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Article

Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov.

by
Hui Zhou
1,
Jieni Qu
1,
Xu Lin
1,
Ning Wang
1,
Zihan Jiang
1,
Qiliang Lai
2 and
Hong Xu
1,*
1
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China
2
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
*
Author to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 481; https://doi.org/10.3390/microorganisms14020481
Submission received: 14 January 2026 / Revised: 11 February 2026 / Accepted: 13 February 2026 / Published: 16 February 2026
(This article belongs to the Section Environmental Microbiology)
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Abstract

Two sulfur-oxidizing bacterial strains, FCG-A2T and FCG-A23T, were isolated from coastal sediments collected in Fangchenggang, Guangxi Province, China. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences placed strain FCG-A2T within the genus Pseudothioclava and strain FCG-A23T within the genus Terasakiella. Genomic relatedness (ANI, AAI, dDDH, and POCP) to the closest described taxa was below the accepted species thresholds, demonstrating that both isolates represent novel species. Strain FCG-A2T grew at 15–35 °C (optimum 25–30 °C), at pH 5.0–10.0 (optimum pH 8.0), and with 1–4% (w/v) NaCl concentrations (optimum 3%). Strain FCG-A23T grew at 20–33 °C (optimum 25–30 °C), at pH 6.0–9.0 (optimum, pH 8.0), and with 2–6% (w/v) NaCl (optimum 2%). For both strains, ubiquinone-10 was the major respiratory quinone, and the predominant fatty acids were summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c); strain FCG-A2T additionally contained C16:0 as a major fatty acid. Both strains oxidized thiosulfate to sulfate, consistent with the presence of genes encoding the Sox system and assimilatory sulfate reduction pathways. Comparative genome annotation further suggested a broader carbohydrate-degradation potential in FCG-A2T than in FCG-A23T, implying a wider ecological distribution and greater opportunities for FCG-A2T to perform sulfur oxidation across habitats. The draft genomes had G + C contents of 62.09% (FCG-A2T) and 49.06% (FCG-A23T). Based on these results, we propose Pseudothioclava alba sp. nov. (type strain FCG-A2T = MCCC 1K08969T = KCTC 8462T) and Terasakiella sediminum sp. nov. (type strain FCG-A23T = MCCC 1K08972T = KCTC 8464T).

1. Introduction

Sulfur is a vital element whose complex biogeochemical cycle is predominantly driven by microorganisms [1,2]. These microbes, including sulfur-oxidizing and sulfate-reducing prokaryotes, are fundamental to global energy flow and nutrient cycling [1,2,3,4]. They support entire ecosystems and play pivotal roles in processes ranging from organic matter decomposition to bioremediation and biocorrosion [3,4,5]. Despite their profound ecological and economic importance, the full taxonomic and functional diversity of sulfur-metabolizing microorganisms remains largely uncharted, particularly within dynamic environments such as marine coastal sediments where intense sulfur cycling occurs [6]. The discovery and characterization of novel species are therefore imperative. Each new isolate expands our understanding of the phylogenetic and metabolic breadth of sulfur transformations, provides a new model system for physiological and genomic investigation, and may harbor unique enzymes or pathways with significant biotechnological potential in pollution control or bio-industrial processes [7]. Consequently, modern taxonomic efforts rely on a polyphasic approach—integrating genotypic, phenotypic, and chemotaxonomic data—to formally describe new taxa [6]. This approach is particularly valuable for exploring under-characterized yet functionally significant genera known to participate in the sulfur cycle, thereby revealing their hidden diversity and ecological contributions. Among these, the marine-associated genera Thioclava, Terasakiella, and the recently proposed genus Pseudothioclava represent compelling targets for the discovery of novel sulfur-metabolizing species.
The genus Thioclava, a member of the family Rhodobacteraceae, was initially proposed by Sorokin et al. [8]. Members of this genus have been isolated from diverse marine environments and are characterized as Gram-negative, short, rod-shaped bacteria with diverse metabolic capabilities, particularly those involved in sulfur cycles [8,9,10,11,12,13,14,15,16]. Certain members of this genus are capable of oxidizing elemental sulfur (S0) to produce sulfate [14], a trait with potential applications in acid mine wastewater treatment and marine sulfur pollution remediation. In addition, some strains play significant roles in crude oil degradation [17] and power generation [18], suggesting that these bacteria are integral to the biogeochemical cycles of sulfur and carbon. To date, nine species with validly published names have been reported within the genus Thioclava, including Thioclava pacifica [8], Thioclava atlantica [12], Thioclava dalianensis [11], Thioclava indica [10], Thioclava marina [13], Thioclava sediminum [13], Thioclava nitratireducens [9], Thioclava litoralis [15] and Thioclava arctica [19]. However, recent advances in phylogenomic analysis have led to significant taxonomic refinement of this group. The genus Pseudothioclava was established by Kim et al. [20] to accommodate lineages that are phenotypically similar to, yet phylogenetically distinct from, Thioclava. The name Pseudothioclav is derived from the Greek pseudês (false) and Thioclava, implying a ‘false Thioclava’. Currently, the genus Pseudothioclava comprises two species with validly published names: the type species Pseudothioclava arenosa (basonym: Thioclava arenosa) and Pseudothioclava nitratireducens (basonym: Defluviimonas nitratireducens), both of which were reclassified based on genome-based phylogeny [20,21]. Exploring the diversity within Pseudothioclava is essential for delineating the taxonomic boundaries and ecological functions of these sulfur-oxidizing bacteria.
The genus Terasakiella, a member of the order Rhodospirillales, was first proposed by Satomi et al. [22] and subsequently reclassified into the family Terasakiellaceae by Hördt et al. [23]. The family Terasakiellaceae was first proposed in 2002 [22], and currently consists solely of the genus Terasakiella, with limited reports on its metabolic capabilities. Therefore, the exploration of new species within the family Terasakiellaceae is of significant importance for expanding our knowledge of bacterial resources and their functional potential. At the time of writing, the genus Terasakiella comprises three species with validly published names, according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN, https://lpsn.dsmz.de/genus/terasakiella, accessed on 10 January 2026). These species are Terasakiella pusilla IF6T [22], Terasakiella brassicae B3T [24] and Terasakiella salincola KMU-80T [25], isolated from a putrid infusion of marine shellfish, pickle-processing factory wastewater and a harbor seawater sample, respectively. These species share several phenotypic characteristics: they are Gram-negative, S-shaped, helical bacteria with DNA G + C contents ranging from 40 to 50 mol%. The major respiratory quinone and predominant fatty acid of the genus Terasakiella are ubiquinone-10 (Q-10) and summed feature 8 (C18:1ω7c and/or C18:1ω6c), respectively.
In the present study, two strains of sulfur-metabolizing bacteria, designated FCG-A2T and FCG-A23T, were isolated from coastal sediments in the South China Sea. They were taxonomically characterized as novel species within the genera Pseudothioclava and Terasakiella, respectively, using a polyphasic approach that included the assessment of phenotypic and chemotaxonomic properties as well as phylogenetic analyses based on 16S rRNA gene sequences and whole-genome sequences. The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain FCG-A2T (= MCCC1K08969T = KCTC 8462T) are OR192614 and JAWLVI000000000, respectively. The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain FCG-A23T (= MCCC1K08972T = KCTC 8464T) are OR206505 and JAWLVJ000000000, respectively. To better understand the metabolic divergence between the two isolates, we further investigated their carbon acquisition strategies by comparing the CAZyme repertoires and predicting the utilizable substrates inferred from the genomes of strains FCG-A2T and FCG-A23T.

