Taxonomic identification and temperature stress tolerance mechanisms of Aequorivita scotiaensis sp. nov.

doi:10.21203/rs.3.rs-3139410/v1

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Taxonomic identification and temperature stress tolerance mechanisms of Aequorivita scotiaensis sp. nov.

https://doi.org/10.21203/rs.3.rs-3139410/v1

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published 21 Nov, 2023

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The deep sea harbours microorganisms with unique life characteristics and life activities due to its special environmental conditions, but the limited sample collection and pure culture techniques available constrain the study of deep-sea microorganisms. In this study, strain Ant34-E75 was isolated from Antarctic deep-sea sediment samples and showed the highest 16S rDNA gene sequence similarity of 97.18%. Polyphasic taxonomy results suggested that strain Ant34-E75 is a new member of the genus Aequorivita. Strain Ant34-E75 belongs to the psychrotrophs and can effectively increase the cold tolerance of Chlamydomonas reininatus. Subsequent transcriptome analysis revealed multiple mechanisms involved in the Ant34-E75 response to temperature stress, and weighted gene co-expression network analysis (WGCNA) showed that the peptidoglycan synthesis pathway was the key component. Overall, this study provides insights into the characteristics of deep-sea microorganisms, elucidates the mechanism of temperature adaptation at the molecular level. Furthermore, it enriches our understanding of the species and genetic diversity of deep-sea microorganisms.

Biological sciences/Microbiology/Bacteria/Marine microbiology

Biological sciences/Microbiology/Bacteria/Bacterial evolution

Biological sciences/Microbiology/Environmental microbiology/Water microbiology

Biological sciences/Microbiology/Bacteria/Bacterial transcription

The ocean covers almost 75% of the Earth's surface and has an average depth of 3800 m¹. The deep sea, accounting for 95% of the total volume of the ocean, is one of the largest and least explored biomes of Earth’s biosphere and is dominated by communities of bacteria, archaea, protists, and unicellular fungi². The extreme conditions in the deep sea, including a lack of light and oxygen, temperatures ranging from 0 to 400℃, and high hydrostatic pressure, make it challenging for life to exist in these habitats³. To adapt to these extreme conditions, organisms in the deep sea have evolved various adaptation and cell signalling mechanisms⁴. In recent decades, the biotechnological potential of cold-adapted microorganisms and their enzymes has been well documented⁵. A wide variety of commercial and industrially important enzymes, including amylases, lipases, and proteases, have been sourced from cold-adapted microorganisms⁵. Cold-resistant bacteria are also utilized in sewage treatment⁶. Marine organisms are more likely to possess novel chemical structures than their terrestrial counterparts⁷. Over the last 50 years, more than 30,000 marine natural products have been discovered, approximately 2% of which have been isolated from deep-sea organisms⁸. These natural products derived from deep-sea microorganisms have the potential to serve as new agents for the development of drugs to treat cancer, infectious diseases, and other human ailments⁸. Despite its vast potential, the deep ocean remains poorly studied due to difficulties in sampling and culturing its microbiota⁷. There are a large number of rare species in the deep-sea environment, where more than half of species are new to science, and more than 95% of the species in some taxa are undescribed⁷. In this study, a new species of the genus Aeqourivita was identified from deep-sea sediment at a depth of 2,560 m in the Scotia Sea.

Cytophaga–Flavobacterium group could be found in all marine and freshwater samples investigated and usually formed the largest bacterial group in marine water, with a median of 18%⁹. The genus Aeqourivita, a member of the Cytophaga–Flavobacterium division, was first described by Bowman & Nichols in 2002 as a novel member of the family Flavobacteriaceae family, within the order Flavobacteriales¹⁰, and it has since been modified by Park¹¹. Additionally, the genus was later reclassified with bacteria in the Vitellibacter genus and moved to the genus Aequorivita in 2016¹². Aequorivita comprises aerobic, Gram-negative, non-spore-forming bacteria, and its major fatty acids are anteiso-C_15:0, iso-C_15:0 and iso-C_17:03-OH, with menaquinone-6 (MK-6) as the major respiratory quinone. The genus currently comprises 16 species with valid published names (https://lpsn.dsmz.de/genus/aequorivita), which have been isolated from terrestrial and marine Antarctic habitats¹⁰, samples of marine algae collected from the South Sea in the Republic of Korea¹¹, estuarine sediments of the Pearl River in China¹³, and the intertidal zone and sediment of the East China Sea^14,15. However, previous studies on Aequorivita have only addressed their taxonomy, fatty acid composition and DNA G + C content. In 2018, the first comprehensive study of the chemistry and biological activity of Aequorivita was conducted. An extract of Aequorivita sp. was found to possess antimicrobial and anthelmintic properties affecting multidrug-resistant bacteria and the nematode Caenorhabditis elegans¹⁶. Through a combination of LC‒MS²-based dereplication and traditional MRSA activity-guided fractionation, seven N-terminal glycine- or serine-bearing iso-fatty acid amides were isolated from Aequorivita sp., three of which were newly discovered¹⁷. To date, there are relatively few studies of this genus, but they have shown that this genus is worthy of study. Therefore, after the identification of the new species, we explored the function of the strain based on experimental evaluations in this study.

