Characterization of a Boron-Tolerant Nocardia niigatensis Isolated from Boron-Rich Soils: Physiological, Enzymatic, and Genomic Insights

TL;DR

A boron-tolerant Nocardia niigatensis strain was isolated from boron-rich soils and shown to have potential for bioremediation and industrial applications.

Contribution

The study provides a comprehensive characterization of a boron-tolerant Nocardia niigatensis strain with physiological, enzymatic, and genomic insights.

Findings

The strain exhibited robust growth at boron concentrations up to 50 mM, indicating potential for bioremediation.

The strain showed positive L-glutaminase activity and a broad enzymatic profile, suggesting industrial relevance.

Metagenomic analysis revealed differences in microbial communities linked to mineral types in boron-rich soils.

Abstract

In this study, a Nocardia niigatensis strain was isolated from boron-rich mining soils in the Bigadiç region of Türkiye and comprehensively characterized. The primary aim of this study was to isolate boron-tolerant Nocardia species and evaluate their physiological, enzymatic, and biochemical profiles. Selective isolation techniques were employed to obtain Nocardia isolates, and species-level identification was achieved using both 16S rRNA gene sequencing and MALDI-TOF MS analysis, which consistently confirmed the isolate as N. niigatensis. In addition to molecular identification, the morphological, physiological, and biochemical characteristics of the strain were extensively investigated. The strain demonstrated notable boron tolerance, exhibiting robust growth at concentrations up to 50 mM, highlighting its potential applicability in the bioremediation of boron-contaminated environments. Physiological assays further revealed moderate halotolerance and a mesophilic growth profile, with optimal growth observed at 27–37 °C. Enzymatic screening indicated positive L-glutaminase activity, an enzyme of considerable industrial relevance. Moreover, API ZYM profiling revealed a broad enzymatic spectrum, including esterases, arylamidases, phosphatases, and glucosidases, suggesting substantial metabolic versatility. Antibiotic susceptibility testing showed sensitivity to doxycycline, tobramycin, and erythromycin, whereas resistance was observed against imipenem and several β-lactam antibiotics. Metagenomic analysis of boron-rich soils from two distinct mining sites revealed marked differences in microbial community composition, with variations in Actinobacteria abundance associated with mineral type. Overall, these findings emphasize the adaptive capacity and biotechnological potential of environmental Nocardia strains inhabiting chemically stressful ecosystems, warranting further genomic and metabolomic investigations.

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1. Introduction

Nocardia is a genus of Actinobacteria comprising Gram-positive, branched, obligate aerobic, and partially acid-fast bacteria that are isolated from both environmental sources and clinical specimens [1]. Members of this genus are classified as both saprophytic and opportunistic pathogens, and are commonly found in various natural habitats, particularly soil, decaying organic matter, and water [2]. Nocardiosis is a medically important disease that primarily affects immunocompromised patients, though it can also occur in healthy individuals. As soilborne opportunistic pathogens, Nocardia species present significant clinical challenges due to their varied clinical presentations, limited prevention methods, and a treatment process that often requires prolonged antimicrobial therapy over months or years [3]. Nocardia species are of interest not only due to their pathogenic potential but also because of their ability to produce secondary metabolites, tolerate environmental stress conditions, and perform rare enzymatic activities [4].

The presence of microorganisms in environments contaminated with heavy metals or metalloids such as boron is of growing interest in microbial ecology and environmental biotechnology. Boron can accumulate in soil at toxic concentrations, particularly in regions with intense industrial or agricultural activity [5]. As one of the world’s major boron reserve holders, Turkey represents a strategically significant region for such investigations [6]. Microorganisms isolated from these boron-rich areas are valuable for their boron tolerance and potential bioremediation capabilities. The ability of actinobacteria to adapt to boron stress has been proposed in a limited number of studies [7].

Recent research has shown that certain Nocardia species possess the enzymatic capacity to produce industrially relevant enzymes such as lipase, esterase, and glutaminase [4,8]. These features highlight their potential utility in the biotransformation and recovery of organic pollutants. Moreover, their resistance profiles to various antibiotics and the genetic mechanisms underlying this resistance underscore their clinical relevance [9]. However, environmental Nocardia strains have not been studied as extensively as their clinical counterparts.

