Diversity and Potential Metabolic Characteristics of Culturable Copiotrophic Bacteria That Can Grow on Low-Nutrient Medium in Zhenbei Seamount in the South China Sea

Abstract

Oligotrophs are predominant in nutrient-poor environments, but copiotrophic bacteria may tolerate conditions of low energy and can also survive and thrive in these nutrient-limited conditions. In the present study, we isolated 648 strains using a dilution plating method after enrichment for low-nutrient conditions. We collected 150 seawater samples at 21 stations in different parts of the water column at the Zhenbei Seamount in the South China Sea. The 648 isolated copiotrophic strains that could grow on low-nutrient medium were in 21 genera and 42 species. A total of 99.4% (644/648) of the bacteria were in the phylum Pseudomonadota, with 73.3% (472/644) in the class Gammaproteobacteria and 26.7% (172/644) in the class Alphaproteobacteria. Among the 42 representative isolates, Pseudoalteromonas arabiensis, Roseibium aggregatum, and Vibrio neocaledonicus were present in all layers of seawater and at almost all of the stations. Almost half of these species (20/42) contained genes that performed nitrate reduction, with confirmation by nitrate reduction testing. These isolates also contained genes that functioned in sulfur metabolism, including sulfate reduction, thiosulfate oxidation, thiosulfate disproportionation, and dimethylsulfoniopropionate degradation. GH23, CBM50, GT4, GT2, and GT51 were the main carbohydrate-active enzymes (CAZymes), and these five enzymes were present in all or almost all of the isolated strains. The most abundant classes of CAZymes were those associated with the degradation of chitin, starch, and cellulose. Collectively, our study of marine copiotrophic bacteria capable of growing on low-nutrient medium demonstrated the diversity of these species and their potential metabolic characteristics.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00248-024-02475-z.

Introduction

The r-K selection theory provides a traditional framework for classifying microbial life history strategies, which highlights a fundamental trade-off between the rate of growth and reproduction and the efficiency of carbon and nutrient utilization [1]. The r-strategists (copiotrophic species) normally only grow in nutrient-rich environments and exhibit rapid growth when there are high levels of organic matter [2, 3]. It is currently believed that slow-growing oligotrophic bacteria (K-strategists) are the primary organisms in microbial communities found in nutrient-poor environments [3]. However, many copiotrophic organisms have evolved to be highly adapted to nutrient-poor environments, allowing them to thrive in conditions with insufficient nutrient supply, much like oligotrophs [4]. For example, Vibrio spp. are ubiquitous copiotrophs and some of them have been demonstrated to be capable of forming very small cells with obligate oligotrophic characteristics in low-nutrient conditions [5].

The South China Sea (SCS) is one of the largest marginal basins in the Western Pacific and has many large seamounts that rise from the ocean seafloor and are important parts of the oceanic lithosphere. For example, the Zhenbei Seamount is at the westernmost end of the Zhenbei-Huangyan Seamount Chain, in the center of the East Sub-basin of the SCS [6]. The SCS is considered to be oligotrophic, and its ecosystem productivity is primarily limited by the availability of nitrogen [7, 8]. More recent studies of the bacterial community in the SCS used culture-independent methods, such as 454 pyrosequencing of 16S rRNA or partial 16S rRNA gene regions (V1–V3), and fluorescent in situ hybridization (Micro-FISH) [9–11]. Although many marine bacteria have been cultured from marine sediments, sponges, and coral of the SCS [12–18], we still have limited knowledge about copiotrophic marine bacteria that could grow at low nutrient concentrations in the SCS. Cultivation-based studies enable researchers to perform physiological tests and predict potential metabolic characteristics from bacterial genomes.

Here, we sought to isolate copiotrophic bacteria capable of growing on low-nutrient media from the water column of different sites at the Zhenbei Seamount in the SCS by using selective media. We then applied genomic analyses to predict their potential metabolic characteristics, especially regarding their cycling of nitrogen, sulfur, and carbon. These results could provide valuable information for understanding the key metabolic pathways of culturable copiotrophic bacteria capable of growing on low-nutrient media in the oligotrophic ocean.