2. Materials and Methods

2.1. Isolation, Purification and Maintenance of Bacterial Strains

A coastal sediment sample was collected from Hongshabei (21.63° N, 108.42° E) in Fangchenggang, Guangxi Province, China, in April 2023. Aliquots of the sediment were transferred into a 50 mL centrifuge tube containing 25 mL of sterilized natural seawater and shaken overnight at 80 rpm. The resulting supernatant was serially diluted and spread onto marine agar 2216 medium (MA, containing 5 g/L peptone, 1 g/L yeast extract, 74.4 mg/L KH2PO4, 2 mg/L Fe2(SO4)3 and 15 g/L agar, prepared with seawater, pH 7.2). After incubation at 30 °C for 7 days under aerobic conditions, single colonies were picked and purified by repeated streaking on MA at least three times. Pure cultures were routinely maintained on MA at 30 °C and preserved at −80 °C in MA broth supplemented with 20% (v/v) glycerol.
For phenotypic characterization and fatty acid analysis, Pseudothioclava arenosa CAU1312T (KCTC 52190), Pseudothioclava nitratireducens DL5-4T (MCCC 1A06995) and Thioclava pacifica TL2T (DSM 10166) were used as reference type strains for comparison with strain FCG-A2T. For strain FCG-A23T, the two closest type strains, Terasakiella pusilla IF6T (NBRC 13613) and Terasakiella brassicae B3T (KCTC 42652) were used as reference strains. Unless otherwise stated, all strains were cultivated on MA (pH 7.2) at 30 °C.

2.2. 16S rRNA Gene Sequencing and Phylogenetic Analysis

Genomic DNA was extracted using a Bacterial Genomic DNA Extraction Kit (TaKaRa, Beijing, China). The 16S rRNA gene was amplified by PCR using the universal primers 27F and 1492R [26]. PCR products were cloned into the pMD-19T vector and subsequently sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Pairwise 16S rRNA gene sequence similarities between strains FCG-A2T and FCG-A23T and related type strains were calculated using the EzBioCloud Identify tool (https://www.ezbiocloud.net/identify, accessed on 20 December 2025) [27] and the BLASTn searches against the NCBI database (BLAST: Basic Local Alignment Search Tool (nih.gov), accessed on 20 December 2025). Phylogenetic analyses were conducted using MEGA version 11 [28] following multiple sequence alignment with the CLUSTAL-W algorithm [29]. Phylogenetic trees were reconstructed using the neighbor-joining (NJ) [30], minimum-evolution (ME) [31] and maximum-likelihood (ML) [32] methods. Bootstrap values were calculated from 1000 replicates [33]. Taxonomic identification was further confirmed using the Type Strain Genome Server (TYGS, https://tygs.dsmz.de/, accessed on 3 February 2026) [34].

2.3. Genome Sequencing and Genomic Analyses

Whole-genome sequencing was performed by BGI Genomics (Shenzhen, China) on the Illumina HiSeq platform. Sequence reads were assembled using SOAPdenovo v2.04 [35]. Draft genome annotation was conducted using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [36]. Functional annotation of metabolic pathways and genes was performed using the Kyoto Encyclopedia of Genes and Genomes database (KEGG, https://www.kegg.jp, accessed on 20 December 2025). Carbohydrate-active enzymes (CAZymes) were annotated using dbCAN3 (https://bcb.unl.edu/dbCAN2/blast.php, accessed on 15 December 2025) [37]. Only CAZymes supported by at least two of the three dbCAN3 methods (HMMER, Diamond, and dbCAN-sub) were retained for downstream analyses. Ecological distribution was inferred using Protologger (https://protologger.bi.denbi.de/, accessed on 9 December 2025) based on genome sequences and 16S rRNA gene sequences. Specifically, the 16S rRNA gene sequences were compared against 19,000 amplicon datasets representing 19 environments (1000 samples per environment) to estimate prevalence and mean relative abundance [38].
Average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated using PyANI (https://github.com/widdowquinn/pyani, accessed on 16 December 2025) [39] and CompareM (https://github.com/dparks1134/CompareM, accessed on 16 December 2025), respectively, using default parameters. Digital DNA–DNA hybridization (dDDH) values were computed using the Genome-to-Genome Distance Calculator (GGDC 3.0; https://ggdc.dsmz.de/ggdc.php, accessed on 12 December 2025) with the recommended BLAST+ method and formula 2 [40,41]. Genome-based phylogenomic analysis was performed using the DOE Systems Biology Knowledgebase (KBase, http://kbase.us, accessed on 6 January 2026) [42] using the apps “Annotate Multiple Microbial Assemblies with RASTtk” (version 1.073) and “Insert Genome Into Species Tree” (version 2.2.0). The percentage of conserved proteins (POCP) was computed as described by Qin et al. [43]. Genomes of related strains were retrieved from the NCBI GenBank database. Stappia stellulata TS101T and Acidithiobacillus acidisediminis S30A2T were used as outgroups for the corresponding phylogenies.

2.4. Morphological, Physiological, and Biochemical Characterization

Cell morphology was examined by scanning electron microscopy (Thermo Fisher Helios 5 UC, Brno, Czech Republic) and transmission electron microscopy (Hitachi HT-7800, Tokyo, Japan). Gram-staining was performed following standard methods [44]. Growth at varying temperatures (4, 15, 20, 25, 30, 37, 40 and 45 °C) was assessed on MA plates. The influence of pH on growth was determined in marine broth 2216 medium (MB, containing 5 g/L peptone, 1 g/L yeast extract, 74.4 mg/L KH2PO4, and 2 mg/L Fe2(SO4)3, prepared with seawater), adjusted to pH 3.0–10.0 (at intervals of 1.0 pH unit) using 1 M HCl or NaOH. Salinity tolerance was assessed using a modified MB prepared with distilled water and supplemented with NaCl at final concentrations of 0–12.0% (w/v) at 1.0% intervals. Catalase and oxidase activities were determined according to the methodology described by Lányi [45]. Antibiotic susceptibility was evaluated by the disc diffusion method [46] on MA plates using the following antibiotics (μg per disc unless otherwise indicated): streptomycin (10), cefoperazone (75), ampicillin (20), erythromycin (15), novobiocin (30), kanamycin (30), chloramphenicol (30), cefazolin (30), gentamicin (10), penicillin G (10 U), vancomycin (30), and neomycin (30). Enzyme activities and substrate utilization profiles were determined using API ZYM, API 50CH, and API 20NE strips (bioMérieux, Lyon, France) according to the manufacturer’s instructions, with the exception that inocula were prepared by suspending bacterial cells in sterile seawater.

2.5. Chemotaxonomic Characterization

For polar lipid and respiratory quinone analyses, biomass was harvested from MA plates after incubation at 30 °C for 5 days. For fatty acid analysis, cells were grown in MB at 30 °C for 5 days, harvested, and freeze-dried. Isoprenoid quinones were extracted using chloroform/methanol (2:1, v/v) and analyzed by reversed-phase HPLC as described previously [47]. Polar lipids were extracted following the protocol established by Minnikin et al. [47] and separated using two-dimensional thin-layer chromatography (TLC). The solvent systems used were chloroform/methanol/water (65:24:4, by vol.) for the first dimension and chloroform/glacial acetic acid/methanol/water (80:15:12:4, by vol.) for the second dimension. Fatty acids were saponified, methylated, and extracted according to Kuykendall et al. [48]. Fatty acid methyl esters (FAMEs) were identified and quantified using the Sherlock Microbial Identification System (MIS; MIDI version 6.0) [49].

2.6. Thiosulfate Oxidation Assay

Metabolic pathways were reconstructed based on genome annotation results. The thiosulfate oxidation pathway was predicted by mapping protein sequences to the KEGG database. The presence of the sulfur oxidation (Sox) gene cluster was further validated by performing BLASTp searches against the NCBI nr database and analyzing the gene neighborhood (operon structure) to ensure functional completeness. To assess sulfur-oxidizing activity, strains FCG-A2T and FCG- A23T were tested for their ability to oxidize thiosulfate. For strain FCG-A2T, thiosulfate oxidation was tested on saltwater base media (SWB) agar plates (containing 30 mM sodium sulfate, 342 mM sodium chloride, 14.8 mM magnesium chloride hexahydrate, 0.1 mM calcium chloride dihydrate, 10 mM ammonium chloride, 1 mM potassium phosphate, and 1.5% agar) supplemented with 25 mM sodium thiosulfate and 0.002% bromothymol blue [14,18]. For FCG-A23T, the same medium was supplemented with 1% (w/v) glucose. Thiosulfate oxidation was inferred from a color change in the pH indicator from blue to yellow, indicating acidification associated with thiosulfate oxidation.

