Isolation and Characterization of a Novel Sulfur-Oxidizing Stutzerimonas Species from Hydrothermal Sediments and Its Adaptation to the Hydrothermal Environment

TL;DR

A new sulfur-oxidizing Stutzerimonas species was isolated from hydrothermal sediments and shown to adapt to extreme environments through specific genes and mineral interactions.

Contribution

The study isolates and characterizes a novel Stutzerimonas species with sulfur-oxidizing capabilities and identifies its adaptation mechanisms in hydrothermal environments.

Findings

Strain 381-2T oxidizes thiosulfate to tetrathionate and encodes the tsdA gene.

The strain influences sulfide mineral weathering and metal ion reprecipitation.

Genomic analysis shows widespread tsdA and metal resistance genes in Stutzerimonas.

Abstract

Stutzerimonas, a genus newly separated from the Pseudomonadaceae family in 2022, has attracted considerable attention due to its diverse metabolic capabilities and environmental adaptability. However, the mechanisms underlying its sulfur-oxidizing capacity and survival strategies in extreme environments remain poorly understood. Clarifying potential sulfur-oxidizing microbial groups contributes to a more accurate understanding of energy flow and elemental cycling in hydrothermal ecosystems. In this study, we isolated and identified a sulfur-oxidizing strain, designated 381-2T, from sediments in the Tianxiu hydrothermal field of the northwest Indian Ocean, and proposed it as a new species of Stutzerimonas. Physiological characterizations demonstrated that strain 381-2T could oxidize thiosulfate to tetrathionate and encoded the key sulfur oxidation gene tsdA. Cultivation with sulfide minerals showed that strain 381-2T could influence sulfide mineral weathering through metabolic activities, such as pH regulation, and potentially promote the reprecipitation of metal ions on the microbial surface. Comparative genomic analysis of 322 Stutzerimonas genomes further revealed the widespread presence of the tsdA gene and metal resistance genes, suggesting potential adaptive strategies for survival in hydrothermal environments. This study expands the understanding of Stutzerimonas species and provides insights into their ecological roles in hydrothermal systems.

Figures

Full text

1. Introduction

Deep-sea hydrothermal vent ecosystems represent one of the most unique habitats on Earth [1,2]. In these sulfide-rich environments, the oxidation of reduced sulfides is a primary energy source for microbial communities [3,4,5,6]. Sulfur-oxidizing microorganisms utilize these reduced sulfides from hydrothermal fluids, playing a crucial role in driving the global sulfur cycle [7,8,9]. Therefore, understanding the diversity and metabolic versatility of these microorganisms in deep-sea ecosystems is essential for elucidating the microbially mediated processes of elemental cycling and mineral formation in such a distinctive habitat.

Diverse pathways involved in the oxidation of sulfur compounds were discovered [10,11,12]. In the S_4_I pathway, thiosulfate is not directly oxidized to sulfate. Instead, it is first converted to the intermediate tetrathionate (S_4_O_6_^2−^) through an oxidative coupling reaction [13,14]. This pathway is widespread in many chemoautotrophs, mainly bacteria in the classes Betaproteobacteria and Gammaproteobacteria [15,16]. In chemolithoautotrophic sulfur-oxidizing bacteria (SOBs), several key enzymes that catalyze tetrathionate formation have been identified and characterized, including thiosulfate: quinol oxidoreductase (DoxDA) and thiosulfate dehydrogenase (TsdA) [17,18,19,20]. Among these, TsdA is a cytochrome c-type dehydrogenase with a high affinity for thiosulfate [21]. However, the ability of heterotrophic microorganisms to oxidize thiosulfate to tetrathionate has often been overlooked. Recently, Du et al. [22] investigated the genome and sulfur metabolic capabilities of a heterotrophic Halomonas strain isolated from a hydrothermal environment. Their study demonstrated that this strain encodes TsdA, which oxidizes thiosulfate to tetrathionate, and utilizes thiosulfate as a supplementary inorganic energy source, thereby actively participating in the sulfur cycle.

Stutzerimonas was proposed in 2022 by Lalucat et al. [23] to belong to the family Pseudomonadaceae, based on genome-based taxonomy. This genus currently includes 16 validly named species [24], which are widely distributed in saline–alkali soils [25], contaminated marine sediments [26], and deep-sea sediments of the Mariana Trench [27]. Most strains show potential for pollutant degradation and nitrate reduction [25,26]. However, few studies have focused on their sulfur-oxidizing capacity, and their adaptation mechanisms to extreme deep-sea hydrothermal environments remain unclear, which limits our knowledge of the genetic basis underlying their environmental adaptation.

The Tianxiu hydrothermal field is the first ultramafic-hosted hydrothermal system identified in the northwest Indian Ocean, located on the slow-spreading Carlsberg Ridge [28]. In this study, a heterotrophic sulfur-oxidizing bacterium was isolated from the Tianxiu hydrothermal sediment. Polyphasic taxonomic analyses indicated that this strain, designated as 381-2^T^, represents a novel species of the genus Stutzerimonas. Cultivation in the presence of minerals suggests its potential role in promoting mineral weathering processes. Comparative genomic analyses further reveal adaptation strategies of Stutzerimonas to the hydrothermal environment, including the capacity for sulfur oxidation.

2. Materials and Methods

2.1. Sampling and Mineralogical Analysis

The sediment sample BC12 used in this study was collected from the Tianxiu hydrothermal field on the Carlsberg Ridge in the northwest Indian Ocean (Figure 1). It was obtained using a box corer during the Chinese Ocean Science Expedition Cruise 72 (DY72) in June 2022. The sampling site is located at 3.691895° N, 63.831765° E, with a water depth of 3400 m.