Materials and Methods

Study Area, Sample Collection, and Isolation and Identification of Bacteria

A total of 150 seawater samples were collected from 21 stations, ranging from the surface to the bottom layer, using Niskin bottles that were attached to a rosette sampling system. These samples were collected during an expedition on the Zhenbei seamount by the Institute of Oceanology of the Chinese Academy of Sciences from July to August 2022 (Figure S1 and Table S1). On board the boat, incubation of samples in the enrichment culture (25 °C for 30 days) was performed in 15-mL glass test tubes that had 7 mL of seawater-based enrichment medium and 3 mL of the seawater sample. This enrichment medium was modified from the medium described by Mu et al. [19] and consisted of the following ingredients (w/v) in autoclaved seawater adjusted to pH 7.0: 0.01% (NH₄)₂SO₄, 0.02% CH₃COONa, 0.002% MgSO₄·7H₂O, 0.01% EDTA-Na₂, and 0.011% sodium pyruvate. In addition, a 10% (w/v) NaHCO₃ solution was subjected to filtration, and a 2% (w/v) KH₂PO₄ solution was autoclaved, and each of these solutions was also incorporated into the autoclaved enrichment medium (1 mL/L). The enrichment cultures (1 mL) were then diluted with 9 mL of a sterile 0.9% sodium chloride solution and spread onto enrichment medium agar plates. After incubation at 25 °C for 7 days, single colonies were selected, purified, and cultured in the enrichment medium. Genomic DNA was extracted using the UltraClean Microbial DNA isolation kit (Mo Bio Laboratories). The 16S rRNA gene was amplified using primers 27F and 1541R, and then sequenced as described previously [20]. An initial examination was used to analyze closely related strains that had valid published names using the EzBioCloud server (www.ezbiocloud.net) [21]. Each strain was identified as a species based on a threshold of 99% similarity in the 16S rRNA gene sequences. Phylogenetic trees of the 16S rRNA gene sequences were reconstructed using a maximum-likelihood method (MEGA X) [22].

Screening of Bacteria Capable of Growth on Low-Nutrient Medium

The isolates representing different species were incubated separately in triplicate on low-nutrient (LN) medium. For the LN medium, glucose (0.0005, 0.0025, 0.015, 0.0375 g/L), sodium acetate (0.0013, 0.0065, 0.0412, 0.1030 g/L), and sodium pyruvate (0.0006, 0.0030, 0.0184, 0.0460 g/L) were separately added to a basal salt medium that provided the sole carbon source, resulting in final concentration of the organic carbon of 0.2, 1, 6, and 15 mg C/L, respectively. The basal salt medium contained 0.1 g (NH₄)₂SO₄ and 0.02 g MgSO₄·7H₂O in 1 L of deionized water. As above, a 10% (w/v) NaHCO₃ solution was subjected to filtration, and a 2% (w/v) KH₂PO₄ solution was autoclaved, and each solution was then incorporated into the autoclaved medium (1 mL/L).

Genome Sequencing, Assembly, and Annotation

Genome sequencing and assembly were performed as described previously [23]. Briefly, for each genome, a paired-end library was constructed with an insert size of 350 bp, and sequencing was conducted on the Illumina NovaSeq 6000 platform. The raw reads were subjected to quality filtering to eliminate low-quality bases and adaptors using Trimmomatic version 0.36 [24], and were then assembled using SPAdes version 3.15.2. Quality was evaluated using BUSCO version 5.0.0 [25]. Coding sequence (CDS) prediction was performed using the Prokka version 1.14.6 [26], while functional annotation of the genes was carried out using the application of EggNog-mapper(v2) [27]. The genes related to nitrogen and sulfur were searched for over a broader range using the NcycDB [28] and ScycDB [29] databases. All annotated proteins were aligned against the databases using the blastp, with the parameter threshold set to e-value < 10⁻⁵. The gene IDs corresponding to the alignment results were manually incorporated into the alignment information. Furthermore, a tailored script was deployed to identify and extract genes related to nitrogen metabolism and sulfur compound degradation processes. The KEGG pathways analysis was performed, and the resulting protein sequences in FASTA format were aligned using the BlastKOALA online tool with reference to the prokaryote database [30]. The annotated protein results in KOALA format were analyzed and visualized for pathway completeness using the Python package KEGG-Decoder [31].