3. Results

3.1. Morphological Observation and 16S rRNA Gene-Based Phylogenetic Analysis

Strain FCG-A2T grew slowly and formed small, white, circular, smooth, and moist colonies with a diameter of 0.3–1.0 mm on MA plates after incubation at 30 °C for 7 days (Figure 1a). The cells were rod-shaped, measuring 0.4–0.6 µm in width and 2.0–8.0 µm in length (Figure 1b,c). Based on 16S rRNA gene sequence analysis (1389 bp; GenBank accession no. OR192614), strain FCG-A2T was shown to be affiliated with the family Rhodobacteraceae, showing close relationships to members of the genera Thioclava, Rhodobacter, and Pseudothioclava. Specifically, it shared the highest sequence similarity with Thioclava pacifica TL2T (98.1%), followed by Rhodobacter xanthinilyticus LPB0142T (97.5%), Thioclava atlantica 13D2W-2T (97.3%), Rhodobacter lacus JA826T (97.2%), and Pseudothioclava arenosa CAU1312T (97.1%). Phylogenetic trees constructed using the NJ and ME algorithms consistently placed strain FCG-A2T within the Thioclava clade (Figure S1a,b). However, the ML tree indicated that FCG-A2T forms a distinct lineage that is closely related to, yet separate from, both the Thioclava and Pseudothioclava genera (Figure 1d). This distinct phylogenetic position was further supported by TYGS analysis (Figure S1c), which confirmed its separation from the genera Thioclava and Pseudothioclava. Consequently, the precise taxonomic status of FCG-A2T required confirmation through further genomic analysis.
Strain FCG-A23T formed circular, smooth, moist, and beige colonies (0.5–1.0 mm in diameter; Figure 2a). Cells were S-shaped, measuring approximately 0.3–0.5 µm in width and 2.0–5.0 µm in length (Figure 2b,c). The 16S rRNA gene sequence (1410 bp, GenBank accession No. OR206505) showed the highest similarity to Terasakiella pusilla IF6T (97.3%), followed by Terasakiella brassicae B3T (97.1%) and Terasakiella salincola KMU-80T (96.0%); similarities to other members of the order Rhodospirillales were below 90%. Phylogenetic trees constructed using the NJ, ME, and ML algorithms revealed that strain FCG-A23T formed a stable cluster with species of the genus Terasakiella (Figure 2d and Figure S2a,b). This placement within the genus was further corroborated by TYGS analysis (Figure S2c).

3.2. Genome Properties, Taxonomy, and Whole-Genome Phylogeny

Quality control of the raw reads showed high accuracy for both strains (Table S1), with Q30 values of 94.56% for strain FCG-A2T and 93.89% for strain FCG-A23T. The genomes were sequenced at high coverage depths of 413× and 273×, respectively, ensuring robust data quality. The final assembly of FCG-A2T comprised 34 contigs with an N50 value of 413,269 bp, while that of strain FCG-A23T comprised 94 contigs with an N50 value of 114,333 bp. Genome completeness was evaluated using BUSCO, yielding scores of 99.2% for FCG-A2T and 100% for FCG-A23T. These high-quality metrics confirm the reliability of the genomic data used for taxonomic identification.
The draft genome of FCG-A2T (GenBank accession no. JAWLVI000000000) consists of 23 scaffolds, with a total length of 3,113,148 bp. The genomic DNA G + C content is 62.09 mol%. Although this value is slightly lower than the range currently reported for the genus Pseudothioclava (63.8–64.7 mol%) [16,50], it falls well within the broader range observed in the closely related genus Thioclava (60.3–65.3 mol%) [8,9,10,11,12,13,14,15,16]. In contrast to the 16S rRNA gene-based analysis, genome-based phylogenetic reconstruction placed strain FCG-A2T within the Pseudothioclava clade (Figure 3a). This topology is fully consistent with the genome-based phylogenetic tree generated by TYGS, which independently positioned strain FCG-A2T within Pseudothioclava. This phylogenetic placement is substantiated by POCP analysis (Figure 3b), which revealed that strain FCG-A2T shares high POCP values of 81% and 76% with the two reference Pseudothioclava species, well above the 50% genus demarcation threshold [43]. Conversely, POCP values between strain FCG-A2T and members of the closely related genus Thioclava were consistently below 70%. These collective genomic metrics strongly support the classification of strain FCG-A2T within the genus Pseudothioclava.
To confirm this taxonomic assignment, comparative genomic analyses were performed (Table 1). The ANI, AAI, and dDDH values between strain FCG-A2T and the type strains of both Pseudothioclava and Thioclava were well below the recommended species boundaries of 95–96% for ANI [51] and 70% for dDDH [52]. Notably, strain FCG-A2T exhibited higher genomic similarity to Pseudothioclava than to Thioclava. Specifically, the ANI values against Pseudothioclava species ranged from 77.92% to 78.53%, whereas those against Thioclava species were lower (72.88–76.29%). Similarly, AAI and dDDH values were consistently higher for Pseudothioclava (AAI: 78.22–79.46%; dDDH: 21–21.3%) compared to Thioclava (AAI: 64.06–73.43%; dDDH: 18.7–20.3%). Collectively, the phylogenomic tree and comparative indices (ANI, AAI, dDDH, and POCP) support the proposal that strain FCG-A2T represents a novel species within the genus Pseudothioclava.
The draft genome of strain FCG-A23T (GenBank accession No. JAWLVJ000000000) comprises 82 scaffolds, with a total length of 4,189,928 bp. The genomic DNA G + C content of strain FCG-A23T is 49.06 mol%, falling well within the range reported for the genus Terasakiella (40–50 mol%). In the genome-based phylogenetic tree, strain FCG-A23T clusters robustly within the Terasakiella clade (Figure 3c), supporting its placement in this genus. This affiliation is further corroborated by POCP analysis (Figure 3d), in which strain FCG-A23T shared high values (70%) with reference Terasakiella species, whereas values against members of other genera remained strictly below the 50% genus demarcation threshold (Figure 3d) [43]. These results conclusively assign strain FCG-A23T to the genus Terasakiella.
Genome-relatedness indices (Table 2) further demonstrated that strain FCG-A23T represents a novel species within the genus Terasakiella (family Terasakiellaceae). The ANI values between strain FCG-A23T and Terasakiella pusilla IF6T and Terasakiella brassicae B3T were 76.71% and 74.92%, respectively. These values are significantly higher than those calculated against species of other genera, supporting genus-level assignment to Terasakiella; however, both ANI values were far below the 95–96% species threshold [51]. Consistently, dDDH values between strain FCG-A23T and T. pusilla IF6T and T. brassicae B3T were 19.7% and 19.0%, respectively, which are also below the 70% cutoff for species circumscription [52]. Therefore, strain FCG-A23T is proposed to represent a novel species within the genus Terasakiella.