Accurate classification of Nocardia species depends critically on molecular identification techniques, especially 16S rRNA gene sequencing and MALDI-TOF MS. While 16S rRNA analysis offers high-resolution phylogenetic insights, MALDI-TOF MS provides rapid and cost-effective species-level identification [10,11]. Nevertheless, in some cases, the resolution of 16S rRNA is insufficient, necessitating multilocus sequence analysis (MLSA) involving genes such as gyrB and rpoB [12]. The gyrB and rpoB genes are universal housekeeping genes essential for the fundamental cellular life cycle across all bacteria. Functionally, gyrB encodes the β-subunit of DNA gyrase, which is critical for DNA replication, whereas rpoB encodes the β-subunit of RNA polymerase, which is responsible for the transcription process [12].

The aim of this study was to isolate boron-tolerant Nocardia species from the boron-rich soils of the Bigadiç region in Türkiye and to characterize their biological and genomic potential using a multidisciplinary approach. In this context, the isolate’s morphological, physiological, and biochemical profiles were evaluated, along with its tolerance to boron, salt, and temperature stress. Species-level identification was performed via 16S rRNA gene sequencing and MALDI-TOF MS. Additionally, metagenomic analyses were conducted to assess the impact of boron mining on microbial diversity.

This work emphasizes the importance of evaluating Nocardia species not only from a clinical perspective but also in the context of environmental and industrial microbiology. It expands our understanding of their adaptive capabilities and biotechnological potential in stress-prone ecosystems such as boron-contaminated environments.

2. Materials and Methods

2.1. Study Site and Sample Collection

Samples were collected from two distinct borate mine sites in the Bigadiç region of Balikesir, Türkiye: the ulexite-rich BR1 (37°16′39.1″ N 43°44′44.1″ E) and the colemanite-rich BR2 (37°14′39.9″ N 43°38′36.4″ E). Approximately 100 g of soil was collected from each site at a depth of about 10 cm after removing surface organic matter. For comprehensive bacterial analysis, samples were placed in separate sterile plastic bags, stored at 4 °C, and utilized for a dual-purpose approach: the culture-dependent isolation of boron-tolerant strains and culture-independent 16S rRNA gene amplicon sequencing to profile the total microbial community. Representative images of the sampling locations are presented in Figure 1.

2.2. Isolation and Purification of Nocardia Species

In this study, the classical dilution and surface spreading method was employed for the isolation of Nocardia species. Portions of 25 g were taken from soil samples and transferred to Erlenmeyer flasks containing 250 mL of 0.9% sodium chloride solution to prepare 10^−1^ suspensions. To separate microorganism spores and mycelia adhered to soil colloids, these solutions were incubated in a shaking water bath at 60 °C and 100 rpm for 1 h, which also reduced contamination from vegetative forms. Serial dilutions were performed up to 10^−4^, and aliquots from the 10^−3^ and 10^−4^ dilutions were inoculated onto Streptomyces Agar, Actinomycetes Isolation Agar, and Medium 65 Agar (GYM Streptomyces Medium; DSMZ (Braunschweig, Germany)) supplemented with cycloheximide (50 μg/mL), nystatin (50 μg/mL), and novobiocin (0.5 μg/mL). Following incubation at 27 °C, developing colonies were examined and counted. The isolated strain displayed typical Nocardia morphology: Gram-positive, branching filaments, and forming dry, wrinkled colonies with cream-colored aerial mycelia. These features are consistent with previously described Nocardia strains, such as those reported by Kageyama et al. [13]. Cultural purity was ensured through at least three successive reseedings using the streak plate method and verified by consistent colony morphology and microscopic examination. All isolation and purification procedures were performed in triplicate to ensure reproducibility.

For the long-term preservation of the purified isolates, a cryopreservation protocol was followed. Two loopfuls of pure culture were harvested from the agar surface and suspended in cryotubes containing sterilized 20% (v/v) glycerol solution prepared with distilled water. These cryopreserved stocks were stored at −20 °C for subsequent studies. Prior to their use in experimental assays, the isolates were reactivated by streaking onto Medium 65 agar and incubating at 27 °C for 7 days.

Morphological characterization of the isolate was performed at two levels using light microscopy. Colony morphology was examined under a light microscope (40× magnification). Gram staining was carried out according to standard protocols, and slides were observed under a compound stereomicroscope (10×) equipped with an oil immersion objective (1000× total magnification). Aerial mycelium, branching patterns, and spore chain structures were visualized and documented using a mounted digital camera.

2.3. Soil DNA Extraction and 16S rRNA Gene Amplicon Sequencing

Total microbial DNA extraction and Illumina-based metagenomic sequencing were conducted by BM Labosis (Ankara, Türkiye), and raw sequencing data were obtained in FASTQ format.