Phenotypic Characteristics

Considering that nitrate is the main bioavailable nitrogen compound utilized by oceanic microorganisms, the ability of the isolates representing different species to reduce nitrate to nitrite was determined as previously described [32]. Briefly, strains were inoculated into nitrate liquid medium (0.1% KNO₃ and 1% peptone) and cultured at 25 °C for 7 days. Then, 2 drops of Griess A reagent (sulfanilamide with 5 mol/L acetic acid) and Griess B reagent (N-[1-naphthyl] ethylenediamine with 5 mol/L acetic acid) were added. If the color changed to red, pink, or orange, this indicated the presence of nitrite (positive for nitrate reductase). If the color did not change (nitrite-negative), zinc dust was then added to reduce the nitrate to nitrite. If the color did not change after adding zinc dust, this indicated no residual nitrate (positive for nitrate reductase); if the color did change after adding zinc dust, this indicated the strain was incapable of nitrate reduction (negative for nitrate reductase).

To assess the ability of different isolates representing different species to oxidize thiosulfate, the different strains were grown on modified DSMZ 142 liquid medium (0.1% [NH₄]₂SO₄, 0.15% MgSO₄·7H₂O, 0.42% CaCl₂^.2H₂O, 0.05% K₂HPO₄, 0.1% Na₂S₂O₃^.5H₂O, vitamins, trace elements, 2% agar, sterile seawater) containing phenol red (0.3 mg/L) at 25 °C. If the liquid changed from red to yellow, this demonstrated a decreased pH and that the strain oxidized thiosulfate [32].

Results and Discussion

Isolation, Identification, and Screening of Bacteria Capable of Growth on Low-Nutrient Medium

To screen for bacteria capable of growth on low-nutrient medium, we initially enriched the culture medium of seawater samples by adding sodium pyruvate and sodium acetate as carbon sources, and allowed growth for 30 days. This method allowed us to isolate 954 strains using dilution plating. After removing duplicate strains from the same sample (based on colony morphology) and using a 100% identity cutoff in 16S rRNA gene sequences, we obtained 648 strains (Table S2). The 648 isolated strains were in 21 genera and 42 species based on 16S rRNA gene sequencing. A total of 99.4% (644/648) of the bacteria we identified were in the phylum Pseudomonadota, with 73.3% (472/644) in the class Gammaproteobacteria and 26.7% (172/644) in the class Alphaproteobacteria. Maximum-likelihood tree of the 42 representative isolates, based on 16S rRNA gene sequence data, is shown in Figure S2.

We observed that all 42 representative isolates except for Bacterioplanoides pacificum ZBN-115–2 and Idiomarina loihiensis ZBN-478 could grow with glucose, sodium pyruvate, and sodium acetate as the sole carbon sources at final organic carbon concentrations of 0.2 and 1 mg C/L, respectively. All 42 representative isolates could grow using glucose, sodium pyruvate, and sodium acetate as the sole carbon sources at final organic carbon concentrations of 6 and 15 mg C/L, respectively. Interestingly, we found that these strains grew faster in medium with sodium pyruvate as the sole carbon source, rather than medium with glucose and sodium acetate as the carbon sources. Notably, the definitions of oligotrophy have been a matter of debate for many decades [3, 33]. Oligotrophic bacteria, such as SAR11 and Prochlorococcus, grow much more slowly compared to copiotrophic bacteria [34]. It is widely acknowledged that one common trait of oligotrophic bacteria is their ability to grow in low-nutrient medium (0.2 to 16.8 mg/L or 0.5 to 15 mg/L) [4, 35]. Although 42 representative isolates can grow at low organic carbon concentrations as mentioned above, all of them could grow faster and better on the nutrient-rich medium (marine 2216E agar, MA) than on LN medium. Therefore, we classified 42 representative isolates as copiotrophic bacteria capable of growing on low-nutrient medium.