3.3. Physiological and Biochemical Characteristics

Strain FCG-A2T exhibited growth at 15–35 °C (optimum, 25–30 °C), at pH 5.0–10.0 (optimum, pH 8.0), and in the presence of 1–4% (w/v) NaCl (optimum, 3%). Overall, these physiological characteristics (Table 3) are comparable to those of the reference strains Pseudothioclava arenosa CAU1312T, Pseudothioclava nitratireducens DL5-4T, Thioclava pacifica TL2T, Thioclava litoralis FTW29T, and Thioclava indica DT23-4T, all of which exhibit relatively broad temperature tolerances. Notably, the optimal NaCl concentration for the growth of strain FCG-A2T (3%) is identical to that of P. nitratireducens DL5-4T, T. litoralis FTW29T, and T. indica DT23-4T. Furthermore, strain FCG-A2T showed sulfur-oxidizing capability and was catalase-positive, traits shared with T. pacifica TL2T and T. indica DT23-4T (Table 3).
Despite these general similarities, the metabolic profile of strain FCG-A2T strongly supports its assignment to the genus Pseudothioclava. In API 20NE tests, the strain did not utilize D-mannose, D-glucose, or adipic acid. This pattern closely resembles those of P. arenosa CAU1312T and P. nitratireducens DL5-4T while distinctly differentiating it from Thioclava species, which are predominantly positive for these substrates (Table 3). In API ZYM assays, strain FCG-A2T was positive for cystine arylamidase, naphthol-AS-BI-phosphohydrolase (weakly), and β-glucosidase, which is consistent with the reference strains P. arenosa CAU1312T and P. nitratireducens DL5-4T. Additionally, the absence of β-galactosidase activity in strain FCG-A2T further aligns it with the Pseudothioclava phenotype, differentiating it from Thioclava members such as T. pacifica and T. indica, which possess this enzyme.
In antibiotic susceptibility tests, strain FCG-A2T was susceptible to ampicillin, gentamicin, penicillin G, vancomycin, and neomycin but resistant to streptomycin, cefoperazone, erythromycin, novobiocin, kanamycin, chloramphenicol, and cefazolin. Taken together, the overall phenotypic profile supports its placement in Pseudothioclava, while the distinguishing features indicate that it represents a novel species.
Strain FCG-A23T grew at 20–33 °C (optimum, 25–30 °C), at pH values ranging from 6.0 to 9.0 (optimum, pH 8.0) and at NaCl concentrations ranging from 2% to 6% (w/v) (optimum, 2%). These growth conditions are comparable to those of Terasakiella pusilla IF6T, Terasakiella brassicae B3T, and Terasakiella salincola KMU-80T. While all three reference strains are capable of growth within the 18–33 °C range, they generally exhibit broader temperature tolerances (Table 4). The optimal salinity for strain FCG-A23T is identical to that of T. salincola KMU-80T, whereas its optimal pH matches that of T. pusilla IF6T. Moreover, strain FCG-A23T shares many phenotypic characteristics with T. pusilla IF6T and T. brassicae B3T in API assays (Table S3). Similar to these reference strains, strain FCG-A23T was positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, and acid phosphatase, but negative for α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase (Table S3). In API ZYM, API 20NE and API 50CH tests, strain FCG-A23T was positive for denitrification, indole production, arginine dihydrolase, and urease, but negative for gelatin hydrolysis, and the utilization of D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, capric acid, and phenylacetic acid. These results are consistent with those observed for T. pusilla IF6T and T. brassicae B3T. Collectively, these shared features support the assignment of strain FCG-A23T to the genus Terasakiella.
Despite these similarities, strain FCG-A23T can be distinguished from its closest relatives by several phenotypic traits. For example, strain FCG-A23T was negative for malic acid utilization, D-glucose fermentation, and cystine arylamidase activity, whereas T. pusilla IF6T and T. brassicae B3T were positive for these characteristics (Table 4). In addition, in contrast to T. pusilla IF6T, strain FCG-A23T was negative for catalase activity and trisodium citrate utilization (Table 4). Strain FCG-A23T was susceptible to all antibiotics tested, including ampicillin, gentamicin, penicillin G, neomycin, streptomycin, erythromycin, kanamycin, chloramphenicol, vancomycin, cefoperazone, novobiocin and cefazolin. Taken together, these phenotypic differences indicate that strain FCG-A23T represents a novel species within the genus Terasakiella.

3.4. Chemotaxonomic Characteristics

The respiratory quinones of strain FCG-A2T consisted of ubiquinone-10 (Q-10, 67.6%) and ubiquinone-9 (Q-9, 32.4%) (Table 3). Notably, the presence of Q-9 alongside Q-10 as a major respiratory quinone serves as a unique chemotaxonomic marker, distinguishing strain FCG-A2T from other members of the genera Pseudothioclava and Thioclava, which typically contain Q-10 exclusively. The polar lipid profile of strain FCG-A2T was similar to those of reference strains of the genera Pseudothioclava and Thioclava, all of which contain phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) as major components (Table 3; Figure S3). The cellular fatty acid composition of strain FCG-A2T was compared with that of reference strains of Pseudothioclava and Thioclava. As shown in Table 5, the predominant fatty acids in strain FCG-A2T were summed feature 3 (C16:1ω7c and/or C16:1ω6c; 21.1%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c; 29.6%), which are also predominant in the reference strains of the genus Pseudothioclava. Taken together, these chemotaxonomic properties support the affiliation of strain FCG-A2T with the genus Pseudothioclava, while confirming its distinctiveness from currently recognized species.
The predominant isoprenoid quinone in strain FCG-A23T was ubiquinone-10 (Q-10), which is consistent with other members of the genus Terasakiella (Terasakiella pusilla IF6T, Terasakiella brassicae B3T and Terasakiella salincola KMU-80T). The major polar lipids of strain FCG-A23T were PE and PG, with diphosphatidylglycerol (DPG) present in minor amounts; these lipids are also reported for T. pusilla IF6T, T. brassicae B3T and T. salincola KMU-80T (Figure S4; Table 4). Similar to T. pusilla IF6T and T. brassicae B3T, the major fatty acids of strain FCG-A23T (>10% of total fatty acids) were summed feature 8 (C18:1ω7c and/or C18:1ω6c; 36.6%), C16:0 (27.6%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c; 13.8%) (Table 6).
Collectively, these chemotaxonomic features support the taxonomic placement of strains FCG-A2T and FCG-A23T within the genera Pseudothioclava and Terasakiella, respectively.

3.5. Genome Annotation

3.5.1. Thiosulfate Oxidation Metabolism

The genome of strain FCG-A2T comprises 3086 predicted genes, including 49 tRNA genes, 15 sRNA genes and three rRNA genes. Among all predicted genes, 1879 genes were annotated and assigned to functional categories based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The most represented KEGG categories were related to metabolism, including carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, and energy metabolism. The number of genes assigned to each KEGG category is summarized in Figure S5. For strain FCG-A23T, 3963 genes were predicted from the genome, including 44 tRNA genes, 13 sRNA genes and three rRNA genes. In total, 3050 genes were annotated and classified into KEGG functional categories (Figure S6). Metabolism was the largest category, with amino acid metabolism (224 genes), carbohydrate metabolism (202 genes), and metabolism of cofactors and vitamins (184 genes) being the most abundant subcategories.
Comparative genomic analysis indicated that strains FCG-A2T and FCG-A23T, similar to their closest relatives, harbor complete gene sets for both assimilatory sulfate reduction (cysN, cysC, cysD, cysH, cysJ, cysI) and the sulfur oxidation (Sox) system (soxA, soxB, soxC, soxD, soxY and soxZ) (Figure 4a–c). The presence of these pathways suggests that both strains potentially participate in the sulfur cycle. To verify their sulfur-oxidizing capability, thiosulfate oxidation assays were performed. Strain FCG-A2T exhibited chemoautotrophic growth, evidenced by a color change in the pH indicator in the medium from blue to yellow, indicating acidification associated with thiosulfate oxidation (Figure 4d). In contrast, strain FCG-A23T oxidized thiosulfate only in the presence of glucose, as indicated by the same color shift from blue to yellow (Figure 4e). Although the oxidation activity was primarily indicated by pH reduction, the presence of complete sox operons provides a solid genetic basis for this metabolic trait. Together, these results confirm that both strains are capable of thiosulfate oxidation, but they employ distinct metabolic strategies. Strain FCG-A2T appears to adopt a more autotrophy-oriented sulfur-based lifestyle, whereas strain FCG-A23T shows a more heterotrophy-associated sulfur metabolism.

