Physiological and Genomic Characterization of a Novel Obligately Chemolithoautotrophic, Sulfur-Oxidizing Bacterium of Genus Thiomicrorhabdus Isolated from a Coastal Sediment

Thiomicrorhabdus species, belonging to the family Piscirickettsiaceae in the phylum Pseudomonadotav are usually detected in various sulfur-rich marine environments. However, only a few bacteria of Thiomicrorhabdus have been isolated, and their ecological roles and environmental adaptations still require further understanding. Here, we report a novel strain, XGS-01T, isolated from a coastal sediment, which belongs to genus Thiomicrorhabdus and is most closely related to Thiomicrorhabdus hydrogeniphila MAS2T, with a sequence similarity of 97.8%. Phenotypic characterization showed that XGS-01T is a mesophilic, sulfur-oxidizing, obligate chemolithoautotrophy, with carbon dioxide as its sole carbon source and oxygen as its sole electron acceptor. During thiosulfate oxidation, strain XGS-01T can produce extracellular sulfur of elemental α-S8, as confirmed via scanning electron microscopy and Raman spectromicroscopy. Polyphasic taxonomy results indicate that strain XGS-01T represents a novel species of the genus Thiomicrorhabdus, named Thiomicrorhabdus lithotrophica sp. nov. Genomic analysis confirmed that XGS-01T performed thiosulfate oxidation through a sox multienzyme complex, and harbored fcc and sqr genes for sulfide oxidation. Comparative genomics analysis among five available genomes from Thiomicrorhabdus species revealed that carbon fixation via the oxidation of reduced-sulfur compounds coupled with oxygen reduction is conserved metabolic pathways among members of genus Thiomicrorhabdus.


Introduction
The genus Thiomicrorhabdus belonging to the family Piscirickettsiaceae of the phylum Pseudomonadota (formerly named Proteobacteria) was first proposed based on phylogenetic analyses, including the 16S rRNA gene and 53 ribosomal protein sequences, by Boden et al. [1].Members of genus Thiomicrorhabdus were distributed in various habitats, including deep-sea hydrothermal vents, continental shelf sediments, and intertidal mud flats [2][3][4][5].Thiomicrorhabdus species are strict chemolithoautotrophs and have diverse energy metabolic pathways [6,7].They generally oxidize reduced sulfur compounds to fix CO 2 with oxygen or nitrate as electron acceptors and synthesize the primary productivity of ecosystems in the process [2,3,5].
At the time of writing, the genus Thiomicrorhabdus has 12 reported species with validly published names.Thiomicrorhabdus frisia JB-A2 T is the first validly published species for the genus Thiomicrorhabdus and is isolated from marine sediments along the Wadden Sea [5].Subsequently, Thiomicrorhabdus chilensis Ch-1 T was isolated from the coastal shelf of Chilean [2].In 2005, Knittel et al. identified the first cryophilic sulfur-oxidizing bacteria of this genus, Thiomicrorhabdus psychrophile SVAL-D T and Thiomicrorhabdus arctica SVAL-E T [3].Thiomicrorhabdus hydrogeniphila MAS2 T was isolated from a tank containing coastal seawater from Tokyo Bay [8].Thiomicrorhabdus aquaedulcis HaS4 T was isolated from the water of Lake Harutori in Japan [9].Thiomicrorhabdus indica 13-15A T were isolated from the deep-sea hydrothermal vent environment of Southwest Indian Ocean [6].Thiomicrorhabdus sediminis G1 T and Thiomicrorhabdus xiamenensis G2 T were isolated from sediment samples collected from the coast of Xiamen [7].Recently, Thiomicrorhabdus heinhorstiae [10], Thiomicrorhabdus cannonii [10], and Thiomicrorhabdus immobilis [11] were discovered one after another.
Although genus Thiomicrorhabdus have been detected from worldwide marine systems, its physiological diversity and ecological role have not yet been made fully explicit due to the lack of cultivated strains.In this study, we isolated a new strain, named XGS-01 T , from the coastal sediment of Xiamen, China.We characterized the bacterium as a novel species of genus Thiomicrorhabdus using a polyphasic taxonomic approach; meanwhile, we compared genomic analyses among members of the genus Thiomicrorhabdus to gain insights into the ecological roles and environmental adaptation mechanisms underlying their widespread distribution.Furthermore, we determined the characteristics of extracellular sulfur produced by Thiomicrorhabdus bacteria.