Total environmental genomic DNA was extracted from soil samples using 1 g of homogenized soil per sample. Homogenization was performed by grinding the samples in a porcelain mortar under liquid nitrogen. The resulting powdered samples were subjected to DNA isolation using the Eurx GeneMATRIX Soil DNA Purification Kit (Gdansk, Poland) according to the following protocol:

• Prior to isolation, 30 µL of SL solution was added to the DNA binding column for column conditioning.
• Approximately 250 mg of soil sample was transferred to a bead-containing tube, followed by the addition of 60 µL of Lyse SL solution and thorough mixing by inversion.
• Tubes were horizontally fixed on a vortex mixer and vortexed at maximum speed for 10 min.
• The bead tube was centrifuged at 20,000× g for 2 min, and 400 µL of the supernatant was transferred to a new Eppendorf tube. Then, 400 µL of PR solution was added, mixed by vortexing, and incubated on ice for 5 min.
• After centrifugation at maximum speed for 1 min, 600 µL of the supernatant was transferred to a fresh tube. Subsequently, 600 µL of Sol SL solution and 200 µL of 96% ethanol were added, and the mixture was vortexed thoroughly.
• A total of 600 µL of the mixture was loaded onto the binding column and centrifuged at 11,000× g for 1 min. This step was repeated for the remaining liquid.
• The column was washed twice: first with 500 µL of Wash SLX1 and then with 500 µL of Wash SLX2, with centrifugation at 11,000× g for 1 min after each wash.
• Finally, the column was transferred to a clean tube, and 50 µL of Elution Buffer was added. After incubation at room temperature for 2 min, the column was centrifuged at 11,000× g for 1 min. The elution step was repeated once, and the purified DNA was stored at −20 °C.

Following extraction, DNA samples were enzymatically fragmented to appropriate lengths for sequencing platforms. Library preparation was performed using the Illumina DNA Prep Kit (Illumina, San Diego, CA, USA, #20060059) according to the manufacturer’s instructions. During library construction, samples were indexed using IDT for Illumina^®^ DNA/RNA UD Indexes Set C (Illumina, USA, #20042666) to attach Illumina adapter sequences. After PCR amplification, library concentration was quantified using a Qubit fluorometer, and samples were normalized prior to sequencing.

Sequencing was conducted by BM Labosis (Ankara, Türkiye) on a NovaSeq 6000 platform (Illumina, USA) using a NovaSeq 6000 S4 Reagent Kit (Illumina, USA, #20028312). Paired-end sequencing (2 × 150 bp) was performed in accordance with the manufacturer’s guidelines.

Taxonomic profiling was performed using genus-level abundance tables derived from the raw data. All subsequent analyses were carried out in the Python (v3.x) [14] environment utilizing the pandas [15], numpy [16], matplotlib [17], and seaborn [18] libraries. The ten most abundant bacterial genera were identified, and both alpha diversity (Shannon and Simpson indices) and beta diversity (Principal Coordinates Analysis [PCoA] based on Euclidean distance) analyses were conducted. Additionally, genus-level distributions within the phylum Actinobacteriota were visualized using comparative heatmaps to assess inter-sample variation.

For the visual representation of taxonomic hierarchy and distribution, Krona charts were generated via the MG-RAST platform (https://mg-rast.org/; accessed on 16 February 2025) as described by Komuroglu et al. [19]. The two biosamples analyzed in this study were retrieved from the NCBI BioSample database under the accession numbers SAMN53263815 and SAMN53263816.

2.4. Color Grouping

For color grouping, a total of 8 potential Nocardia strains that were purified were inoculated onto an oatmeal agar [20] culture medium. Isolates inoculated by the streak plate method were incubated at 27 °C for 14 days, after which the aerial mycelium color and substrate mycelium colors were determined according to a color chart, and grouping was performed. Consequently, all isolates were grouped by considering the colony color and substrate mycelium color.