3.5.2. CAZymes and Substrates

In light of the distinct sulfur metabolic modes described above, we further assessed whether the carbon acquisition potentials of these strains were similarly differentiated. Therefore, CAZymes were annotated and potential substrate utilization was inferred from the annotated CAZyme families. To place the two isolates in a broader genomic context, we compared their CAZyme repertoires with those of type strains from their respective genera, Pseudothioclava, Thioclava and Terasakiella.
Overall, CAZyme profiling across the genera Pseudothioclava and Terasakiella revealed distinct enzymatic signatures consistent with genome-based phylogenetic clustering (Figure 5a,b). Strain FCG-A2T encodes 56 CAZymes, including 32 glycosyltransferases (GTs), 14 glycoside hydrolases (GHs), four carbohydrate esterases (CEs), five auxiliary activity enzymes (AAs), and one carbohydrate-binding module (CBM). Notably, all three species within genus Pseudothioclava, including strain FCG-A2T, P. arenosa CAU1312T, and P. nitratireducens DL5-4T, share genes encoding CE4, GH13 and CBM50, suggesting that these three CAZyme families may serve as characteristic enzymatic markers for the genus Pseudothioclava. In comparison, while members of the genus Thioclava also typically possess these enzymes, only CE4 and GH13 are conserved across all species (as CBM50 is absent in two Thioclava strains), indicating that CE4 and GH13 represent the defining signature enzymes for Thioclava. The consistent presence of the CE4, GH13 and CBM50 triad in strain FCG-A2T aligns functionally with the Pseudothioclava genus. (Figure 5a). In contrast, strain FCG-A23T encodes 48 CAZymes, comprising 31 GTs, 10 GHs, two CEs, four AAs and one polysaccharide lyase (PL) and clusters with Terasakiella pusilla DSM 6293T and Terasakiella brassicae B3T based on comparable CAZyme profiles, notably including the absence of CBMs (Figure 5b). Thus, the similarity in CAZyme repertoires provides additional support for the phylogenetic placement of strain FCG-A2T in Pseudothioclava and FCG-A23T in Terasakiella. At the genus level, several CAZyme families were consistently observed and may represent genus-associated genomic traits. CE4, GH13 and CBM50 were conserved across the analyzed Pseudothioclava genomes, whereas AA4 and CE11 were consistently present in Terasakiella. Functionally, Pseudothioclava and Thioclava genomes tend to encode a higher diversity and abundance of CAZymes than Terasakiella, suggesting a broad carbohydrate-processing potential and, potentially, high flexibility in carbon utilization.
Consistent with this, substrate inference based on dbCAN-annotated CAZymes indicates divergent carbon-use potential between the two isolates. In strain FCG-A2T, the occurrence of CE4, GH13 and CBM50 suggests potential activity on chitin-derived substrates (e.g., chitooligosaccharides) as well as sucrose and starch (Figure 5a). In contrast, strain FCG-A23T harbors CAZymes such as AA1, AA4, GH13 and CE11, implying the capacity to target plant polymer-related substrates (e.g., lignin-associated components) in addition to sucrose and other polysaccharides (Figure 5b, right). The broader predicted substrate spectrum in strain FCG-A2T provides a plausible genomic basis for its greater metabolic versatility, which—together with the sulfur metabolic differences described above—may ultimately contribute to distinct ecological strategies.
Notably, despite the diverse CAZyme repertoire identified in strain FCG-A2T, API 50CH results showed relatively limited substrate utilization (Table 4 and Table S2). This discrepancy between genomic predictions and phenotypic expression is frequently encountered in bacterial characterization. CAZyme-encoding genes represent metabolic potential rather than constitutive expression, as many carbohydrate-degrading enzymes require specific inducers for expression that may be absent under standard assay conditions [54]. Additionally, the API 50CH system, designed primarily for clinical isolates, may not provide optimal conditions (e.g., salinity, pH) for marine bacteria such as Pseudothioclava and Thioclava species [55]. Furthermore, the API 50CH panel primarily targets simple sugars, whereas marine Pseudothioclava and Thioclava species likely utilize complex marine-derived polysaccharides such as chitin derivatives and algal carbohydrates that are not represented in standard biochemical assays. Thus, the limited substrate utilization observed reflects the inherent limitations of standardized phenotypic tests rather than contradicting the genomic CAZyme potential.

3.6. Ecological Distribution and Habitat Association

Based on Protologger-based ecological prediction [38], strain FCG-A2T exhibits a markedly broader environmental distribution and higher overall prevalence than strain FCG-A23T (Figure 6; Tables S4 and S5). For strain FCG-A2T, the predicted prevalence is non-zero across all 19 environmental categories (Table S4), indicating a wide habitat range. In contrast, strain FCG-A23T is predicted to occur only in a limited set of environments—coral, marine water, marine sediment, plant-associated habitats and freshwater—with relatively low prevalence values of 0.14%, 0.05%, 0.04%, 0.02%, and 0.01%, respectively (Table S5). Notably, strain FCG-A2T shows substantially higher prevalence, exceeding 10% in nine environments, including marine sediment (37.1%), coral (29.8%), rhizosphere (18.7%), activated sludge (14.3%), wastewater (13.8%), and marine water (11.1%). As suggested by the genomic CAZyme analyses above, this difference in predicted habitat breadth may be linked to differences in CAZyme repertoires and associated substrate-processing potential.
Both strains are predicted to utilize sucrose, a common plant-derived carbohydrate, which may be consistent with their predicted occurrence in plant-associated habitats (FCG-A2T, 9.2% and FCG-A23T, 0.02%; Figure 6). Coral environments represent another predicted major habitat. In coral ecosystems, chitin can occur as a structural component in associated organisms, and coral-associated matrices may contain sulfated polysaccharides [56]; moreover, coral surfaces are typically coated with mucus layers rich in polysaccharides [57]. The availability of these polymers is compatible with corals being predicted as major habitats for both strains, with prevalence values of 29.8% for FCG-A2T and 0.14% for FCG-A23T (Figure 6). Beyond corals, chitin–which can be partially degraded into chitooligosaccharides–is widespread in marine environments (e.g., crustacean shells), soil (e.g., fungal cell walls and insect exoskeletons), and rhizosphere systems [58,59]. Accordingly, the predicted capacity of FCG-A2T to utilize chitin-related substrates aligns with its predicted distribution in marine sediment (37.1%), marine water (11.1%), soil (9.4%) and rhizosphere (18.7%) (Figure 6a). Starch is abundant in plant storage tissues and can enter soil through plant litter; in addition, roots release various organic substances into soil, including sugars such as sucrose. Therefore, the predicted capacity of strain FCG-A2T to utilize starch- and sucrose-related substrates further supports its occurrence in plant-, soil- and rhizosphere-associated habitats (Figure 6a).
In contrast, the CAZyme profile of FCG-A23T suggests a potential to target lignin-associated or other plant polymer-related substrates. Lignin is primarily derived from terrestrial plants and can enter soils and peatlands through plant debris, and subsequently be transported to coastal and marine systems through riverine input and shoreline erosion [60,61]. This provides a plausible explanation for the predicted distribution of FCG-A23T in plant-associated habitats (0.02%), marine water (0.05%) and marine sediment (0.04%) (Figure 6b).
Overall, the predicted ecological patterns align with the inferred substrate-processing potential: the broader CAZyme repertoire and wider predicted substrate spectrum of FCG-A2T likely contribute to its broader environmental distribution. Such a wide distribution may increase the opportunities for FCG-A2T to participate in sulfur oxidation across multiple habitats, thereby potentially contributing to sulfur-driven biogeochemical cycling.