The sediment samples were freeze-dried and subsequently ground to a particle size of less than 75 μm (200 mesh). Elemental analysis was performed using X-ray fluorescence (XRF) on an Epsilon 1 instrument (PANalytical, Almelo, The Netherlands), with results reported in oxide form. To determine the mineral composition, X-ray diffraction (XRD) analysis was conducted using a Bruker D8 Discover instrument (Bruker, Karlsruhe, Germany), equipped with a Cu Kα target, operating at 42 kV and 100 mA. The analysis covered a 2θ range of 5–80°, with a step size of 0.02° and a scan speed of 0.2 s/step. All XRD experiments were carried out at the Analytical and Testing Center of Zhejiang University (Hangzhou, China).

2.2. Strain Isolation, Cultivation, and Preservation

The sediment sample was immediately suspended in sterile seawater onboard. Ten-fold serial dilutions were then prepared, and 200 μL of the dilution was spread onto SOB selective agar plates. The plates were incubated at ambient temperature until they were transferred to the onshore laboratory (approximately 28 °C). The SOB selective medium was modified from the artificial seawater (ASW) medium described by Wentzien et al. [29] and had the following composition per liter: 1 L filtered seawater, 0.4 g NH_4_Cl, 0.2 g NaHCO_3_, 0.001 g FeSO_4_·7H_2_O, 0.1 g KH_2_PO_4_, 3 g Na_2_S_2_O_3_·5H_2_O, 1 mL of 1000× trace element solution, 1 mL of 1000× vitamin solution, and 0.004 g phenol red as a pH indicator. The medium was adjusted to pH 7.0. Na_2_S_2_O_3_·5H_2_O, the vitamin solution, and the trace element stock solution were filter-sterilized through 0.22 μm membranes, while the remaining components were autoclaved at 121 °C for 20 min. The solid medium was prepared by supplementing the above mixture with 1.5% (w/v) agar powder. The compositions of the 1000× trace element solution and the 1000× vitamin mix were prepared according to established protocols [30].

The 16S rRNA gene sequence was amplified under the conditions described by Xu et al. [31], using universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACTT-3′). The 16S rRNA gene was sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China, utilizing Sanger sequencing on an Applied Biosystems (ABI) DNA 3730 XL sequencer(Applied Biosystems, Foster, CA, USA). The 16S rRNA gene sequence similarity between the strains and reference strains was analyzed using the EzBioCloud online server (www.ezbiocloud.net/identify, accessed on 7 November 2025) [32]. The strains were stored long-term at −80 °C in 25% (v/v) glycerol.

2.3. Thiosulfate Oxidation of Strain 381-2T

The sulfur-oxidizing capacity of strain 381-2^T^ was detected in modified MMT medium [22] supplemented with and without 10 mM Na_2_S_2_O_3_ as the sole sulfur source. The composition of modified MMT medium per liter was as follows: 23.0 g NaCl, 0.5 g NH_4_Cl, 0.1 g CaCl_2_·2H_2_O, 0.5 g K_2_HPO_4_, 0.4 g MgCl_2_, 0.8 g CH_3_COONa, 0.2 g yeast extract, 1 mL of 1000× trace element solution, and 1 mL of 1000× vitamin solution. The pH was adjusted to 7.0. Bacterial growth was monitored by measuring the optical density at 600 nm using a GENESYS 50 UV–Visible Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of Na_2_S_2_O_3_ in the culture medium was determined by indirect iodometric titration [33,34], and the consumption of thiosulfate was used as an indicator for evaluating the sulfur-oxidizing capacity of the strain. Sulfur oxidation products were analyzed using Arc HPLC (Waters, Milford, MA, USA) equipped with a Syncronis C18 column (250 mm × 4.6 mm, 5 μm) (Thermo Scientific, Waltham, MA, USA). The column was eluted with 50 mM KH_2_PO_4_ (pH adjusted to 2.68 with phosphoric acid) at a flow rate of 0.5 mL/min, and product peaks were identified by UV detection at 215 nm. The chromatographic conditions followed the method described by Zhang et al. [33].

2.4. Microbe–Mineral Interaction Experiments of Strain 381-2T and Sulfide Minerals

Pyrite (FeS_2_), sphalerite (ZnS), and pyrrhotite (Fe_1−x_S) powders, which are common sulfide minerals in hydrothermal environments, were used as mineral substrates. Filtered seawater was employed as the culture medium with an initial pH of 7.0. Strain 381-2^T^ was inoculated into the mineral-containing culture medium, and the uninoculated mineral medium was set as the abiotic control. All cultures were statically incubated at 28 °C for two months. After incubation, the final pH of each culture system was determined, and the morphological changes in sulfide minerals were examined by scanning electron microscopy (SEM). Sulfide mineral particles were observed using a Zeiss Ultra 55 SEM (Zeiss, Oberkochen, Germany) equipped with an energy-dispersive X-ray spectrometer (EDS, Oxford Instruments, Abingdon, UK, Inca X-Max 20, model No. 51-XMX0004) at an accelerating voltage of 15 kV. Prior to SEM-EDS analysis, all samples were sputter-coated with a conductive film of platinum.