DNA Extraction and Genomic Analyses
The genomic DNA of strain XGS-01 T was extracted according to a previously reported method [15].The complete genome of strain XGS-01 T was sequenced using SMRT technology performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd.(Shanghai, China).The sequencing was performed on a Pacific Biosciences (PacBio) sequencing platform via single-molecule real-time (SMRT) technology.The sequenced reads were filtered, and high-quality paired-end reads were assembled to reconstruct the circulating genome by using SOAPdenovo2 software.The G+C content of chromosomal DNA was determined from the complete genome sequences.

16S rRNA Gene Phylogeny
The 16S rRNA gene was PCR-amplified and sequenced with 27F (5 -AGAGTTGATC-MTGCGCTCAG-3 ) and 1492R (3 -TACGYTACGTCTTACGACT-5 ) primers.The almost fulllength 16S rRNA gene sequence of strain XGS-01 T was analyzed using the gapped-blast search algorithm [16].The gene sequences of the strains were edited by DNAMAN and used to identify the 16S rRNA sequence similarities and phylogenetic neighbors by NCBI (https:// www.ncbi.nlm.nih.gov/,accessed on 14 May 2023) and the Ez Biocloud server (https:// www.ezbiocloud.net,accessed on 21 May 2023) [17].The 16S rRNA sequences with taxa closely related to the strain XGS-01 T were selected for downloading.The phylogenetic tree was constructed using MEGA 11.0 software [18] with three methods-the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods [19]-for the target sequences and all downloaded gene sequences.Genetic distance was calculated using the Kimura two-parameter model [20].Bootstrap analysis was calculated based on 1000 replications.

Morphology, Physiology, and Chemotaxonomic Analysis
Morphological observations of the novel isolated XGS-01 T was performed via a transmission electron microscopy (JEM-1400, JEOL, Tokyo, Japan) with cultures grown in MMJS liquid medium at 32 • C for 1 day.The Gram stain test was conducted with a Gram-staining kit (Hangzhou Tianhe Microbiological Reagent Co., Ltd., Hangzhou, China).
Fatty acids of whole cells grown in the MMJS medium at 28 • C for 24 h were saponified, methylated, and extracted using the standard midi protocol (Sherlock Microbial Identification System, version 6.0B).Using the same method, the cellular fatty acid composition of T. sediminis G1 T and T. xiamenensis G2 T was also determined in parallel with that of strain XGS-01 T in this study.

The Determination of Extracellular Sulfur via Scanning Electron Microscopy and Raman Spectromicroscopy
In order to determine the characteristics of extracellular sulfur produced by strain XGS-01 T , strain XGS-01 T was incubated at 32 • C for 12 h, and subsequently, the extracellular sulfur was collected by using 0.2 µm filter membrane and rinsed it with phosphate buffer solution (PBS) before centrifugation, repeating three times to remove residual culture medium on the surface of elemental sulfur.The collected elemental sulfur sample was determined using scanning electron microscopy (SEM) (ZEISS Sigma FE-SEM, Carl Zeiss, Jena, Germany), Raman spectromicroscopy (Horiba LabRAM HR-evolution, Longjumeau, France), and X-ray diffraction analysis (HyPix-400, Rigaku Ultima IV, Tokyo, Japan).

Phylogenetic Analysis Based on 16S rRNA Gene
The phylogenetic tree was constructed based on the NJ method for the complete 16S rRNA gene sequence (1553 bp) of XGS-01 T , showing that strain XGS-01 T was clustered with Thiomicrorhabdus species (Figure 1).The phylogenetic tree based on the ML method (Figure S1) and the MP method (Figure S2) also supports this topology.These results indicated that strain XGS-01 T belonged to genus Thiomicrorhabdus.BLAST comparison showed that strain XGS-01 T had the highest sequence similarity with T. hydrogeniphila MAS2 T [21], with 97.8% similarity, followed by T. psychrophila SVAL-D T (97.4% similarity) [3] and T. frisia JB-A2 T (97.3% similarity) [5].This value is lower than the threshold criterion for prokaryotic species delineation, which is 98.65% for 16S rRNA gene similarity, indicating that strain XGS-01 T represents a potential new species within the genus Thiomicrorhabdus.

Morphology, Physiology, and Chemotaxonomic Analysis
Strain XGS-01 T was rod-shaped, about 0.7-0.8μm wide and 1.6-1.8μm long, and had non-flagella (Figure 2), which was similar to other strains in the genus Thiomicrorhabdus.