4. Conclusions

Two sulfur-metabolizing bacterial strains, FCG-A2T and FCG-A23T, were isolated from the South China Sea. Strain FCG-A2T shares multiple phenotypic and chemotaxonomic characteristics with recognized members of the genus Pseudothioclava, including comparable optimal growth conditions, fatty acid profiles, and respiratory quinone composition. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences consistently placed FCG-A2T within the genus Pseudothioclava. In combination with its genomic relatedness metrics and distinguishing phenotypic features, these results support the conclusion that FCG-A2T represents a novel species within the genus Pseudothioclava, for which the name Pseudothioclava alba sp. nov. is proposed. The formal species description is provided in Table 7.
Strain FCG-A23T similarly exhibited phenotypic and chemotaxonomic traits consistent with members of the genus Terasakiella, including optimal growth conditions, the predominant ubiquinone, major fatty acid composition, and several enzyme activities. Phylogenetic inference based on the 16S rRNA gene and whole-genome analyses placed FCG-A23T within the genus Terasakiella. Together with genomic evidence and differential phenotypic characteristics, these findings indicate that FCG-A23T constitutes a novel species of the genus Terasakiella, for which the name Terasakiella sediminum sp. nov. is proposed. The formal species description is also provided in Table 7.
In addition, comparative genomic analyses revealed differences in CAZyme repertoires between the two strains, suggesting distinct carbohydrate utilization potentials. CAZyme-based substrate prediction indicated that both strains may utilize sucrose, while FCG-A2T shows a broader predicted capacity for processing diverse polysaccharides. Protologger-based ecological inference further indicated a wider predicted habitat range and higher prevalence for FCG-A2T, whereas FCG-A23T was restricted to a narrower range of habitats. This wide distribution may increase opportunities for FCG-A2T to perform sulfur oxidation across multiple habitats, potentially contributing to sulfur-driven biogeochemical cycling.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms14020481/s1, Figure S1: Phylogenetic trees of strain FCG-A2T based on 16S rRNA gene sequences using NJ and ME methods and TYGS, illustrating the phylogenetic positions of Pseudothioclava alba FCG-A2T and related taxa; Figure S2: Phylogenetic trees of strain FCG-A23T based on 16S rRNA gene sequences using NJ, ME methods and TYGS, illustrating the phylogenetic positions of Terasakiella sediminum FCG-A23T and related taxa; Figure S3: Two-dimensional-thin layer chromatograms (TLCs) showing the total polar lipids of strain FCG-A2T alongside the reference strains; Figure S4: Two-dimensional-thin-layer chromatogram illustrating the total polar lipids of strain FCG-A23T alongside the reference strains; Figure S5: Functional classification of Pseudothioclava alba FCG-A2T genes categorized according to the KEGG database; Figure S6: Functional classification of Terasakiella sediminum FCG-A23T genes categorized according to the KEGG database. Table S1: Genome assembly and quality assessment metrics for strains FCG-A2T and FCG-A23T; Table S2: API test results for strain FCG-A2T and reference strains Pseudothioclava arenosa CAU1312T, Pseudothioclava nitratireducens DL5-4T and Thioclava pacifica TL2T; Table S3: API test results for strain FCG-A23T, reference strains Terasakiella brassicae B3T, Terasakiella pusilla IF6T; Table S4: Environmental distribution survey for strain FCG-A2T; Table S5: Environmental distribution survey for strain FCG-A23T.