2.5. Phylogenetic Affiliation

The 16S rRNA gene phylogenetic tree of strain 381-2^T^ was reconstructed using MEGA v12 [35]. Specifically, the 16S rRNA gene sequences of the 30 most closely related and validly published strains were obtained from the EzBioCloud web server and aligned using Clustal W v2.0 [36]. The phylogenetic tree was then constructed using both the neighbor-joining and maximum-likelihood methods, applying the Kimura two-parameter nucleotide substitution model and 1000 bootstrap replications. The phylogenetic tree was constructed using the full-length 16S rRNA gene sequences of each species.

The maximum-likelihood phylogenetic tree of strain 381-2^T^ and its closely related Stutzerimonas reference strains was reconstructed based on 120 bacterial marker genes identified using GTDB-tk v2.4.1 [37]. The tree was built using IQ-TREE v2.2.5 [38] with the parameters “-bb 1000 -MFP” and the best-fit amino acid model Q.insect+F+I+R3. The tree was visualized using tvBOT version 2.6.1 (https://chiplot.online/tvbot.html, accessed on 27 October 2025) [39]. The 16S rRNA gene (accession number: CP000934) and genome (accession number: GCF_000019225.1) of the outgroup Cellvibrio japonicus Ueda107^T^ were obtained from the NCBI GenBank database.

2.6. Distribution of Strain 381-2T in Global Marine Environments

To investigate the global distribution of strain 381-2^T^, the full-length 16S rRNA gene sequence was queried against the Sequence Read Archive (SRA) using the Integrated Microbial Next Generation Sequencing (IMNGS) platform, with a sequence identity threshold of 99% [40,41]. Marine-associated datasets were processed as follows: (1) filtering based on NCBI registration and habitat information; (2) removing duplicates using Batch Entrez; and (3) extracting latitude and longitude coordinates [42].

2.7. Determination of Phenotypic Characteristics

Cell morphology and size were examined using transmission electron microscopy (TEM, JEM-1400Flash HC, JEOL Ltd., Okinawa, Japan) after incubating strain 381-2^T^ on the MB agar plate at 28 °C for three days. Gram staining was performed following standard protocols [43]. The temperature range for growth was assessed by incubating cultures at 4, 10, 15, 20, 25, 28, 30, 37, 40, and 45 °C. The pH range for growth was tested across 16 pH values: 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, 12.0, and 13.0. Different buffer solutions were used: 0.1 M citrate-citrate sodium buffer for pH 3.0–4.0; 40 mM MES for pH 5.0–6.0; 40 mM HEPES for pH 6.5–7.0; 40 mM Tricine for pH 7.5–8.5; and 40 mM CHES for pH 9.0–13.0. Growth in NaCl concentrations ranging from 0 to 12.0% (w/v, with 0.5% increments) was evaluated in sodium-free MB, as described by Zhang et al. [44]. Hydrolytic activities toward starch, Tween 20, and Tween 80 were determined following the methods described by Xu et al. [45]. Anaerobic experiments were conducted using MGC AnaeroPack sachets (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) on MB plates with 20 mM thiosulfate, 5 mM sulfite, 20 mM sulfate, 5 mM nitrite, and 20 mM nitrate provided as potential electron acceptors. Unless otherwise indicated, all physiological and biochemical tests were performed in MB medium at 28 °C. The API 20NE test strips (bioMérieux Inc., Marcy-l’Étoile, France) were used according to the manufacturer’s instructions.

2.8. Determination of Chemotaxonomic Characteristics

The reference strains Stutzerimonas xanthomarina DSM 18231^T^ and Stutzerimonas zhaodongensis DSM 27599^T^ were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). For fatty acid methyl ester (FAME) analysis, cells of strain 381-2^T^ and the reference strains Stutzerimonas xanthomarina DSM 18231^T^ and Stutzerimonas zhaodongensis DSM 27599^T^ were cultivated in MB medium at 28 °C and harvested at the end of exponential growth. Cellular fatty acids were extracted and prepared using saponification, methylation, extraction, and lye washing as described in previous studies [46]. The PAME profile was analyzed by gas chromatography (Agilent 8860, Santa Clara, CA, USA) and the Sherlock Microbial Identification System (MIS) with MIS library generation v6.5. For polar lipids analysis, cells of strain 381-2^T^ were harvested under the same conditions. Subsequently, polar lipids were extracted following the previous description [47] and separated by two-dimensional thin-layer chromatography (TLC) on 60 F254 silica gel plates (10 × 10 cm; Merck Millipore, Darmstadt, Germany). Total lipids, amino lipids, glycolipids, and phospholipids were determined by spraying with phosphomolybdic acid, ninhydrin, molybdenum blue, and α-naphthol and sulfuric acid, respectively [48].

2.9. Genomic Sequencing, Assembly, and Annotation

The cells of strain 381-2^T^ were cultured in Marine Broth 2216 (MB; Difco^TM^, Becton, Dickinson and Company, Sparks, MD, USA) at 28 °C for 24 h and harvested by centrifugation at 12,000× g for 10 min. Genomic DNA was then extracted using the protocol described by Dellaporta et al. [49]. Briefly, DNA was extracted using the sodium dodecyl sulfate (SDS) method. Cells were first rinsed with sterile water, lysed using 50 μL 20% SDS solution, and treated with RNase A to remove RNA, and the DNA was further washed with 70% ethanol. Then, DNA quality and purity were assessed via gel electrophoresis. Subsequently, the DNA library was prepared and subjected to high-throughput paired-end sequencing on an Illumina NovaSeq 6000 platform (PE150). Specifically, approximately 0.2 μg of extracted DNA was fragmented to a size of 350 bp via ultrasonication. The DNA fragments were then end-polished, A-tailed, and ligated with full-length adapters compatible with Illumina sequencing, followed by further PCR amplification. The PCR products were purified and quality assessed using the AMPure XP system (Beverly, Beverly, MA, USA) and Agilent 5400 system (Agilent, Santa Clara, CA, USA), and quantified by QPCR (1.5 nM). The operations for DNA extraction, library construction, and sequencing were all performed at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Raw data was cleaned by Fastp v0.23.1 [50]. The clean data obtained from sequencing were assembled using SPAdes v3.10.1 [51], and quality assessment was performed using CheckM v1.0.7 [52]. Basic genomic information was obtained using SeqKit toolkit v2.8.0 [53], and tRNA and rRNA information for the strain was predicted using Prokka v1.11 [54]. The genome sequences of other Stutzerimonas type strains used in this study were obtained from the NCBI GenBank database, with detailed information provided in Table S1.