Morphology, Physiology, and Chemotaxonomic Analysis
Strain XGS-01 T was rod-shaped, about 0.7-0.8µm wide and 1.6-1.8µm long, and had non-flagella (Figure 2), which was similar to other strains in the genus Thiomicrorhabdus.Cells were Gram stain negative.Strain XGS-01 T could utilize a variety of reduced sulfur compounds as electron donors, including thiosulfate, sulfide, sulfur, and tetrathionate, but not sulfite.The strain XGS-01 T cannot use hydrogen as an electron donor, which differs from that of its closely related species strain MAS2 T .Oxygen is the sole electron acceptor for strain XGS-01 T .In addition, strain XGS-01 T could not utilize all tested organic matters as carbon sources and energy sources.These results indicate that strain XGS-01 T is an obligate chemolithoautotrophic sulfur-oxidizing bacterium (Table 1).For the utilization of nitrogen sources, strain XGS-01 T is able to utilize both inorganic nitrogen sources, such as ammonium, nitrogen and nitrate, and organic nitrogen, such as aminotriacetic acid.Strain XGS-01 T could not use nitrite as the nitrogen source.The result is significantly different from strain MAS2 T , which utilizes ammonium as the nitrogen source.Kanamycin, rifampicin, chloramphenicol, ampicillin, and streptomycin could inhibit the growth of strain XGS-01 T , which was basically consistent with other strain types of this genus.The major fatty acids of strain XGS-01 T are C 18:1 (50.14%),C 16:0 (15.97%), and C 16:1 (6.90%).There are significant differences between strain XGS-01 T and the closely related species, T. hydrogeniphila MAS2 T .The proportion of fatty acid C 16:1 in strain XGS-01 T is 6.90%, whereas it is the main fatty acid in strain MAS2 T , accounting for 44.40%.In addition, some fatty acids detected in strain XGS-01 T were not found in strain MAS2 T .The profiles of fatty acids in strain XGS-01 T were also different from those of strains G1 T and G2 T , such as C 18:0 (Table 2). in strain XGS-01 T were also different from those of strains G1 T and G2 T , such as C18:0 (Table 2).