Author Contributions

Conceptualization, H.X. and J.Q.; methodology, H.Z., J.Q. and Q.L.; software, H.Z. and J.Q.; validation, H.Z. and X.L.; formal analysis, H.Z., J.Q. and H.X.; investigation, H.Z., J.Q. and X.L.; resources, H.X.; data curation, H.Z. and H.X.; writing—original draft preparation, H.Z., J.Q. and H.X.; writing—review and editing, H.Z., N.W. and H.X.; visualization, H.Z., N.W. and Z.J.; supervision, H.X.; project administration, H.X.; funding acquisition, H.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (Grant No. U20B2037), the Fund of the Technology Innovation Center for Exploitation of Marine Biological Resources, MNR, China (Grant No. TICMBR202407) and the XMU Undergraduate Innovation and Entrepreneurship Training Programs (Grant No. 2023X752).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Whole-genome sequencing data of the novel strains Pseudothioclava alba FCG-A2T and Terasakiella sediminum FCG-A23T have been deposited in GenBank under the accession numbers JAWLVI000000000 and JAWLVJ000000000, respectively. The 16S rRNA gene sequence has been deposited under the accession numbers OR192614 and OR206505, respectively.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Microorganisms 14 00481 g001
Figure 1. 16S rRNA gene-based phylogenetic tree and morphological observations of strain FCG-A2T. (a) Colony morphology; (b) scanning electron microscopy (SEM) image; (c) transmission electron microscopy (TEM) image; (d) maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, illustrating the phylogenetic position of strain FCG-A2T and related taxa. Filled circles indicate branches also recovered by the minimum-evolution and neighbor-joining methods. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Stappia stellulata TS101T was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.
Figure 1. 16S rRNA gene-based phylogenetic tree and morphological observations of strain FCG-A2T. (a) Colony morphology; (b) scanning electron microscopy (SEM) image; (c) transmission electron microscopy (TEM) image; (d) maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, illustrating the phylogenetic position of strain FCG-A2T and related taxa. Filled circles indicate branches also recovered by the minimum-evolution and neighbor-joining methods. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Stappia stellulata TS101T was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.
Microorganisms 14 00481 g001
Microorganisms 14 00481 g002
Figure 2. 16S rRNA gene-based phylogenetic tree and morphological observations of strain FCG-A23T. (a) Colony morphology; (b) scanning electron microscopy (SEM) image; (c) transmission electron microscopy (TEM) image; (d) maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, illustrating the phylogenetic position of Terasakiella sediminum FCG-A23T and related taxa. Filled circles indicate branches also recovered by the minimum-evolution and neighbor-joining methods. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Acidithiobacillus acidisediminis S30A2T was used as the outgroup. Bar, 0.05 substitutions per nucleotide position.
Figure 2. 16S rRNA gene-based phylogenetic tree and morphological observations of strain FCG-A23T. (a) Colony morphology; (b) scanning electron microscopy (SEM) image; (c) transmission electron microscopy (TEM) image; (d) maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, illustrating the phylogenetic position of Terasakiella sediminum FCG-A23T and related taxa. Filled circles indicate branches also recovered by the minimum-evolution and neighbor-joining methods. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Acidithiobacillus acidisediminis S30A2T was used as the outgroup. Bar, 0.05 substitutions per nucleotide position.
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Microorganisms 14 00481 g003
Figure 3. Genome-based phylogenetic trees and POCP heatmaps. (a) Phylogenetic tree of strain FCG-A2T depicting its phylogenetic position relative to related taxa. (b) POCP heatmap of strain FCG-A2T. (c) Phylogenetic tree of strain FCG-A23T depicting its phylogenetic position relative to related taxa. (d) POCP heatmap of strain FCG-A23T. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Stappia stellulata TS101T and Acidithiobacillus acidisediminis S30A2T were used as outgroups for (a) and (c), respectively. Bars, 0.050 substitutions per nucleotide position in (a) and 0.10 substitutions per nucleotide position in (c).
Figure 3. Genome-based phylogenetic trees and POCP heatmaps. (a) Phylogenetic tree of strain FCG-A2T depicting its phylogenetic position relative to related taxa. (b) POCP heatmap of strain FCG-A2T. (c) Phylogenetic tree of strain FCG-A23T depicting its phylogenetic position relative to related taxa. (d) POCP heatmap of strain FCG-A23T. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Stappia stellulata TS101T and Acidithiobacillus acidisediminis S30A2T were used as outgroups for (a) and (c), respectively. Bars, 0.050 substitutions per nucleotide position in (a) and 0.10 substitutions per nucleotide position in (c).
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Microorganisms 14 00481 g004
Figure 4. (a) Thiosulfate oxidation pathway and experimental validation. (a) Assimilatory sulfate reduction pathway in strains FCG-A2T and FCG-A23T, as determined using the KEGG database. Genes highlighted in red boxes have been explicitly annotated. (b) Distribution of the Sox system genes among species of the genera Pseudothiocalva and Thioclava. (c) Distribution of the Sox system among species of the genus Terasakiella. (d) Sulfur oxidation from thiosulfate to sulfate by strain FCG-A2T. Negative control: Hyphomonas polymerpha PS-728T; positive control: Thioclava pacifica TL2T. (e) Sulfur oxidation from thiosulfate to sulfate by strain FCG-A23T. Negative control: Hyphomonas polymerpha PS-728T; positive control: Thioclava pacifica TL2T.
Figure 4. (a) Thiosulfate oxidation pathway and experimental validation. (a) Assimilatory sulfate reduction pathway in strains FCG-A2T and FCG-A23T, as determined using the KEGG database. Genes highlighted in red boxes have been explicitly annotated. (b) Distribution of the Sox system genes among species of the genera Pseudothiocalva and Thioclava. (c) Distribution of the Sox system among species of the genus Terasakiella. (d) Sulfur oxidation from thiosulfate to sulfate by strain FCG-A2T. Negative control: Hyphomonas polymerpha PS-728T; positive control: Thioclava pacifica TL2T. (e) Sulfur oxidation from thiosulfate to sulfate by strain FCG-A23T. Negative control: Hyphomonas polymerpha PS-728T; positive control: Thioclava pacifica TL2T.
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Microorganisms 14 00481 g005
Figure 5. Genome-based phylogenetic trees and CAZyme profiles of strains (a) FCG-A2T and (b) FCG-A23T. The total counts and categories of CAZymes (middle panel) include Glycosyl Transferases (GTs, orange), Glycoside Hydrolases (GHs, blue), Carbohydrate Esterases (CEs, red), Carbohydrate-Binding Modules (CBMs, yellow), Auxiliary Activities (AAs, green) and Polysaccharide Lyases (PLs, purple). The numbers and color intensity (right panel) represent the counts and relative abundance of selected CAZyme families.
Figure 5. Genome-based phylogenetic trees and CAZyme profiles of strains (a) FCG-A2T and (b) FCG-A23T. The total counts and categories of CAZymes (middle panel) include Glycosyl Transferases (GTs, orange), Glycoside Hydrolases (GHs, blue), Carbohydrate Esterases (CEs, red), Carbohydrate-Binding Modules (CBMs, yellow), Auxiliary Activities (AAs, green) and Polysaccharide Lyases (PLs, purple). The numbers and color intensity (right panel) represent the counts and relative abundance of selected CAZyme families.
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Microorganisms 14 00481 g006
Figure 6. Predicted Environmental distribution of strains (a) FCG-A2T and (b) FCG-A23T.
Figure 6. Predicted Environmental distribution of strains (a) FCG-A2T and (b) FCG-A23T.
Microorganisms 14 00481 g006
Table 1. Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA–DNA hybridization (dDDH) values between strain FCG-A2T and related type strains.
Table 1. Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA–DNA hybridization (dDDH) values between strain FCG-A2T and related type strains.
StrainsGenBank AccessionANI
(%)		AAI
(%)		dDDH
(%)
Pseudothioclava arenosa CAU1312TNTJD0000000078.5379.4621.30%
Pseudothioclava nitratireducens DL5-4TJARGDK00000000077.9278.2221.00%
Thioclava pacifica TL2TAUND0000000076.2973.4319.70
Thioclava marina 11.10-0-13TMPZS0000000076.1973.2919.90
Thioclava atlantica 13D2W2TAQRC0000000076.2173.0319.40
Thioclava nitratireducens 25B10-4TCP01943776.1772.2519.70
Thioclava sediminum TAW-CT134TMPZV0000000076.1069.5919.50
Thioclava dalianensis DLFJ1-1TJHEH0000000075.4865.8219.10
Thioclava litoralis FTW29TCP13544372.8864.0620.30
Thioclava indica DT23-4TAUNB0000000075.4468.1918.70
Thioclava arctica 15-R06ZXC-3TJBFRYC00000000074.2672.1418.90
Table 2. Average amino acid identity (AAI), average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FCG-A23T and ten closely related type strains of the order Rhodospirillales.
Table 2. Average amino acid identity (AAI), average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FCG-A23T and ten closely related type strains of the order Rhodospirillales.
StrainsGenBank AccessionAAI
(%)		ANI
(%)		dDDH
(%)
Terasakiella pusilla IF6TJHYO0000000078.9976.7119.70
Terasakiella brassicae B3TBMHV0000000075.8474.9219.00
Aestuariispira insulae AH-MY2TQRDW0000000055.6969.7818.10
Thalassospira australica NP3b2TJRJE0000000056.3670.2217.50
Thalassospira povalilytica Zumi95TPGTS0000000056.5270.2818.50
Thalassospira xiamenensis M-5TCP00438856.6370.0221.40
Thalassospira indica PB8BTCP03155556.5370.2724.90
Novispirillum itersonii subsp. itersonii LMG4337TARMX0000000056.3569.7525.50