Subsequently, coding sequences (CDSs) of strain 381-2^T^ and the other Stutzerimonas type strains were predicted using Prodigal v2.6.3 [55] under the “-single” mode. Functional annotation was then carried out in two approaches. First, KofamScan v1.3.0 [56] was employed with HMM profiles from the KEGG database (release 1 October 2025) and default score thresholds. Second, annotation against the Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases was performed using eggNOG-mapper web server version 2 (http://eggnog-mapper.embl.de/, accessed on 1 November 2025) [57].

Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain 381-2^T^ and all Stutzerimonas reference strain genomes were calculated using the locally installed FastANI v1.33 [58] and the online Genome-to-Genome Distance Calculator version 3.0(GGDC, https://ggdc.dsmz.de/home.php, accessed on 14 October 2025) [59]. Orthologous genes were identified using OrthoFinder v2.5.4 [60] for comparative genomics analysis.

2.10. Sulfur-Oxidizing Potential in the Stutzerimonas

To further explore the sulfur oxidation and hydrothermal environment adaptation potential of Stutzerimonas, high-quality genomes of Stutzerimonas strains were obtained from the NCBI Assembly database, in addition to all the type-strain genomes. These genomes met the criteria proposed by Bowers et al. [61] (integrity > 90.0%, contamination rate < 5.0%, presence of 23S, 16S, and 5S rRNA genes, and at least 18 tRNA genes). To ensure the accuracy of the sulfur oxidation potential analysis, all putative tsdA gene sequences (KO number: K19713) obtained from KEGG annotation were individually verified by using the diamond v2.1.8.162 [62] blastp module against the UniProtKB/Swiss-Prot database (Release 2025_04) [63]. This high-quality, manually curated, non-redundant protein database was used to confirm the tsdA genes (D3RVD4, A4VND8, D5WYQ5, and Q4FQB7), which were then selected for subsequent analysis. The other metal resistance genes of Stutzerimonas strains were annotated using the BacMet 2.0 database [64], with results obtained using diamond v2.1.8.162 blastp module, applying thresholds of ≥80% identity and ≥80% coverage.

3. Results

3.1. Mineralogical Analysis

Strain 381-2^T^ was isolated from a surface sediment sample BC12 collected at a depth of 3400 m in the Tianxiu hydrothermal field. The sampling site was located at the periphery of an active hydrothermal vent. The sediment exhibited a dark brown color and a coarse texture, with visible hydrothermal mineral grains. The XRF analysis indicated that sediment BC12 was enriched in hydrothermally derived metallic elements such as Fe, Mg, and Cu (Table S2). The XRD results indicated the presence of minerals such as pyrrhotite and lizardite in BC12 (Figure S1). Both XRF and XRD results support that BC12 is distinct from typical oceanic sediments, exhibiting characteristics of metal-rich sediment derived from deep-sea hydrothermal activity.

3.2. Growth of Strain 381-2T on Thiosulfate-Containing Substrates

Strain 381-2^T^ was isolated and purified from sediment sample BC12, collected in the Tianxiu hydrothermal field, using SOB selective medium. Strain 381-2^T^ exhibited a higher growth rate and achieved a greater cell density in MMT medium supplemented with 10 mM thiosulfate than in the control medium without thiosulfate (Figure 2a). The culture entered the stationary phase after approximately 40 h of incubation (Figure 2a). Concurrently, the pH of the culture medium containing 10 mM thiosulfate increased by approximately 2 units over the 40 h incubation period (Figure 2b). Indirect iodometric titration revealed that about 47% of the initially supplied thiosulfate was consumed after 70 h. HPLC analysis confirmed tetrathionate as the oxidation product of thiosulfate (Figure 2c).

3.3. Strain 381-2T and Mineral Interactions

To investigate the interaction between strain 381-2^T^ and sulfide minerals, a two-month microbe–mineral interaction experiment was conducted. The results showed that the pH decreased in all sulfide mineral cultures. Among them, the pyrite culture system exhibited the lowest pH, with the uninoculated control at 2.13 and the inoculated culture at 2.32. For the sphalerite culture system, the pH values were 5.91 and 6.03 for the control and inoculated culture, respectively. For the pyrrhotite culture system, the pH decreased to 3.42 in the control and 3.67 in the inoculated culture. The significant pH decrease observed in the controls indicated that pyrite and pyrrhotite were highly susceptible to abiotic oxidation, while sphalerite remained relatively stable. In contrast, inoculation with strain 381-2^T^ led to a consistently higher pH in all sulfide mineral cultures compared to their respective controls.

