession number CP199747 and the BioProject number PRJNA1321125.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Partnership Hubert Curien (PHC) Maghreb (PHC 23MAG09) program and\u0026nbsp;by the CNRST Labeled Research Unit N°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., 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"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAit Barka, E., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H.-P., \u0026hellip; van Wezel, G. P. (2015). Taxonomy, Physiology, and Natural Products of Actinobacteria. Microbiology and Molecular Biology Reviews, 80(1), 1\u0026ndash;43. https://doi.org/10.1128/mmbr.00019-15\u003c/li\u003e\n\u003cli\u003eAlanzi, A. R. (2024). 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Genome Biology, 20(1). https://doi.org/10.1186/s13059-019-1891-0\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nocardiopsis, Novel species, Halotolerant, Zima Lake, Polyphasic taxonomy, Comparative analysis","lastPublishedDoi":"10.21203/rs.3.rs-7605207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7605207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSaline and arid ecosystems are recognized as promising reservoirs of novel actinomycetes with unique adaptations and secondary metabolism potential. In this study, a halotolerant actinomycetota strain, designated ZS3416R2A, was isolated from saline soil in the hypersaline wetland of Lake Zima, Morocco. Phylogenetic analysis using the 16S rRNA gene sequence showed 99.03% similarity to \u003cem\u003eNocardiopsis terrae\u003c/em\u003e DSM 45157, indicating a close affiliation with the genus \u003cem\u003eNocardiopsis\u003c/em\u003e. Whole-genome sequencing revealed a 6.1 Mb circular chromosome with a G\u0026thinsp;+\u0026thinsp;C content of 70.48 mol% and 5,855 coding sequences. Genome-based metrics (ANI 95.07%, dDDH 66.4%) supported its classification as a novel species distinct from \u003cem\u003eN. terrae\u003c/em\u003e, while AAI (95.87%) and POCP (82.4%) confirmed its placement within the genus. Comparative analyses revealed unique gene families, biosynthetic gene clusters, and regions of genomic plasticity in ZS3416R2A. Phenotypically, ZS3416R2A grew over a broad range of salinity (0\u0026ndash;15%), temperature (16\u0026ndash;37\u0026deg;C), and pH (7.0\u0026ndash;8.0), with denser aerial mycelium formation than DSM 45157, reflecting adaptation to the arid saline environment of Lake Zima. Chemotaxonomic characterization identified MK-10(H\u003csub\u003e6\u003c/sub\u003e), MK-10(H\u003csub\u003e4\u003c/sub\u003e) and MK-9(H\u003csub\u003e4\u003c/sub\u003e) as predominant menaquinones, iso-C16:0 and 10-methyl-C18:0 as major fatty acids, and diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol as major polar lipids. Based on these polyphasic evidences, strain ZS3416R2A represents a novel species within the genus \u003cem\u003eNocardiopsis\u003c/em\u003e, for which the name \u003cem\u003eNocardiopsis moroccensis\u003c/em\u003e sp. nov. is proposed. The type strain is ZS3416R2A\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;CCMM B1332\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 120542\u003csup\u003eT\u003c/sup\u003e).\u003c/p\u003e","manuscriptTitle":"Genome Sequencing and Comparative Analysis of Nocardiopsis moroccensis nov., a Halotolerant Actinomycetota from Moroccan Hypersaline Ecosystem","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 16:25:58","doi":"10.21203/rs.3.rs-7605207/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d86947a6-c042-42ad-8ed7-341ad0370dbb","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-03T17:23:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-28 16:25:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7605207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7605207","identity":"rs-7605207","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}