Genome Features and Central Metabolism among Genus Thiomicrorhabdus
The genome size of strain XGS-01 T was 2.5 Mb, and the G+C content of strain XGS-01 T was 39.1 mol%, which is similar to those of T. hydrogeniphila MAS2 T (39.6 mol%) [8] and T. frisia.JB-A2 T (39.6 mol%) [5] and lower than that of T. chilensis DSM 12352 T (49.9 mol%) [2].The average nucleotide identity (ANI) values between strain XGS-01 T and other species were below the thresholds for species delineation (95-96%) [22], indicating that strain XGS-01 T represents a novel species of the genus Thiomicrorhabdus.None of them have plasmids.
Central metabolic pathways, including sulfur oxidation, nitrogen, and carbon metabolism, were further analyzed among members of genus Thiomicrorhabdus, including the complete genome of strain XGS-01 T obtained in this study, and T. frisia Kp2 T , T. arctica DSM 13458 T , T. chilensis DSM 12352 T , and Thiomicrorhabdus sp.Milos-T2 T , genomes of which are currently available in the NCBI database.The genome of strain XGS-01 T contains a complete set of soxABCDXYZ genes encoding the Sox multienzyme complex, which performs thiosulfate oxidation.The gene cluster is ubiquitously present in all species of the genus Thiomicrorhabdus.This result shows that thiosulfate oxidation performed via a Sox multienzyme complex is highly conserved among Thiomicrorhabdus species.Strain XGS-01 T has sqr and fcc genes for sulfide oxidation, which were also found in other species, except T. arctica SVAL-E T , which lacks the fcc gene (Table 3).There are no complete sulfate-reducing genes and dsr genes in the genome of strain XGS-01 T .All genes encoding the carboxyl oxygenase (Rubisco) and ribulose diphosphate (RuBP), essential for carbon fixation, are present in the genome of strain XGS-01 T .In addition, strain XGS-01 T possesses two carbonic anhydrases, including α-type and γ-type.There are some differences in carbonic anhydrase among these species; for example, T. frisia JB-A2 T , T. arctica DSM 134585 T , and Thiomicrorhabdus sp.Milos-T2 T have α-type, β-type, and γ-type, whereas T. chilensis DSM 12352 T has α-type and γ-type carbonic anhydrase.Strain XGS-01 T has an incomplete nitrogen metabolism pathway.The genome has no genes involved in the denitrification pathway.The genes encoding nitrate reductase and nitrite reductase are absent in the genome of strain XGS-01 T .The incomplete nitrogen metabolism pathway was also found in the other species, except T. frisia JB-A2 T .Elemental sulfur was present in the culture during the mid-exponential growth phase when strain XGS-01 T was incubated with thiosulfate and oxygen.Previous studies showed that the accumulation of elemental sulfur as an intermediate metabolite of thiosulfate oxidation only occurred in microorganisms lacking both SoxCD and dsr operons [23][24][25].In this study, strain XGS-01 T can not only oxidize thiosulfate to produce sulfate through the Sox system; it can also secrete extracellular sulfur during this process despite the presence of SoxCD genes in its genomes.The reason for extracellular sulfur production may be the inhibition of SoxCD.The expression of SoxCD usually depends on environmental pH, and the optimal pH value for the sulfite dehydrogenase (SoxCD) activity is 8.0 [26].During the thiosulfate oxidation process of strain XGS-01 T , the pH of medium will gradually decrease due to the production of metabolites, such as sulfate, and then inhibit the expression of SoxCD.
The extracellular sulfur was further characterized via SEM, XRD, and Raman spectroscopy.Morphological observations under SEM showed sulfur particles in two shapes: rod and globule.The length of rod-shaped sulfur can reach up to 30 µm, and some rodshaped sulfur has short branches.The diameter of sulfur globules ranges from several hundred nanometers to 8 um, mostly 2-3 µm (Figure 3a,b).The XRD patterns of biogenic S 0 matched well with the diffraction patterns of α-S 8 in the database (Figure 3c).In addition, the Raman spectroscopy of S 0 particles also showed the presence of α-S 8 compared with a commercial S 0 standard (Figure 3d).These results suggest that the elemental sulfur produced by strain XGS-01 T are aggregates of nanocrystalline α-S 8 , which is similar to most currently reported extracellular sulfur types, such as Allochromatium vinosum, Chlorobaculum tempidum, and Sulfurimonas hydrogeniphila [27][28][29].The produced a-S 8 has been found in very diverse environments, such as marine sediments, water columns, sulfidic caves, and hydrothermal vents, and the pattern is the most thermodynamically stable form of elemental sulfur at ambient temperature and pressure [23][24][25].The crystalline sulfur could serve as an important intermediate in the biogeochemical sulfur cycle and could be further consumed by a wide diversity of microorganisms, such as sulfur oxidizers, sulfur reducers, or sulfur disproportionators.

Conclusions
A novel Thiomicrorhabuds species was characterized from coastal sediments, whic contributed to our understanding of the ecological role and environmental adaptation o Thiomicrorhabuds bacteria in marine environments.The result of comparisons of the com plete 16S rRNA gene sequence showed that the sequence of strain XGS-01 T was at lea 2.2% different from other Thiomicrorhabdus members described previously.Pairwise AN values between strain XGS-01 T and other species of the genus Thiomicrorhabdus were 70.0 72.0%.According to the generally recognized criteria for delineating bacterial specie strains with a 16S rRNA gene sequence dissimilarity of greater than 2% and ANI value of less than 95-96% are considered to belong to separate species.Thus, strain XGS-01 does not belong to any presently described species.Although strain XGS-01 T shared th highest 16S rRNA gene sequence similarity to T. hydrogeniphila MAS2 T , many of the phy iological characteristics of strain XGS-01 T are different from strain MAS2 T (Table 1).Th growth conditions and maximum growth rate differed between the two strains.The util zation patterns of electron donors were also different in that strain XGS-01 T could not us hydrogen as sole energy source, whereas strain MAS2 T could.In addition, strain XGS-01 is able to utilize diverse inorganic nitrogen and organic nitrogen, whereas strain MAS2 only uses ammonium as a nitrogen source.There are also significant differences betwee strain XGS-01 T and MAS2 T in the composition of fatty acids.These results clearly indicat that strain XGS-01 T can be differentiated from T. hydrogeniphila MAS2 T at the species leve