SEM analysis showed rod-shaped microbial cells attached to the surface of sphalerite (Figure 3a). In addition, rod-shaped pits, likely resulting from microbial dissolution, were observed on the pyrrhotite surfaces at the micron scale (Figure 3b,c). EDS analysis of the rod-shaped microbial structure in Figure 3a revealed a primary elemental composition of S, Zn, P, C, and O (Figure 3d). Analysis of another red cross-marked region on the same sphalerite sample detected Zn and S (Figure 3e). Further EDS analysis of the red cross-marked zone in Figure 3c revealed that its dominant elements were S, Fe, C, and O (Figure 3f).

3.4. Phylogenomic Affiliation of Strain 381-2T

The 16S rRNA gene sequence of strain 381-2^T^ showed the highest similarity (99.52%) to that of Stutzerimonas zhaodongensis NEAU-ST5-21^T^, a strain isolated from saline–alkaline soils in Zhaodong city [25]. In both the neighbor-joining phylogenetic tree (Figure 4) and maximum-likelihood phylogenetic tree (Figure S2) based on 16S rRNA gene sequences, strain 381-2^T^ clustered tightly with Stutzerimonas zhaodongensis NEAU-ST5-21^T^ with a branch bootstrap value exceeding 70%, indicating a robust and reliable phylogenetic relationship.

Genome-based phylogenetic analysis further revealed that strain 381-2^T^, Stutzerimonas xanthomarina DSM 18231^T^, Stutzerimonas zhaodongensis NEAU-ST5-21^T^, Stutzerimonas mariansis PS1^T^, and Stutzerimonas nitritolerans GL14^T^ form a highly supported clade (bootstrap value > 90%, Figure 5), indicating that they constitute a monophyletic lineage, which is distinct from other type strains of Stutzerimonas.

3.5. Distribution of Strain 381-2T in Global Marine Environments

The species closely related to strain 381-2^T^ (16S rRNA gene identity ≥ 99%) are widely distributed across global marine environments (Figure 6). Strain 381-2^T^ is prevalent in both coastal and open ocean ecosystems, with a broad latitudinal range extending from equatorial to polar regions (Figure 6).

3.6. Morphological, Physiological, and Chemotaxonomic Characteristics of Strain 381-2T

Strain 381-2^T^ is a Gram-negative bacterium capable of growth under both aerobic and anaerobic conditions on MB agar plates. After 3 days of incubation at 28 °C on MB agar, colonies were typically irregular, beige, wrinkled, and dry, measuring 2–3 mm in diameter; round colonies were occasionally observed. TEM revealed that the cells are rod-shaped, measuring 3.2–3.3 µm in length and 1.3–1.5 µm in width (Figure S3). Strain 381-2^T^ grew at temperatures ranging from 4 to 40 °C, with an optimum at 28 °C. The pH range for growth is 5.5–9.5, with an optimum at pH 7.0. The strain can grow in the presence of 0–10% (w/v) NaCl, with an optimum NaCl concentration of 4.5% (w/v). Notably, strain 381-2^T^ exhibited a greater NaCl tolerance than the other Stutzerimonas type strains (Table 1).

The API 20NE test indicated that, in contrast to Stutzerimonas zhaodongensis NEAU-ST5-21^T^, strain 381-2^T^ was unable to utilize L-arabinose or arginine, could not hydrolyze urea, and was negative for gelatinase activity. Strain 381-2^T^ could be further distinguished from Stutzerimonas zhaodongensis NEAU-ST5-21^T^ by multiple phenotypic characteristics, including the hydrolytic capabilities toward starch, Tween 20 and Tween 80. Additionally, strain 381-2^T^ and Stutzerimonas xanthomarina DSM 18231^T^ differed in their ability to assimilate capric acid (Table 1).

The major fatty acids (>10%) of strain 381-2^T^ were C_16:1_ω7c and/or C_16:1_ω6c (34.36%), C_18:1_ω7c and/or C_18:1_ω6c (23.87%), and C_16:0_ (21.11%). The detailed fatty acid profiles of strain 381-2^T^ and the two closely related Stutzerimonas type strains are listed in Table 2. The only respiratory quinone of strain 381-2^T^ was ubiquinone-9. The major polar lipids of strain 381-2^T^ consisted of phosphatidylglycerol, phosphatidylethanolamine, and one unidentified phospholipid. Additional minor polar lipids included two unidentified glycolipids, two unidentified phospholipids, and three unidentified lipids (Figure 7).

3.7. Genome Characteristics of Strain 381-2T

The genome of strain 381-2^T^ showed 100.00% completeness and 1.01% contamination, meeting the high-quality standards proposed by Bowers et al. [61]. The assembled genome consists of 36 contigs, with a genome size of 4,898,288 bp and a G+C content of 60.2%. Gene prediction using Prokka identified 3 rRNA genes and 56 tRNA genes. Prodigal predicted 4508 CDSs, of which 4021 (89.20%) were assigned to COGs, 1163 CDSs (25.80%) were annotated with GOs, and 2754 CDSs (61.09%) were functionally annotated in the KEGG database.

The ANI and isDDH values between strain 381-2^T^ and Stutzerimonas xanthomarina DSM 18231^T^ were 88.18% and 33.90%, respectively (Table S3). In addition, the ANI and isDDH values between strain 381-2^T^ and other Stutzerimonas species ranged from 79.12% to 88.18% and from 20.20% to 33.90%, respectively (Table S3). These values were below the species delineation thresholds of ANI (95–96%) and isDDH (70%) [66], supporting the proposal that strain 381-2^T^ represents a novel species within the genus Stutzerimonas.