Diversity of Copiotrophic Bacteria Capable of Growing on Low-Nutrient Medium

We analyzed the distribution and abundance of these 42 species according to seawater depth and sampling site (Figure S3). We isolated nine strains from six or seven of all seven seawater depths: Roseibium aggregatum (7/7), Idiomarina aquatica (7/7), Paracoccus homiensis (6/7), Pseudoalteromonas shioyasakiensis (7/7), Pseudoalteromonas arabiensis (7/7), Pseudoalteromonas gelatinilytica (6/7), Tritonibacter mobilis (6/7), Vibrio alginolyticus (7/7), and Vibrio neocaledonicus (7/7). Pseudoalteromonas arabiensis (20/22 stations), Roseibium aggregatum (21/22 stations), and Vibrio neocaledonicus (21/22 stations) were found at almost all stations. Our recent work showed that Roseibium aggregatum, which can denitrify and oxidize sulfur, was present in all sampled seawater layers of the photic zone of the Western North Pacific Ocean [32]. Our present results also identified Roseibium aggregatum in all seawater layers and in almost all stations of the Zhenbei Seamount in the South China Sea.

It remains uncertain whether a clear demarcation line between oligotrophic and copiotrophic bacteria exists [33]. Based on previous studies, many genera among the 42 representative copiotrophic bacteria that we identified can survive in oligotrophic culture media or environments. The facultatively oligotrophic bacterium Aeromonas sp. No. 6 was isolated using F5 agar medium (polypeptone 1 g, yeast extract 0.1 g, agar 15 g in 1000 mL of distilled water) from an oligotrophic area of Northern Lake Biwa in the center of Japan [36]. Ivanova et al. [37] proposed that two strains isolated from seawater samples from the north-western Pacific Ocean at a depth of 4000 to 5000 m were novel species-Idiomarina abyssalis and Idiomarina zobellii, and demonstrated that they could grow on oligotrophic medium and on Trypticase Soy Agar (TSA; Difco). Paracoccus sp. strain KS1 is able to grow in oligotrophic minimal media with olivine as the sole Fe³⁺ source [38]. Species in the genus Pseudoalteromonas are common in oligotrophic ecosystems (low absolute concentrations of nutrients), such as the deep sea and polar areas [39–41]. Under conditions of nutrient deprivation, many Gram-negative bacteria, including Pseudomonas and Vibrio spp., can enter the VBNC state [42] and survive for a long time in oligotrophic environments. The members of the Vibrio, Rhodobacter, and Flavobacterium lineages were identified as culturable oligotrophic bacteria within naturally occurring bacterioplankton communities of the Ligurian Sea by using 16S rRNA sequencing and probing [43].

Potential Metabolic Characteristics of Copiotrophic Bacteria Capable of Growing on Low-Nutrient Medium

Based on the KEGG pathway comparison analysis, the identified pathways can be roughly categorized into modules (Figure S4, Table S3). Regarding energy metabolism pathways, all 42 representative isolates possessed enzymes for a complete TCA cycle. Interestingly, we detected Paracoccus homiensis ZBS-68, Salipiger bermudensis ZBS-10, and Salipiger thiooxidans ZBS-95 contained enzymes for a complete Calvin-Benson-Bassham (CBB) cycle, indicating that these stains may utilize CBB cycle to transform CO₂ into the organic matter [44]. All bacteria had enzymes involved in the 3-hydroxypropionate (3-HP) pathway; however, none of these bacteria had been found to contain a complete set of metabolic proteins for this pathway [45]. In addition, none of these bacteria had the enzymes associated with photosynthesis pathways. In transporter pathways, the bacteria studied here exhibited a rich diversity of transporters, and most strains contained necessary transport proteins required for the uptake and utilization of specific metabolites, such as phosphate, phosphonate, urea, thiamin, and vitamin B12.