Gimibacter soli 6D33TCP11680554.3369.6727.30
Thalassospira xianhensis P-4TJPWA0000000056.5870.1019.30
Table 3. Phenotypic characteristics distinguishing strain FCG-A2T from closely related species.
Table 3. Phenotypic characteristics distinguishing strain FCG-A2T from closely related species.
Characteristic123456
Temperature range for growth (°C) (Optimum)15–35
(25–30)		20–40
(30) #		8–40
(28–30) &		15–47
(35) *		15–42
(28–30)		10–40
(28–32)
NaCl range for growth (%, w/v) (Optimum)1–4 (3)1–4 (2) #0–7 (1–3) &1–9 (3.5) *0.5–10 (3)0–18 (3)
pH range for growth (Optimum)5–10 (8)6–9 (6.5) #6–9 (7) &6.5–8.5 (8) *4.0–9.0
(5.5–7.5)		6–10
Catalase activity++ #+ &+ *w+
Oxidase activity−+ #+ &+ *w+
Sulfur oxidation to sulfate+NANA+ *NA+
Hydrolysis of (API 20NE):
 D-glucose fermentation−−++−−
 Gelatin hydrolysis−w−+−−
 β-galactosidase−−−+−+
 D-glucose−−++++
 D-mannose−−−++w
 D-mannitol−−++++
 Adipic acid−−−+−−
 Malic acid−+++−+
Enzyme activity (API ZYM)
 Cystine arylamidase++w+w w
 α-chymotrypsinw−−−w −
 Naphtol-AS-BI-phosphohydrolasew+ww−w
 β-glucosidase+w++−+
Utilization of (API 50CH):
 Salicin−ww+NANA
 D-Cellobiose−−++NANA
 Glycogen−−w+NANA
 L-fucose+−−−NANA
Major polar lipidsPG, PE, DPGPE, PG, PN, PGL, PLDPG, PE, PG, AL, PLPG, PE, DPG, PGL *DPG, PE, PGPG, PE
Major respiratory quinoneQ-9, Q-10Q-10 #Q-10 &Q-10 *Q-9, Q-10Q-10
DNA G + C content (mol%)62.0964.7 #63.8 &63.1 *61.460.3
Taxa: 1, FCG-A2T; 2, Pseudothioclava arenosa CAU 1312T; 3, Pseudothioclava nitratireducens DL5-4T; 4, Thioclava pacifica TL2T; 5, Thioclava litoralis FTW29T; 6, Thioclava indica DT23-4T. +, Positive; w, weakly positive; −, negative; NA, not available. PE: phosphatidylethanolamine, PG: phosphatidylglycerol, DPG: diphosphatidylglycerol, PGL: phosphoglycolipid, AL: aminolipid, PL: phospholipid. # Data from Thongphrom et al. [16]. & Data from Liu et al. [50]. * Data from Sorokin et al. [8]. All data for strain 5 are from Chen et al. [15]. All data for strain 6 are from Liu et al. [10].
Table 4. Differential characteristics of strain FCG-A23T and other closely related type strains.
Table 4. Differential characteristics of strain FCG-A23T and other closely related type strains.
Characteristic1234
Temperature range for growth (°C)20–336–40 §15–37 ‡10–40 (25)
NaCl range for growth (%, w/v)2–6 1–8 §0.5–8 ‡2–6
pH range for growth 6.0–9.06.0–9.0 §5.5–9.0 ‡5.5–9.5 (7.0)
Catalase activity−w−−
Oxidase activity++++
Utilization of (API 50CH):
 Glycerolw+−−
 Inulin−−+−
 D-Raffinose−−+−
 Potassium 2-Ketogluconate +−−NA
Hydrolysis of (API 20NE):
 Tryptophan (Indole production)w++−
 Adipic acid−−+NA
 Malic acid−++−
 Trisodium citrate−+−NA
Enzyme activity (API ZYM)
 Reduction of nitrate to nitrite+−+−
 D-Glucose fermentation−++NA
 Cystine arylamidase−++−
 Naphthol-AS-BI-phosphohydrolasew+w+
Major polar lipidsPE, PG, DPGPE, PG, DPG, APL, PS ‡PE, PG, DPG, APL, PS ‡PE, PG, DPG
Major isoprenoid quinoneQ-10Q-10 †Q-10 ‡Q-10
DNA G + C content (mol%)49.0648 †42.3 ‡48.3
Taxa: 1, FCG-A23T; 2, Terasakiella pusilla IF6T; 3, Terasakiella brassicae B3T; 4, Terasakiella salincola KMU-80T. +, Positive; −, Negative; w, weakly positive; NA: not available. All species are Gram-negative. PE: phosphatidylethanolamine, PG: phosphatidylglycerol, DPG: diphosphatidylglycerol, APL: aminophospholipid, PS: phosphatidylserine. § Data from Jaewoo et al. [25]. † Data from Satomi et al. [22]. ‡ Data from Han et al. [24]. All data for strain 4 are from Jaewoo et al. [25].
Table 5. Cellular fatty acid composition (%) of strain FCG-A2T and relevant type strains.
Table 5. Cellular fatty acid composition (%) of strain FCG-A2T and relevant type strains.
123456
Straight-chain
C11:01.31.42TRTRTRTR
C12:02.1TRTRTRTRTR
C14:01.3TRTRTR1.2TR
C16:017.89.195.082.417.23.7
C17:06.92.57TRTRTRTR
C18:07.910.777.43.91.12.0
Unsaturated
Cyclo C19:0 ω8cTRTRTR5.6TRTR
Hydroxy
C10:0 2-OH1.21.681.26TRTRTR
C10:0 3-OH2.93.282.72TR2.2TR
Iso-C11:0 3-OH1.11.591.04TRTRTR
C11:0 3-OH1.51.771.4TRTRTR
C18:0 3-OHTRTRTR5TRTR
10-methyl C19:0TRTRTR1.2TRTR
Summed features *
1TRTRTRTRTRTR
2TR1.05TRTR1.5TR
321.16.743.17TR1.67.39
4TRTRTRTRTRTR
5TRTRTRTRTRTR
829.652.7974.5577.166.581.9
Taxa: 1, FCG-A2T; 2, Pseudothioclava arenosa CAU1312T; 3, Pseudothioclava nitratireducens DL5-4T; 4, Thioclava pacifica TL2T; 5, Thioclava litoralis FTW29T (data from Lai et al. [15]); 6, Thioclava indica DT23-4T (data from Liu et al. [10]). Fatty acids present at less than 1.0% in all strains are not listed. TR, trace amount (<1.0%). * Summed features are fatty acids that cannot be resolved reliably from other fatty acids using the chosen chromatographic conditions. The MIDI system groups these fatty acids together as one feature with a single percentage of the total [53]. Summed feature 1, C13:0 3-OH and/or iso-C15:1 I; Summed feature 2, C14:0 3-OH and/or iso-C16:1 I; Summed feature 3, C16:1ω7c and/or C16:1ω6c; Summed feature 4, iso-C17:1 I and/or anteiso-C17:1 B; Summed feature 5, C18:0 ante and/or C18:2 ω6,9c; Summed feature 8, C18:1ω7c and/or C18:1ω6c.
Table 6. Cellular fatty acid composition (%) of strain FCG-A23T and relevant type strains.
Table 6. Cellular fatty acid composition (%) of strain FCG-A23T and relevant type strains.
1234
Straight-chain
C12:01.13.63.32.8
C14:04.01.33.6TR
C16:027.612.618.413.4
C18:01.01.41.7TR
Unsaturated
C16:1ω5c2.91.4TR1.7
C17:1ω6c1.6TR1.3TR
C18:1ω5cTR1.01TR1
C18:1ω7cTRTRTR60.2
C20:1ω7cTRTRTRTR
Hydroxy
C16:0 3-OHTRTRTR1
C18:0 3-OHTRTRTRTR
Summed features *
27.06.46.35.3
313.815.925.011.1
836.652.336.0TR
Taxa: 1, FCG-A23T; 2, Terasakiella pusilla IF6T; 3, Terasakiella brassicae B3T; 4, Terasakiella salincola KMU-80T (Data from Jaewoo et al. [25]). Fatty acids present at less than 1.0% in all strains are not listed. TR, Trace amounts (<1.0%). * Summed features are groups of two or three fatty acids that could not be separated by the MIDI system. Summed feature 2, C14:0 3-OH and/or iso-C16:1 I; Summed feature 3, C16:1ω7c and/or C16:1ω6c; Summed feature 8, C18:1ω7c and/or C18:1ω6c.
Table 7. Descriptions of Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov.
Table 7. Descriptions of Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov.
Genus namePseudothioclavaTerasakiella
Species namePseudothioclava albaTerasakiella sediminum
Species statussp. nov.sp. nov.
Species etymologyal’ba. L. fem. adj. alba, whitese.di’mi.num. L. gen. pl. n. sediminum, of sediments
Description of the new taxon and diagnostic traitsCells are Gram-negative, facultatively autotrophic, rod-shaped (0.4–0.6 × 2.0–8.0 µm). Catalase-positive and oxidase-negative. Colonies on MA are circular, smooth, moist and white. Growth occurs at 15–35 °C (optimum, 25–30 °C), at pH 5.0–10.0 (optimum, pH 8.0) and in the presence of 1–4% (w/v) NaCl (optimum, 3%). The principal respiratory quinone is Q-10. Major fatty acids (>10%) include summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. Polar lipids comprise phosphatidylethanolamine and phosphatidylglycerol. In the API ZYM strip, enzymatic activity is detected for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, α-glucosidase, and β-glucosidase. In the API 20NE system, positive for arginine dihydrolase and Urease. In the API 50CH system, acid is produced from D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, potassium 2-ketogluconate, potassium 5-ketogluconate; weakly positive for D-sorbitol, L-arabitol and hydrolysis of esculin.Cells are aerobic, Gram-negative, non-flagellated, S-shaped (0.3–0.5 × 2.0–5.0 µm). Colonies on marine agar 2216E are circular and beige after 3 days at 30 °C. Growth occurs at 20–33 °C (optimum, 25–30 °C), at pH 6.0–9.0 (optimum, pH 8.0) and in the presence of 2–6% (w/v) NaCl (optimum, 2%). Oxidase-positive and catalase-negative. The major fatty acids are summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The major respiratory quinone is Q-10. Polar lipids consist of phosphatidylethanolamine and phosphatidylglycerol. In the API ZYM strip, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, and acid phosphatase. In the API 20NEsystem, positive for nitrate reduction, denitrification, arginine dihydrolase, urease, L-arabinose and D-maltose. In the API 50CH system, acid is produced from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, methyl-β-D-xylopyranoside, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, amygdalin, D-cellobiose, D-maltose, D-lactose, gentiobiose, D-turanose, D-lyxose, potassium 2-ketogluconate and potassium 5-ketogluconate.
Country of originChinaChina
Region of originGuangxi ProvinceGuangxi Province
Date of isolation31 July 202331 July 2023
Source of isolationsedimentsediment
Sampling date31 May 202331 May 2023
Latitude21.63° N21.63° N
Longitude108.42° E108.42° E
16S rRNA gene accession numberOR192614OR206505
Genome accession numberJAWLVI000000000JAWLVJ000000000
Genome size (Mb)3.14.2
G + C content (%)62.0949.06
Designation of the Type strainFCG-A2TFCG-A23T
Strain collection NumbersMCCC 1K08969T = KCTC 8462TMCCC 1K08972T = KCTC 8464T
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Zhou, H.; Qu, J.; Lin, X.; Wang, N.; Jiang, Z.; Lai, Q.; Xu, H. Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov. Microorganisms 2026, 14, 481. https://doi.org/10.3390/microorganisms14020481

AMA Style

Zhou H, Qu J, Lin X, Wang N, Jiang Z, Lai Q, Xu H. Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov. Microorganisms. 2026; 14(2):481. https://doi.org/10.3390/microorganisms14020481

Chicago/Turabian Style

Zhou, Hui, Jieni Qu, Xu Lin, Ning Wang, Zihan Jiang, Qiliang Lai, and Hong Xu. 2026. "Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov." Microorganisms 14, no. 2: 481. https://doi.org/10.3390/microorganisms14020481

APA Style

Zhou, H., Qu, J., Lin, X., Wang, N., Jiang, Z., Lai, Q., & Xu, H. (2026). Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov. Microorganisms, 14(2), 481. https://doi.org/10.3390/microorganisms14020481

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