Comparative genomic analysis revealed that the genome of strain 381-2^T^ contains 174 unique orthologous groups, which are absent in the genomes of 16 other Stutzerimonas type strains (Table S4). Among these, 51 orthologous groups were identified in the COG database under the following functional categories: C (energy production and conversion), E (amino acid transport and metabolism), G (carbohydrate transport and metabolism), K (transcription), L (replication, recombination and repair), N (cell motility), O (posttranslational modification, protein turnover, chaperones), P (inorganic ion transport and metabolism), S (function unknown), U (intracellular trafficking, secretion, and vesicular transport), and V (defense mechanisms) (Table S4). Additionally, 24 unique orthologous groups in 381-2^T^ were annotated in KEGG as conjugative transfer-related proteins (TraABEFGHIKLNUVW), DNA recombinase (spoIVCA), recombination protein (RecT), plasmid segregation protein (ParM), putative transposase, transport system small permease protein (DctQ), formate dehydrogenase subunit delta (FdsD), alginate O-acetyltransferase complex protein (AlgFJ), cobaltochelatase (CobT), mercuric ion transport protein (MerC), exodeoxyribonuclease VII small subunit (XseB), and uncharacterized proteins (Table S4).

3.8. Genomic Analysis of Sulfur Oxidation and Metal Resistance Potential in Stutzerimonas

Functional gene annotation revealed that the genomes of 12 Stutzerimonas type strains, including strain 381-2^T^, encode the tsdA gene, which is the most abundant sulfur oxidation gene in this genus (Figure 8; Table S5). Additionally, 322 high-quality Stutzerimonas genomes retrieved from the NCBI Assembly database (Table S6) were further analyzed, with 287 (89.13%) of these genomes containing at least one tsdA gene (Figure 9). Among them, 28 genomes encoded two or more copies of the tsdA gene, indicating the multi-copy presence of the tsdA gene in certain Stutzerimonas genomes (Table S7). Further validation revealed that the tsdA encoded by Stutzerimonas belongs to two Uniprot accession numbers (A4VND8 and Q4FQB7). The amino acid sequence identities of TsdA proteins ranged from 39.3% to 100.0%, with an average identity of 86.13% (Table S7). Notably, we found that strain 381-2^T^ and other Stutzerimonas type strains commonly encode genes associated with the denitrification pathway and oxidative phosphorylation (Figure 8).

Analysis of the isolation environments of Stutzerimonas genomes revealed that members of this genus are widely distributed in diverse habitats, including soil, marine, contaminated environments, rhizosphere, and clinical samples (Figure 9; Table S6). Notably, 16 genomes were derived from hydrothermal ecosystems, and all of these genomes encoded the tsdA gene (Figure 9; Table S7). Phylogenomic analysis based on strain 381-2^T^ and 322 high-quality Stutzerimonas genomes further demonstrated that strain 381-2^T^ is closely related to Stutzerimonas genomes derived from marine and hydrothermal environments (Figure 9).

The results of the metal resistance gene analysis indicate that 381-2^T^ encodes 14 metal resistance or metal transport genes, enabling it to resist various metals, including Cu, Cr, Te, Se, Hg, Ni, Cd, Zn, and Co through multiple mechanisms (Figure 10; Tables S5 and S8). For mercury resistance, 381-2^T^ encodes merT for uptaking Hg^2+^ [67], merA for reducing Hg^2+^ to volatile Hg^0^ [68], and the efflux protein MerE [69], with additional regulatory genes merD, merP, and merR involved in this process [70]. Regarding copper resistance, strain 381-2^T^ encodes two copR genes, which sense intracellular copper ion concentrations and regulate copper-related resistance pathways [71]. The genes ruvB and recG contribute to repairing DNA oxidative damage caused by Cr, Te, and Se [72,73], while chrA exports Cr^6+^ from the cell [68]. Additionally, mrdH and mreA are associated with the efflux of Zn^2+^, Cd^2+^, Ni^2+^, and Co^2+^ [73]. Among the other 322 high-quality Stutzerimonas genomes, the number of metal resistance genes ranged from 3 to 26, with an average of 8 genes per genome (Table S8).

4. Discussion

4.1. Isolation and Description of Stutzerimonas sp. nov. 381-2T

In this study, a novel Stutzerimonas strain was isolated from a deep-sea hydrothermal sediment sample. The 16S rRNA gene sequence of strain 381-2^T^ shared 99.52% identity with that of Stutzerimonas zhaodongensis NEAU-ST5-21^T^. However, genome-relatedness indices, including ANI and isDDH values, between 381-2^T^ and Stutzerimonas type strains were below the proposed species delineation thresholds of 95.0–96.0% and 70.0%, respectively. The maximum-likelihood phylogenomic tree based on the GTDB database showed that strain 381-2^T^ was solidly clustered in a clade with Stutzerimonas xanthomarina DSM 18231^T^. Phenotypically, strain 381-2^T^ could be clearly differentiated from related type strains. Notably, it exhibited a broader NaCl tolerance range (0–10% w/v) and a distinct optimal NaCl concentration for growth compared to Stutzerimonas zhaodongensis NEAU-ST5-21^T^ and Stutzerimonas xanthomarina DSM 18231^T^. In API 20NE tests, strain 381-2^T^ differed from Stutzerimonas zhaodongensis NEAU-ST5-21^T^ by its inability to utilize L-arabinose or arginine, hydrolyze urea, or produce gelatinase. Further phenotypic distinctions from Stutzerimonas zhaodongensis NEAU-ST5-21^T^ included the hydrolysis of starch, Tween 20, and Tween 80. Additionally, strain 381-2^T^ differed from Stutzerimonas xanthomarina DSM 18231^T^ in its assimilation of capric acid. Based on those genetic, genomic, phylogenomic, biochemical, and chemotaxonomic characteristics, strain 381-2^T^ could be identified as a novel Stutzerimonas species, for which the name Stutzerimonas tianxiuensis sp. nov. 381-2^T^ is proposed.