Cold-adapted microorganisms can be classified into two categories according to their growth temperature: psychrophiles and psychrotrophs. Psychrophiles can grow and reproduce at temperatures below 0°C, with an optimum growth temperature below 15°C and a maximum growth temperature not exceeding 20°C. On the other hand, psychrotrophs can grow and reproduce at 0 ~ 5°C, with an optimum growth temperature above 15°C and a maximum growth temperature exceeding 20°C^18–20. In this study, the experimental strain of Aequorivita was shown to be capable of growing between 5–37°C, with an optimum growth temperature of 27°C, and was identified as a psychrotroph. Moreover, we discovered that quite a few Aequorivita bacteria were isolated from algae; therefore, the experimental strain exhibited algal-bacterial symbiosis. Chlamydomonas reinhardtii was chosen for culture with this strain, since this species is often used as a model organism in various studies due to its short growth cycle, moderate cell size and clear genetic background^21–23. The experimental results showed that the strain not only tolerated low temperatures but also increased the freezing tolerance of Chlamydomonas reinhardtii. Consequently, the mechanisms of tolerance to temperature stress were investigated in this train by transcriptome analysis, and taxonomic identification was performed.

In summary, we completed the taxonomic identification of a new strain, Ant34-E75, obtained from Antarctic deep-sea sediment, and proposed that it belongs to the genus Aequorivita, with the name Aequorivita scotiaensis Ant34-E75. This work increases the understanding of deep-sea microorganisms through a systematic description of their characteristics. Transcriptomic analysis revealed the mechanisms of the temperature stress response in the strain, such as peptidoglycan synthesis, the ABC transport system, two-component system, amino acid composition adjustment in proteins, nitrogen metabolism, oxidative phosphorylation, and nicotinate and nicotinamide metabolism. Weighted gene co-expression network analysis (WGCNA) identified the peptidoglycan synthesis pathway as a key module related to temperature stress. Thus, we provide insights into the mechanisms of microorganismal adaptation to deep-sea environments.

Polyphasic taxonomy of the strain

According to polyphasic taxonomy, the properties of strain Ant34-E75 were generally consistent with the description of Aequorivita, including the morphological characteristics, respiratory quinones, major fatty acids, and G + C content of the whole genome of the strain¹⁰. Furthermore, the results showed that Ant34-E75 clustered under Aequorivita in the phylogenetic tree (Fig. 1a). However, compared with other strains of the same genus, there were many differences in enzymatic properties and other physical and chemical properties (Tables 1 and 2). The 16S rDNA sequencing results showed that strain Ant34-E75 presented a high similarity of 97.18% to the reference strain Aequorivita viscosa 8-1b^T and showed less than 97.0% similarity with other species of the genus Aequorivita. Additionally, the maximum similarity (Supplementary Table 1) of average nucleotide identity was 78.06%, which was far below the level indicative of relatedness at the species level (95%)²⁴. Based on phenotypic and genotypic characterization, strain Ant34-E75 represents a novel species of the genus Aequorivita, for which the name Aequorivita scotiaensis sp. nov. is proposed. The type strain is strain Ant34-E75.

Description of Aequorivita scotiaensis sp. nov.

The strain is Gram-negative, oxidase-negative, catalase-positive, rod-shaped, and 0.2–0.4 x 0.8–1.6 µm in size (Fig. 1b). It grows optimally in the presence of 2–4% NaCl, at 27°C, in a pH range of 6–8. It hydrolyses gelatine, but not sodium alginate, casein, starch, Tween 80, urea or xylan. It is capable of hydrolysing arginine, urea, esculin, gelatine and p-nitro-β-D-methylgalactose (API 20NE). Positivity for lipoesterase C8, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase and β-fucosidase are present (API ZYM), pH 6, 1% NaCl, 4% NaCl, 1% sodium lactate, D-serine, rifamycin SV, gelatine, Glycyl-L-proline, L-aspartic acid, L-glutamic acid, lithium chloride, potassium tellurite, Tween 40, acetoacetic acid, acetic acid, aztreonam and sodium butyrate is observed in Biolog GENIII. The main respiratory quinone is menaquinone-6, and the major fatty acids are iso-C_15:1G, iso-C_15:0 and iso-C_17:03-OH (Table 2). The polar lipid profile contains phosphatidylethanolamine, three unidentified aminolipids and one unidentified lipid. The genome size is 3,451,112 bp, with a G + C content of 38.41mol%, and its complete genome sequence has been deposited in the NCBI database under accession number CP122379.