2.5. Antimicrobial Activity Studies

The antimicrobial activity of the purified and cryopreserved (20% glycerol, −20 °C) Nocardia isolate was evaluated against a panel of five reference microorganisms including pathogenic and non-pathogenic strains using the cross-streak method as described by Ertaş and Özdemir (2025) [21]. The test organisms included the following standard strains and clinical isolates: Escherichia coli ATCC 25922, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853. Prior to the assay, each Nocardia isolate was reactivated by streaking onto Mueller–Hinton Agar (MHA) plates and incubating at 27 °C for 7 days. For the antimicrobial activity test, a single streak of each activated Nocardia isolate was inoculated along the center of a fresh MHA plate. The plates were then incubated at 27 °C for 7 days to allow adequate growth and potential production of antimicrobial metabolites. After this incubation, the indicator bacterial strains were streaked perpendicularly to the Nocardia growth, starting from the edge of the Nocardia streak toward the periphery of the plate. The plates were further incubated at 37 °C for 18–24 h to allow growth of the test organisms. Antimicrobial activity was assessed by measuring the presence and width of any inhibition zone (clear area) around the test organism streaks. A visible zone of inhibition was interpreted as sensitivity, while the absence of such a zone indicated resistance.

2.6. Determination of Temperature and Salt Tolerances

ISP-2 medium was used to determine the temperature and salt tolerances of the purified Nocardia isolates [22]. Inoculations of the isolates were performed using the streak plate method. For temperature tolerance experiments, the inoculated Nocardia strains were incubated at temperatures of 4, 10, 27, 37, 45, 50, and 55 °C for 7 days. For salt tolerance, NaCl was added to each prepared ISP-2 medium at concentrations of 0, 1, 2.5, 5, 7.5, 10, 15, and 20% (w/v), and the plates were incubated at 27 °C for 7 days. Cultures showing growth after incubation were considered positive results.

2.7. Evaluation of Extracellular Hydrolytic Enzyme Potential

2.7.1. Amylase

For amylase enzyme production, Bennett’s agar was prepared with the addition of 2% starch and incubated at 27 °C for 4 days. After incubation, Lugol’s iodine solution was added to the medium, and zones of clearing around the colonies were selected as positive results [23,24].

2.7.2. Cellulase

For cellulase enzyme production, Bennett’s agar was prepared with the addition of 1% carboxymethyl cellulose (dissolved in distilled water) and incubated at 27 °C for 4 days. A 1% Congo red solution was added to the medium, and the formation of clear yellow zones around the colonies was selected as a positive result [25].

2.7.3. L-Asparaginase

For L-asparaginase enzyme production, M9 agar was prepared with the addition of 1% asparagine and incubated at 27 °C for 4 days. Three drops of phenolphthalein solution were added to the medium, and colonies exhibiting a pink color change were selected as positive results [26].

2.7.4. L-Glutaminase

For L-glutaminase enzyme production, M9 agar was prepared with the addition of 1% glutamine and 1% phenol red solution, and the pH was adjusted until the color turned yellow. The medium was incubated at 27 °C for 4 days. Colonies exhibiting a pink color change around the medium were selected as positive results [27].

2.7.5. Protease

For protease enzyme production, Bennett’s agar was prepared with the addition of 1% skim milk powder and incubated at 27 °C for 4 days. Zones of clearing around the colonies on the medium were selected as positive results [24].

2.7.6. Lipase

For lipase enzyme production, Bennett’s agar was prepared with the addition of 2% Tween 80 and incubated at 27 °C for 4 days. A 0.001% Rhodamine B solution was added to the medium, and colonies exhibiting zones of clearing around them were selected as positive results [24].

2.7.7. Xylanase

For xylanase enzyme production, Bennett’s agar was prepared with the addition of 1% xylan and incubated at 27 °C for 4 days. Zones of clearing around the colonies on the medium were selected as positive results [24].

2.8. API-ZYM Tests

The enzymatic profile of Nocardia sp. soil isolates was determined using the API-ZYM system (bioMérieux, Marcy-l’Étoile, France). This system comprises 20 microtubes, containing 19 enzyme substrates and one control. The substrates included alkaline phosphatase, esterases, lipases, arylamidases, trypsin, chymotrypsin, acid phosphatase, phosphoramidase, galactosidases, glucuronidase, glucosidases, N-acetyl-β-glucosaminidase, mannosidase, and fucosidase. Test strain suspensions were prepared in sterile distilled water and adjusted to a McFarland No. 5 standard. Tubes were inoculated according to the manufacturer’s instructions and incubated at 37 °C for 4 h. Following reagent addition, color intensity was recorded on a scale of 0–5 points. An index of 2 or more was considered positive. All strains were tested in duplicate to ensure result reproducibility.