Stutzerimonas tianxiuensis (tian.xiu.en’sis. N.L. fem. adj. tianxiuensis pertaining to Tianxiu, the hydrothermal vent name where the type strain was isolated).

Cells are rod-shaped, 3.2–3.3 µm long and 1.3–1.5 µm wide, Gram-negative, and facultatively anaerobic. Growth occurs at 10–40 °C (optimum 28 °C), pH 5.5–9.5 (optimum 7.0), and in the presence of 0–10% (w/v) NaCl (optimum 4.5%). The strain is capable of oxidizing thiosulfate to tetrathionate. In the carbon source assimilation assay using API 20NE tests with 12 substrates, the strain assimilates glucose, D-mannitol, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and citrate, but does not utilize L-arabinose, D-mannose, N-acetylglucosamine, or phenylacetic acid. Nitrate reductase activity (NO_3_^−^ reduction) is positive, but tryptophanase, arginine dihydrolase, urease, α-glucosidase, protease, and β-galactosidase activities are consistently negative. The major fatty acids (>10 %) of strain 381-2^T^ are C_16:1_ω7c and/or C_16:1_ω6c, C_18:1_ω7c and/or C_18:1_ω6c and C_16:0_. The major polar lipids comprise phosphatidylglycerol, phosphatidylethanolamine, and one unidentified glycolipid. The only respiratory quinone is identified as ubiquinone-9.

The type strain, 381-2^T^ (= CCTCC AB 2025277^T^ = MCCC M32512^T^), was isolated from Tianxiu hydrothermal sediments collected from the Carlsberg Ridge in the Northwestern Indian Ocean. The genomic DNA G+C content of the type strain is 60.2%. The NCBI GenBank accession numbers for the 16S rRNA gene and genome sequences of strain 381-2^T^ are PX706103 and JBPVWS000000000, respectively.

4.2. Strain 381-2T Has the Potential to Participate in Sulfide Mineral Transformation

Strain 381-2^T^ was isolated from hydrothermal sediments, which are characterized by elevated concentrations of Fe, Mg, Cu and other metals, in addition to hydrothermal sulfide minerals. Through microbe–mineral interaction experiments of strain 381-2^T^ with various sulfide minerals, this study demonstrates that its metabolic activity affects the weathering process of sulfide minerals, which is manifested by the regulation of system pH [74], attachment to mineral surfaces, and potential promotion of mineral reprecipitation on microbial surfaces.

The difference in pH between the experimental and control groups directly reflects the influence of acid–base balance of the system by microbial activity. In the abiotic controls, the pH decreased in all sulfide mineral systems resulting from the chemical oxidation of sulfides. The pyrite and pyrrhotite systems exhibited the largest pH drop, reaching strongly acidic conditions, which confirms their higher susceptibility to abiotic oxidation. In contrast, inoculation with 381-2^T^ resulted in higher pH in all mineral systems compared to the controls. Considering the sulfur oxidation capacity of strain 381-2^T^, we deduced that this strain may utilize intermediate sulfur species (e.g., S_2_O_3_^2−^) produced during sulfide mineral dissolution, generating energy through an alkalogenic sulfur oxidation process (S_4_I), which in turn raises the system pH. However, while strain 381-2^T^ can counteract some degree of acidification through this process, it cannot fully prevent the system from becoming acidic due to the ongoing abiotic processes that drive pH reduction.

Mineralized cells observed on the sphalerite surface via SEM-EDS provided direct evidence of microbe–mineral interactions (Figure 3). The elemental signals of C, O, P, and S detected by EDS strongly indicated the presence of microbial cells and their extracellular polymeric substances (EPS). Genomic analysis of strain 381-2^T^ identified nine genes related to exopolysaccharide biosynthesis (Table S5), which are involved in the synthesis of various EPS (Colanic acid, Alginate, Psl polysaccharide, Poly-N-acetyl-glucosamine (PNAG), and Vibrio polysaccharide) and microbial biofilm formation. Consistent with this, in situ cultivation on pyrite also demonstrated that colonizing cells secrete polysaccharide-rich EPS on mineral surfaces [75]. EPS are biomacromolecular polymers secreted by microorganisms during growth [76]. Not only do they serve as key biological adhesives for microbial attachment to mineral surfaces [77,78], but their negatively charged functional groups (e.g., carboxyl and phosphate groups) can also effectively adsorb metal cations from the environment [79]. This adsorption results in local supersaturation of metal ions, thereby providing nucleation sites for mineral precipitation [80,81]. We hypothesize that the Zn and S signals detected on the surface of attached cells may reflect the reprecipitation of Zn^2+^ and S^2−^/S_n_^2−^ from dissolved sphalerite within the EPS matrix, or the formation of other zinc-bearing secondary phases.

Additionally, genomic analysis of strain 381-2^T^ revealed the presence of various metal resistance genes, including a mer operon for mercury detoxification, heavy metal efflux pumps for copper and zinc, and genes for oxidative stress resistance. Similarly, a variety of metal transporters and resistance genes have been annotated in other high-quality Stutzerimonas genomes. These genetic determinants help Stutzerimonas species cope with metal stress in hydrothermal environments and enhance their adaptability to these heavy-metal-rich conditions [82].