1. Nitrogen metabolism.

Among the 42 representative isolates we identified, 20 had the ability to reduce nitrate to nitrite based on the nitrate reduction test, consistent with their possession of genes responsible for dissimilatory nitrate reduction (narGHI/napAB) (Fig. 1, Figure S5, Table S4). Among these 20 isolates, all but 4 (Pseudoalteromonas shioyasakiensis ZBS-96, Pseudoalteromonas arabiensis ZBS-357, Pseudoalteromonas lipolytica ZBS-149, and Idiomarina aquatica ZBS-64–1) also contained genes for dissimilatory nitrate reduction to ammonium (DNRA), which code for proteins that reduce nitrate to ammonium (napAB/narGHI, nirBD, nrfA). Complete denitrification is catalyzed by four nitrogen reductases that function in sequence: nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase [46]. Denitrification is widely acknowledged as a key component of the global nitrogen cycle, driven by a wide array of microorganisms. We found that Bacterioplanoides pacificum ZBN-115–2, Marinobacter nauticus ZBS-94–1, Marinobacter shengliensis ZBS-703, Pseudomonas zhaodongensis ZBN-608, Roseibium aggregatum ZBS-8, and Halomonas meridiana ZBS-587 could convert nitrate to nitrogen gas based on the nitrate reduction test (addition of Zn dust) (Figure S5). This observation is in accordance with our identification of specific genes (napAB/narGHI, nirS/nirK, norBC, nosZ) in these 6 isolates. Denitrification removes nitrogen from seawater, thereby constraining biological growth. Denitrification and DNRA are linked to the levels of bioavailable nitrogen in the ocean, ultimately influencing marine productivity [47].

• (2)Sulfur metabolism.

Fig. 1.

Genes that function in nitrogen metabolism among the 42 representative isolates. Colored circle: present, empty circle: absent. DNRA: dissimilatory nitrate reduction to ammonium

Microbial cycling of inorganic sulfur compounds is an ecologically important biogeochemical process [48]. Thiosulfate is a key intermediate in marine environments, because it facilitates the integration of reductive, oxidative, and disproportionation processes within the sulfur cycle [49]. All of our isolates except Planococcus citreus ZBN-491 grew on the modified DSMZ 142 agar plates.

Assimilatory sulfate reduction (ASR) is a common pathway for sulfate reduction in various environments, but sulfate is first converted to sulfide in steps catalyzed by various enzymes (Sat, CysND, CysC, CycH, CysJI, etc.) [50]. All of our isolates except Brachybacterium paraconglomeratum ZBN-604, Idiomarina loihiensis ZBN-478, and Planococcus citreus ZBN-491 contained genes in the ASR pathway (Fig. 2, Table S5).

Fig. 2.

Genes that function in sulfur metabolism among the 42 representative isolates. Colored circle: present, empty circle: absent. ASR, assimilatory sulfate reduction; Sox, sulfur oxidation; S₄I, tetrathionate intermediate thiosulfate oxidation; TD, thiosulfate disproportionation; DMSP, dimethylsulfoniopropionate