2.9. Identification of Microorganism Species by MALDI-TOF MS

The identification of bacterial isolates was performed using the MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry, Bruker Daltonics GmbH, Bremen, Germany) Microbial Identification system at the Hatay Mustafa Kemal University Plant Health Clinic Application and Research Center. In this procedure, pre-extraction for protein isolation from pure bacterial cultures was conducted using the ethanol-formic acid method. Subsequently, the spectra obtained with the device’s flex control software (Biotyper 3.0; Microflex LT; Bruker Daltonics GmbH, Bremen, Germany) were compared using the Maldi Biotyper Real-Time Classification (RTC) (Version 4.1) software to proceed with identification. The analysis results were evaluated using the following score ranges: high-probability species identification, 2000–3000 (green); genus-level and probable species-level identification, 1700–1999 (yellow) [28].

2.10. Antibiotic Susceptibility Test

The antibiogram potential of the isolates was determined using the modified Kirby-Bauer disk diffusion method on Muller-Hinton agar, in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI Performance Standards for Antimicrobial Susceptibility Testing) [29]. Each isolate was spread onto a separate nutrient agar plate, an antibiotic disk was placed on top, and the plates were incubated at 37 °C for 24 h. The antibiotic disks were distributed onto the medium using a sterile forcep, and each disk was gently pressed onto the agar surface.

Thirteen different antimicrobial agents were utilized, and the results were documented in accordance with CLSI guidelines. These agents included Doxycycline 30 mcg/disk (DO 30), Vancomycin 30 mcg/disk (VA 30), Rifampicin 30 mcg/disk (RF 30), Tobramycin 10 mcg/disk (TOB 10), Enoxacin 10 mcg/disk (EN 10), Imipenem 10 mcg/disk (IPM 10), Sulbactam-Ampicillin 10–10 mcg/disk (SAM 20), Neomycin 10 mcg/disk (N 10), Clindamycin 5 mcg/disk (CC 5), Nitrofurantoin 30 mcg/disk (FM 30), Penicillin 10 U/disk (P 10), Oleandomycin 15 mcg/disk (OL 15), and Erythromycin 15 mcg/disk (E 15).

2.11. Determination of the Boron Tolerance

Boron tolerance of the selected mining isolates was carried out on Medium 65 (GYM Streptomyces Medium) supplemented with different concentration of boron. The concentration range of boron was selected from 2 to 300 mM. The working concentrations of boron were prepared from 1 M stock solution of boric acid. The stock solution of boron was prepared in sterilized deionized water. Due to the heat-labile nature of boric acid, the stock solution was not autoclaved; instead, it was sterilized using a 0.22 mµ pore size membrane filter and subsequently added to Medium 65 under aseptic conditions [5]. Minimum inhibitory concentration (MIC) was evaluated until the selected mining isolates were unable to grow on boron containing Medium 65. Based on this evaluation, MIC was determined at 27 °C in 3 days.

2.12. DNA Isolation, Polymerase Chain Reaction (PCR), and 16S rRNA Gene Sequencing

DNA isolation was performed according to the method described by Sharef et al. [30]. The phylogenetic analysis of the 16S rRNA gene region involved a multi-step process. Following sequencing, the resulting ab1 chromatogram files were meticulously examined using Codon Code Aligner V.6.0.2. Low-quality base sequences, particularly those at the ends of the reads and marked with ‘N’, were meticulously trimmed to generate high-quality sequences. Subsequently, the 16S rDNA nucleotide sequences of all isolates were subjected to BLAST (version 2.16.0) analysis against the NCBI GenBank database to identify closely related type strains, whose accession codes were then retrieved.

For phylogenetic tree construction, both the generated isolate sequences and the selected reference sequences from GenBank were imported into MegaX (version 10.2.6) software in FASTA format. A Clustal W multiple sequence alignment was then performed to align homologous regions. The most appropriate evolutionary model for phylogenetic inference was determined using the JModelTest tool within MegaX (version 10.2.6). Finally, a phylogenetic tree was constructed using the Maximum Likelihood algorithm implemented in the MegaX (version 10.2.6) software package, providing a robust representation of the evolutionary relationships among the analyzed isolates [31]. The 16S rRNA gene sequence of the isolate BR005 has been deposited in the GenBank database under the accession number PX831500.

3. Results and Discussion

3.1. Microbiological Characteristics of Isolated Strains

Eight strains potentially belonging to the genus Nocardia were isolated from two soil samples based on their initial colony morphology. Preliminary morphological and biochemical screenings revealed consistent phenotypic profiles across all eight isolates, suggesting that they belonged to the same species (Figure 2). Microscopic examination of the representative strain, BR005, showed Gram-positive, branching filamentous structures and the formation of spore chains (Figure 3). When cultured on oatmeal agar, the colonies appeared dry and wrinkled, with both aerial and substrate mycelia displaying a distinct cream coloration.