4.3. Adaptation Properties of Stutzerimonas to Deep-Sea Hydrothermal Fields

Stutzerimonas (formerly known as the Pseudomonas stutzeri complex) is a newly reclassified genus within the family Pseudomonadaceae, exhibiting substantial genotypic and phenotypic diversity [26]. In this study, comparative genomics analyses were conducted to explore the adaptive mechanisms of Stutzerimonas to the deep-sea hydrothermal environment.

Members of the genus Stutzerimonas generally exhibit sulfur-oxidizing potential. A comprehensive analysis of 322 high-quality Stutzerimonas genomes revealed that the majority of Stutzerimonas strains (89.13%) harbor the sulfur-oxidizing gene tsdA (Figure 9). Notably, all 16 strains isolated from hydrothermal environments carried the tsdA gene, which represents the highest proportion of tsdA-encoding strains among all the sampled environments (Figure 9 and Figure S4). In this study, the tsdA gene was identified in strain 381-2^T^, which was isolated from hydrothermal sediment. Experimental validation confirmed that strain 381-2^T^ oxidized thiosulfate to tetrathionate, thereby promoting its growth. These findings suggest that Stutzerimonas species may utilize the tsdA-mediated sulfur oxidation pathway (S_4_I) to utilize the abundant reduced sulfur compounds in hydrothermal vents, facilitating their colonization of these sulfur-rich niches [83].

Additionally, Stutzerimonas possesses diverse metabolic pathways and serves as an important driver of sulfur, nitrogen, and carbon cycles in deep-sea hydrothermal ecosystems. Stutzerimonas is frequently reported to perform denitrification, reducing nitrate to nitrogen under anaerobic conditions [26,27]. In the oxygen-limited zones of deep-sea hydrothermal vents, Stutzerimonas can utilize nitrate as an electron acceptor to support anaerobic respiration [84]. In this study, the sulfur-oxidizing strain 381-2^T^ was found to encode a complete denitrification pathway (Figure 8; Table S5). Meanwhile, its genome also contains multiple carboxylase genes, such as pyruvate carboxylase and acetyl-CoA carboxylase (Table S5). These findings indicate that 381-2^T^ has the potential to drive anaplerotic CO_2_ fixation [85,86] using energy derived from sulfur oxidation and denitrification, thereby replenishing intermediates of the tricarboxylic acid (TCA) cycle. This mechanism may enable the strain to sustain growth in environments with limited organic carbon. Thus, the sulfur-oxidizing and denitrification abilities of Stutzerimonas provide a flexible energy utilization strategy, enhancing its adaptability in dynamic hydrothermal chemical conditions and playing a critical role in the sulfur, nitrogen, and carbon cycles in these ecosystems.

The genus Stutzerimonas likely occupies microaerobic and facultative anaerobic niches in hydrothermal environments. Strain 381-2^T^ and other type strains of Stutzerimonas possess a complete set of respiratory chain complexes (including Complexes I, II, III, IV and V) along with a notably diverse terminal oxidase system (Figure 8; Table S5). This system includes the cbb3-type cytochrome c oxidase (CcoN, CcoO, CcoP), which exhibits high affinity for oxygen and supports efficient respiration under microaerobic conditions [87]. It also includes oxidases such as the bd-type quinol oxidase (CydA, CydB), which is expressed under low-oxygen conditions and is insensitive to inhibitors like cyanide, thereby enhancing its competitiveness in oxygen-limited environments [88,89]. The presence of multiple terminal oxidases allows Stutzerimonas to finely regulate its metabolic processes, marking this genus as a classic facultative anaerobe. It can flexibly switch its respiratory mode in response to dynamic changes in environmental oxygen partial pressure, allowing it to colonize and thrive in hydrothermal settings with steep redox gradients [90].

In addition, Stutzerimonas species are widely distributed in marine environments. Apart from deep-sea hydrothermal areas, they are also found in surface seawater [91], contaminated marine sediment [26], deep-sea sediments from the Mariana Trench [27], and marine organisms [92]. This broad distribution is closely associated with their versatile metabolic capabilities, which enable adaptation to various marine niches and contribute to the biogeochemical cycling of sulfur and nitrogen in the oceans.

5. Conclusions

In conclusion, this study described the isolation and characterization of a novel sulfur-oxidizing bacterium, strain 381-2^T^, which is proposed as Stutzerimonas tianxiuensis sp. nov., from metal-rich sediments in the Tianxiu hydrothermal field. Phylogenetic and taxonomic analyses, including 16S rRNA gene sequencing and comparative genomic analysis, confirmed that strain 381-2^T^ represents a new species within the genus Stutzerimonas. Physiological experiments revealed that strain 381-2^T^ oxidized thiosulfate to tetrathionate, promoting its growth. Furthermore, genomic analysis identified the key sulfur oxidation gene tsdA, involved in the S_4_I pathway. Microbe–mineral interaction experiments suggested that strain 381-2^T^ can influence pH, adhere to mineral surfaces, and potentially promote mineral reprecipitation, thereby contributing to the weathering of sulfide minerals. The widespread presence of the tsdA gene in the genus Stutzerimonas and the prevalence of metal resistance or transport genes highlight the adaptability of these bacteria to metal-rich hydrothermal environments. Overall, Stutzerimonas species employ diverse survival strategies, including sulfur compound utilization, to thrive in extreme marine environments, offering valuable insights into their genomic and physiological adaptations in hydrothermal vent ecosystems.