There are two common thiosulfate oxidation pathways: the sulfur oxidization (Sox) system and the tetrathionate intermediate thiosulfate oxidation (S₄I) pathway [51]. The Sox system consists of typical periplasmic multi-enzymes encoded by soxYZ, soxXA, soxB, and soxCD, and this system can convert thiosulfate, sulfide, sulfite, and elemental sulfur into sulfate as the ultimate end product [52]. Only 6 of our isolates (Paracoccus homiensis ZBS-68, Roseibium aggregatum ZBS-8, Roseovarius atlanticus ZBS-192, Roseovarius nubinhibens ZBS-326–2, Salipiger bermudensis ZBS-10, and Salipiger thiooxidans ZBS-95) encoded Sox genes (soxXA, soxB, soxC, soxYZ). Thus, growth of these strains in the modified DSMZ 142 liquid medium changed the color from red to yellow (Figure S6), indicating oxidation of thiosulfate, consistent with the genomic analysis. Under neutral or slightly acidic growth conditions, thiosulfate dehydrogenase (TsdA) facilitates the conversion of a portion of the thiosulfate into tetrathionate [53]. Many prokaryotes can oxidize thiosulfate to tetrathionate is, including many obligately chemo-lithoautotrophic sulfur-oxidizing bacteria and chemo-organoheterotrophic bacteria [54]. Among our isolates, we speculate that Pseudoalteromonas tetraodonis ZBN-167, Vibrio azureus ZBN-312, Alcanivorax xenomutans ZBS-261, Halomonas hydrothermalis ZBS-735–2, Marinobacter nauticus ZBS-94–1, and Vibrio alginolyticus ZBS-471 may have a functional S₄I pathway because they all had the TsdA gene.

Thiosulfate disproportionation plays a crucial role in sulfur transformation in aquatic systems, and many microorganisms have this function [55]. The genes for 3-mercaptopyruvate sulfurtransferase (sseA) and thiosulfate sulfurtransferase (glpE), which encode proteins for thiosulfate disproportionation to tetrathionate and sulfite, were present in 10 of our isolates (Pseudoalteromonas lipolytica ZBS-149, Tritonibacter mobilis ZBS-29, Pseudoalteromonas spongiae ZBN-172, Pseudoalteromonas tetraodonis ZBN-167, Pseudoalteromonas ruthenica ZBN-534, Vibrio azureus ZBN-312, Brachybacterium paraconglomeratum ZBN-604, Roseibium aggregatum ZBS-8, Vibrio neocaledonicus ZBS-97, and Vibrio alginolyticus ZBS-471), suggesting they function in thiosulfate disproportionation.

Many diverse bacteria utilize dimethylsulfoniopropionate (DMSP) as a source of sulfur and carbon [56]. There is evidence that a significant portion of DMSP is metabolized via a demethylation pathway, emphasizing the significance of this pathway in marine ecosystems [57, 58]. Three of our isolates (Idiomarina loihiensis ZBN-478, Pseudomonas zhaodongensis ZBN-608, and Nitratireductor kimnyeongensis ZBN-30) contained the dmdB/dmdC genes. Tritonibacter mobilis ZBS-29, Roseovarius atlanticus ZBS-192, and Roseovarius nubinhibens ZBS-326–2 contained the dmdA gene based on KEGG pathway analysis. These genes encode the key enzymes for catabolization of 3-methylmercaptopropionate (MMPA) to methylthioacrylyl-CoA (MTA-CoA) as part of the biochemical pathways for DMSP degradation [58].

• (3)Organic carbon metabolism.

In carbohydrate metabolism pathways, the glycolysis pathway was present in 42 representative isolates with complete sets of metabolic enzymes (Figure S4). More than half of the isolates (24/42) possessed a complete gluconeogenic metabolic pathway. We investigated the presence of carbohydrate-active enzymes (CAZymes) in the 42 representative isolates to examine their potential for degradation of complex carbohydrates. This analysis led to the identification of 3836 genes that matched with CAZyme database, with genes in all six of the CAZyme classes (Figure S7, Table S6). The distributions of these CAZyme classes were relatively homogeneous among the 42 isolates. GHs (glycoside hydrolases) were in 40.6% of isolates, followed by GTs (glycosyl transferases, 36.6%), CBMs (carbohydrate-binding modules, 12.9%), CEs (carbohydrate esterases, 5.1%), AAs (auxiliary activities, 4.7%), and PLs (polysaccharide lyases, 4.7%). GH23 (chitinase), CBM50, GT4, GT2 (chitin transglycosylase), and GT51 were the main CAZymes and were present in all or almost all of the 42 strains.