These morphological features are in complete accordance with the descriptions provided by Kageyama et al. (2004) [13] for the type strain of Nocardia niigatensis. Although Kageyama et al. (2004) [13] originally characterized this species from clinical specimens, the classical phenotypic traits exhibited by the environmental isolate BR005 confirm the taxonomic identification at the morphological level. Subsequent species-level identification via 16S rRNA gene sequencing and MALDI-TOF MS further confirmed that all eight strains belonged to N. niigatensis. Given this genetic and phenotypic uniformity, BR005 was selected as the representative strain for comprehensive physiological, enzymatic, and genomic characterization to ensure methodological efficiency and clarity in data presentation.

3.2. Enzymatic Profile and Metabolic Potential

The enzymatic profile of N. niigatensis BR005, as determined by API ZYM tests and specific activity assays, reflects a broad metabolic capacity, including positive results for butyrate esterase, esterase-lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, phosphohydrolase, α-glucosidase, and β-glucosidase (Table 1). This enzymatic repertoire suggests the strain can degrade a wide range of substrates, a characteristic often linked to the metabolic versatility of soil-dwelling Actinobacteria and their role in nutrient cycling [4,8]. The positive detection of L-glutaminase (Figure 4) further indicates biotechnological relevance, consistent with previous reports of Nocardia species producing industrially valuable enzymes, such as lipases applicable in biodiesel production and wastewater treatment [32]. Moreover, the presence of arylamidases points to efficient nitrogen cycling through protein degradation, while α- and β-glucosidase activities reflect an ability to metabolize complex carbohydrate substrates commonly associated with Actinobacteria adapted to nutrient-poor or chemically stressed environments [32]. This enzymatic versatility, coupled with its notable boron tolerance, positions N. niigatensis BR005 as a promising candidate for environmental bioremediation and various biotechnological applications, particularly in heavy-metal- or metalloid-contaminated ecosystems [7,33].

3.3. Tolerance Characteristics

Regarding boron tolerance, the isolate’s “very strong” growth at 10–50 mM boron concentrations suggests this strain’s potential for survival and even bioremediation of this element in boron-contaminated environments. The role of actinomycetes like Nocardia in mitigating boron contamination has been previously emphasized [33]. However, the absence of growth at 150–200 mM boron concentrations indicates that tolerance is within specific limits (Table 2).

In salt tolerance tests, the strain’s growth at 1%, 2.5%, and 5% salt concentrations suggests that this Nocardia strain is moderately halotolerant (Table 2). The lack of growth at 7.5–20% salt concentrations indicates a limited adaptation capacity to extremely saline environments. This finding may be consistent with the environmental conditions of the strain’s isolation source.

Upon examining temperature tolerance, the Nocardia strain’s growth at 27 °C and 37 °C confirms its mesophilic nature (Table 2). The absence of growth at low and high temperatures such as 4 °C, 10 °C, 45 °C, 50 °C and 55 °C reveals that the strain exhibits optimal activity within a narrow temperature range. This characteristic necessitates strict control of temperature for the successful laboratory cultivation of the strain and for maximizing its potential applications.

3.4. Antimicrobial Activity and Antibiotic Susceptibility

The isolated Nocardia strain exhibited no antimicrobial activity against any of the tested pathogenic microorganisms (Escherichia coli ATCC 25922, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853), suggesting its limited potential for direct antibiotic production under the tested conditions (Table 3). However, this lack of antibacterial activity does not preclude its potential to produce other bioactive metabolites. The genus Nocardia is well-documented as a prolific source of diverse secondary metabolites, including antifungals, immunomodulators, and anticancer agents, highlighting its metabolic potential beyond antimicrobial activity [4]. Therefore, more detailed metabolomic analyses could reveal whether this specific strain produces compounds with different biological activities.

From a clinical perspective, Nocardia species are recognized as important opportunistic pathogens, primarily causing infections in immunocompromised individuals, such as those with HIV, organ transplants, or chronic lung disease, but they can also affect immunocompetent hosts, leading to pulmonary, cutaneous, or disseminated nocardiosis [34]. The pathogenicity is significant, with mortality rates for disseminated infections reported to be as high as 85% [34,35].