Conclusions
A novel Thiomicrorhabuds species was characterized from coastal sediments, which contributed to our understanding of the ecological role and environmental adaptation of Thiomicrorhabuds bacteria in marine environments.The result of comparisons of the complete 16S rRNA gene sequence showed that the sequence of strain XGS-01 T was at least 2.2% different from other Thiomicrorhabdus members described previously.Pairwise ANI values between strain XGS-01 T and other species of the genus Thiomicrorhabdus were 70.0-72.0%.According to the generally recognized criteria for delineating bacterial species, strains with a 16S rRNA gene sequence dissimilarity of greater than 2% and ANI values of less than 95-96% are considered to belong to separate species.Thus, strain XGS-01 T does not belong to any presently described species.Although strain XGS-01 T shared the highest 16S rRNA gene sequence similarity to T. hydrogeniphila MAS2 T , many of the physiological characteristics of strain XGS-01 T are different from strain MAS2 T (Table 1).The growth conditions and maximum growth rate differed between the two strains.The utilization patterns of electron donors were also different in that strain XGS-01 T could not use hydrogen as sole energy source, whereas strain MAS2 T could.In addition, strain XGS-01 T is able to utilize diverse inorganic nitrogen and organic nitrogen, whereas strain MAS2 T only uses ammonium as a nitrogen source.There are also significant differences between strain XGS-01 T and MAS2 T in the composition of fatty acids.These results clearly indicate that strain XGS-01 T can be differentiated from T. hydrogeniphila MAS2 T at the species level.Correspondingly, genomic analysis confirmed that strain XGS-01 T performed thiosulfate oxidation through a sox multienzyme complex and harbored fcc and sqr genes to catalyze sulfide oxidation.Comparative genomics further revealed that carbon fixation via the oxidation of reduced sulfur compounds coupled with oxygen reduction is conserved metabolic pathways among members of genus Thiomicrohabdus, indicating they play an important role in the sulfur oxidation process in aerobic marine environments.In addition, our study used strain XGS-01 T as the model with which to first characterize the extracellular sulfur produced by genus Thiomicrorhabdus.The produced a-S 8 , as the most thermodynamically stable form of elemental sulfur, could serve as an important intermediate in the biogeochemical sulfur cycle and could be further consumed by a wide diversity of microorganisms.Our results provide new insight into the potential ecological significance of extracellular S 0 produced by Thiomicrorhabdus in coastal marine ecosystems.
Type strain XGS-01 T (=MCCC 1A18865 T ) was isolated from the coastal sediments of Xiamen, China.The GenBank accession number for the genome sequence of Thiomicrorhabdus lithotrophica XGS-01 T is CP102381, and the 16S rRNA sequence number is OQ978225.

Figure 1 .
Figure 1.Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain XGS-01 T and members of the genus Thiomicrorhabdus.Bootstrap values (>50%) based on 1000 replicates are shown at branch nodes.Bar, 0.01 substitutions per nucleotide position.

Figure 1 .
Figure 1.Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain XGS-01 T and members of the genus Thiomicrorhabdus.Bootstrap values (>50%) based on 1000 replicates are shown at branch nodes.Bar, 0.01 substitutions per nucleotide position.

Figure 2 .
Figure 2. Transmission electron microscopy of a cell of strain XGS-01 T .

Figure 3 .
Figure 3.The morphology and structure of extracellular sulfur.(a,b) Extracellular sulfur morpho ogy under scanning electron microscopy; (c) XRD pattern of extracellular sulfur; (d) Raman spe troscopy of extracellular sulfur.

Figure 3 .
Figure 3.The morphology and structure of extracellular sulfur.(a,b) Extracellular sulfur morphology under scanning electron microscopy; (c) XRD pattern of extracellular sulfur; (d) Raman spectroscopy of extracellular sulfur.
: Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences; Figure S2: Maximum-Parsimony phylogenetic tree based on 16S rRNA gene sequences.Author Contributions: Conceptualization, Y.G., H.Z. and L.J.; Data curation, Y.G. and H.Z.; Methodology, Y.G., H.Z. and J.W.; Resources, Z.S., S.W. and R.C.; Supervision, R.W. and R.C.; Writing-original draft, H.Z.; Writing-review and editing, L.J. and R.W.All authors have read and agreed to the published version of the manuscript.Funding: This work was financially supported by the National Natural Science Foundation of China (No. 42176134), Scientific Research Foundation of Third Institute of Oceanography (No. 2019021), and National Natural Science Foundation of China (No. 42006088).Institutional Review Board Statement: The present study does not include any experimental work with humans or vertebrates.Informed Consent Statement: Not applicable.