This analysis also predicted the presence of various classes of enzymes that degrade complex polysaccharides, and the most abundant classes were those associated with the degradation of chitin, starch, and cellulose (Fig. 3). The greatest number of genes was in the class of chitin degradation, and the family GT2 was most abundant, followed by GH23 and CBM5 (chitin-binding). Chitin is among the most abundant naturally occurring organic substances, and previous research estimated that more than 10¹¹ tons of chitin are generated annually in marine environments [59]. The presence of multiple genes for chitin degradation in our isolates suggests they can use chitin as a major source of carbon and nitrogen, thereby facilitating their survival in this oligotrophic ocean environment. Among the 42 representative strains, many bacterial genera (Pseudoalteromonas, Pseudomonas, Alteromonas, Aquimarina, Shewanella, and Vibrio) have been reported as capable of degrading chitin [60–62]. In our study, the highest number of chitinase genes was found in strains belonging to the genera Vibrio and Pseudoalteromonas (Figure S4). Through genomic comparisons, it was found that strains Vibrio alginolyticus ZBS-471 and Vibrio azureus ZBN-312 possessed the most complete chitin metabolic pathways, including the sensor gene ChiS and the two-component regulatory system CdsR and CdsS for chitin catabolism [63, 64] (Figure S8). All strains within the genera Vibrio and Pseudoalteromonas except for Pseudoalteromonas shioyasakiensis ZBS-96 and Vibrio neocaledonicus ZBS-97, contained chitinases, indicating their capacity for chitin metabolism.

Fig. 3.

Number of genes encoding polysaccharide degradation enzymes in the 42 representative isolates

Conclusions

The results of our investigation indicated that culturable copiotrophic bacteria that can grow on low-nutrient medium were widely distributed in the oligotrophic ocean waters of the Zhenbei Seamount, particularly in water from the surface to a depth of 3000 m. The DNRA process, which generates ammonium as the end-product, was the predominant pathway for nitrate reduction. This indicates that these culturable copiotrophic bacteria that could grow on low-nutrient medium may play an important role in the retention of nitrogen in the ocean. These bacteria also have the potential to function in ASR, thiosulfate reduction, thiosulfate oxidation, disproportionation reactions of thiosulfate, and DMSP degradation. Most of the strains we identified also contained genes that functioned in chitin degradation, suggesting that chitin may be an important source of carbon and nitrogen. The metabolic potential for biogeochemical cycling of nitrogen, sulfur, and carbon might provide important advantages for copiotrophic bacteria, capable of growing on low-nutrient medium, which allow them to inhabit oligotrophic sites in the ocean.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ Contributions

ZDC and SZL designed this research, ZZQ collected the seawater samples, isolated the strains and performed 16S rDNA amplification and DNA sequencing. ZZQ, LSZ, and JS performed the genome sequencing, assembly and annotation, and analysis of the genomes and metabolism. ZZQ and LSZ drafted the manuscript. ZDC and SZL supervised the study and contributed to text preparation and revised the manuscript. All authors read and approved the final manuscript.

Funding

This research work was supported by the Science and Technology Innovation Project of Laoshan Laboratory (LSKJ202203102), the NSFC Innovative Group Grant (42221005), the National Natural Science Foundation of China (42376143), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB42000000).

Data Availability

The 16S rRNA gene sequences of the 42 representative isolates have been deposited in GenBank database (accession numbers: PP029086–PP029090, PP029092–PP029098, PP029100–PP029108, PP029110–PP029122, PP029124–PP029129, and PP029132–PP029133). The genome sequences have been deposited in the NCBI Sequence Read Archive (SRA) database with the BioProject number PRJNA1061501.

Declarations

Conflict of Interest

The authors declare no competing interests.