The antibiotic susceptibility profile of our environmental Nocardia niigatensis isolate was evaluated to assess its inherent resistance mechanisms and potential clinical relevance. The strain showed ‘very strong’ susceptibility to erythromycin and tobramycin. This aligns with susceptibility patterns reported for some Nocardia species in large-scale studies, though significant interspecies variability exists. For instance, while N. farcinica often shows high resistance to tobramycin, other species like N. cyriacigeorgica and N. otitidiscaviarum can exhibit higher susceptibility rates [35,36]. Conversely, the resistance observed in our strain to imipenem, sulbactam-ampicillin, nitrofurantoin, and penicillin reflects common intrinsic resistance patterns within the genus. This is consistent with the widespread resistance to β-lactams reported in clinical isolates, particularly in species like N. otitidiscaviarum, which often shows pan-β-lactam resistance potentially mediated by genes like blaAST-1 [35]. Furthermore, the susceptibility of our isolate to doxycycline and clindamycin suggests potential alternative treatment options, which is noteworthy given the rising concern of multidrug resistance (MDR) in clinical Nocardia. Recent genomic studies report MDR rates as high as 38.5% among clinical isolates in China, with some species like N. otitidiscaviarum showing 100% MDR rates [35]. These comparisons underscore the critical importance of accurate species identification in predicting antibiotic susceptibility and guiding effective therapy, as resistance profiles are highly species-dependent [35,37,38].

3.5. 16S rRNA Gene Sequencing and Taxonomic Profiling

The 16S rRNA gene amplicon sequencing analysis was performed on soil samples BR1 and BR2, collected from ulexite and colemanite boron deposits, respectively. The results demonstrate a polyphyletic microbial diversity within the investigated soil samples. Taxonomic distribution at the phylum level revealed that Firmicutes was the most dominant group in the BR1 (ulexite) sample, accounting for 49.89%, followed by Bacteroidota (35.98%) and Proteobacteria (5.75%). Actinobacteriota represented 3.7% of the total community in this sample. In the BR2 (colemanite) sample, Firmicutes (43.06%), Bacteroidota (31.5%), and Proteobacteria (15.86%) were the primary phyla, while Actinobacteriota accounted for 3.34%.

These distinct geological structures, characterized by their dominant minerals (colemanite, Ca_2_B_6_O_11_·5H_2_O, and ulexite, NaCaB_5_O_6_(OH)6·5H_2_O), impose significant selective pressures on the soil microbiome. Geochemical variations directly influence dissolved boron concentration, pH, and nutrient bioavailability, thereby shaping microbial community structure [39].

Alpha diversity analysis, including the Shannon and Simpson indices, indicated that the BR1 sample possessed significantly higher diversity values compared to BR2 (Figure 5). This suggests that ulexite mine soil harbors a richer and more balanced microbial community, whereas the limited diversity in BR2 implies that colemanite-specific conditions are highly selective, favoring only a few well-adapted microbial groups [40].

Krona visualizations (Figure 6 and Figure 7) further illustrate that microbial community structure differs between the two samples at both phylum and genus levels, highlighting taxon-specific variability depending on mineral type. Given the study’s focus on boron-tolerant Nocardia (a member of Actinobacteriota), subsequent analysis and the heatmap presentation (Figure 8) specifically targeted this phylum.

The sequencing data substantiate our culture-dependent isolation strategy: the log abundance of the Nocardia genus in the BR1 sample (≈4.8) was approximately twice that in BR2 (≈2.5). This higher metagenomic prevalence in the ulexite sample reinforces the rationale for selecting this environment for targeted isolation of boron-tolerant strains.

The heatmap of Actinobacterial genera (Figure 8) shows that certain genera were more abundant in BR1, suggesting either inherent richness or specific adaptation to ulexite geochemistry. This contrast supports the hypothesis that Actinobacteriota members undergo selection based on the mineral matrix.

Overall, the 16S rRNA gene amplicon sequencing findings validate the culturing approach and highlight additional abundant Actinobacterial genera in BR1 (e.g., Bifidobacterium, Collinsella, Senegalimassilia) as promising targets for future isolation of novel boron-tolerant strains. The community differences reflect the geochemical distinction between ulexite and colemanite deposits, underscoring the profound impact of mineralogy on microbial diversity.

3.6. 16S rRNA Gene Sequencing and MALDI-TOF MS for Species Identification

In this study, bacterial isolate BR005 was identified to the species level using two complementary methods: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA gene region sequencing. Both methods consistently identified the isolate as Nocardia niigatensis, though they differed in terms of precision and confidence of identification.

MALDI-TOF MS offers a rapid and cost-effective method for microbial identification based on protein profiles. In the MALDI-TOF MS analysis of isolate BR005, the identification as Nocardia niigatensis was classified as ‘probable’ with a log (score) of 1.782. This value, according to the manufacturer’s criteria, falls within the 1.700–1.999 range, which signifies genus-level and probable species-level identification’. This result underscores the method’s limited discriminatory power in certain instances. A recent study reported that the Bruker Biotyper 3.1 system identified Nocardia species with 84.2% accuracy, whereas the updated MBT Compass 4.1 software increased this accuracy to 98.7%. These findings clearly demonstrate that the success of MALDI-TOF MS is dependent on the comprehensiveness and recency of the database utilized.

However, MALDI-TOF MS may be limited, particularly for rare species or those not sufficiently represented in the database [11]. Additionally, the reproducibility of results can decrease as protein profiles may vary depending on environmental conditions and the bacterial growth phase [41].

In contrast, 16S rRNA gene region analysis offers more robust and reliable information, especially for classifying phylogenetically complex groups like actinomycetes [42,43] The sequence data for isolate BR005, through BLAST (version 2.16.0) comparisons and a phylogenetic tree constructed with the Maximum Likelihood algorithm, clustered with Nocardia niigatensis reference strains and was supported by high bootstrap values (Figure 9). This result once again demonstrates that 16S rRNA analysis is the gold standard in microorganism systematics [37,44].

While 16S rRNA gene analysis is widely regarded as the “gold standard” in microbial systematics, it may not always provide sufficient discrimination due to high sequence similarity among certain Nocardia species. In such cases, the limited resolution of 16S rRNA analysis necessitates the inclusion of advanced genomic techniques such as Multilocus Sequence Analysis (MLSA), as well as the integration of morphological data. Modern taxonomic standards do not rely solely on molecular data for species delineation; instead, they advocate a polyphasic approach in which molecular findings are substantiated by classical morphological descriptions. In this context, the updated taxonomic framework proposed by Nouioui et al. (2018) [45] underscores the critical role of integrating morphological traits with genomic evidence for the classification of phylogenetically complex genera such as Nocardia. The typical phenotypic features observed in strain BR005 including cream-colored branching filaments and spore chain formation confirm its identity at the morphological level and reinforce the taxonomic reliability established by genetic analyses.

However, for some Nocardia species, the 16S rRNA gene region might not provide sufficient differentiation. In such cases, Multilocus Sequence Analysis (MLSA), based on the analysis of additional gene regions like gyrB, rpoB, and hsp65, can provide more accurate identification [12]. In this study, however, 16S rRNA gene analysis was sufficient for the reliable identification of the BR005 isolate.

4. Conclusions

In this study, a Nocardia niigatensis strain (BR005) was successfully isolated from boron-rich mining soils in the Bigadiç region of Türkiye and subjected to a comprehensive characterization. Polyphasic identification, utilizing both 16S rRNA gene sequencing and MALDI-TOF MS, consistently confirmed the isolate’s identity, with 16S rRNA analysis serving as the “gold standard” for precise phylogenetic placement.

The physiological profile of the strain revealed robust growth at boron concentrations up to 50 mM and moderate halotolerance (up to 5% NaCl), highlighting its adaptive capacity and potential utility in the bioremediation of boron-contaminated or saline environments. Enzymatic screenings demonstrated a broad metabolic repertoire, most notably L-glutaminase activity and a wide range of esterases, arylamidases, and glucosidases. These findings suggest that the strain is well-equipped to degrade complex organic matter and holds significant value for industrial and biotechnological applications.

While the isolate exhibited no direct antimicrobial activity against the tested pathogens, its antibiotic susceptibility profile—characterized by sensitivity to erythromycin and tobramycin but resistance to imipenem and other β-lactams—provides critical insights into the intrinsic resistance mechanisms of environmental Nocardia. Furthermore, metagenomic analysis demonstrated that the mineral matrix (ulexite vs. colemanite) significantly influences microbial diversity, with ulexite deposits supporting a richer and more balanced community where Actinobacteria members undergo selection based on specific geochemical conditions.

Ultimately, the results of this study emphasize the importance of environmental Nocardia as a reservoir of specialized enzymes and stress-tolerance mechanisms. This strain represents a promising candidate for future genomic and metabolomic investigations aimed at discovering novel bioactive compounds and enhancing environmental sustainability.