Algal-bacterial symbiosis

Based on an algal-bacterial symbiosis experiment, strain Ant34-E75 could effectively increase the freezing tolerance of Chlamydomonas reinhardtii. The results are shown in Fig. 1c. Chlamydomonas reinhardtii could grow for 10 min under cold treatment but could not grow for 20 min when the strain was inoculated at 0% or 1%. The strain allowed Chlamydomonas reinhardtii to continue growing even after 20 minutes of cold treatment when it was inoculated at 10% or 20% and increased the cold tolerance time of the algae by 10 minutes after 10% or 20% inoculation. However, the algae did not grow when the cold treatment time was extended to 30 min (time points of 40 min and 50 min were tested). These results suggest that the newly isolated strain Ant34-E75 has the ability to increase the cold tolerance of Chlamydomonas reinhardtii and enhance the growth of the algae under cold stress.

Transcriptome sequencing and differentially expressed genes (DEGs) analysis

To explore the molecular mechanism of the Ant34-E75 response to temperature stress, five groups of bioreactors were used for culture. The strain was cultured to the logarithmic growth phase at 27°C and then cultured at different temperatures (5°C for 2 hours and 6 hours and at 37°C for 2 hours and 6 hours) (Fig. 1d). Transcriptome sequencing resulted in clean data with high quality (a minimum Q20 score of 98.82% and Q30 score of 95.31%) (Supplementary Table 2). The proportion of alignment for clean data mapped to the reference genome was 98.89 to 99.36% (Supplementary Table 3). The results of the differential gene expression analysis, which was performed using the parameters of a |log₂FoldChange|>1 and p value < 0.05, showed that there were more DEGs in the 6-hour groups (538, 536) than in the 2-hour groups (379, 272) (Fig. 2). The DEGs were annotated and classified into various categories, such as glycan biosynthesis and metabolism, lipid metabolism, environmental information processing, genetic information processing, cellular processes, amino acid metabolism and other metabolism, for further discussion (Supplementary Table 4). The gene expression levels in these categories showed obvious differences under different temperature stresses, indicating the possible mechanisms of the strain Ant34-E75 response to different temperature stresses. Among the potential mechanisms, peptidoglycan biosynthesis, ABC transporters, lysine biosynthesis, phenylalanine metabolism, and nicotinamide and nicotinamide metabolism showed a significant increase at low temperature and a significant decrease at high temperature, while two-component systems, arginine and proline metabolism, cysteine and methionine metabolism and oxidative phosphorylation showed the opposite pattern. In addition, nitrogen metabolism increased significantly under both low- and high-temperature conditions. The results of the transcriptome sequencing and differential gene expression analyses provide valuable insights into the molecular mechanism of the Ant34-E75 response to temperature stress.

Weighted gene co-expression network

WGCNA was used to construct a gene co-expression network to identify the key molecular mechanism and identify novel functional genes involved in the response to temperature stress. The results indicated a scale-free topological fit index of 0.8, and a soft threshold of 18 was selected, resulting in an average connectivity of 53 (Fig. 3a). The analysis revealed positive correlations among the OD₆₀₀, OD₄₉₀ and temperature (Fig. 3b). In addition, among the seven modules, the blue module was significantly negatively correlated, while the brown module was significantly positively correlated (Fig. 3c). To further analyse these findings, the top 20 DEGs with the highest weights in the blue and brown modules were selected and are listed in Supplementary Table 5. Most of the DEGs in the blue module were found to show decreased expression at low temperature and increased or unchanged expression at high temperature. In contrast, the brown module was characterized by DEGs that presented increased expression at low temperature and decreased unchanged expression at high temperature. It could be concluded that more genes showed differential expression at low temperature than at high temperature. We noted that some novel hypothetical proteins were highly weighted in the brown module, such as E75_GM000872, E75_GM002528, E75_GM002833, E75_GM003171, and E75_GM000308. Other than these hypothetical proteins, murC, which is an important gene in peptidoglycan biosynthesis, presented the highest weight.

Pathway prediction and qRT-PCR

Further analysis of the DEGs related to peptidoglycan biosynthesis in the strain was conducted based on the annotation results and WGCNA. The results showed that the expression of DEGs involved in peptidoglycan synthesis increased at low temperature and decreased at high temperature (Supplementary Table 4). One of the genes with a high weight in the brown module, murC, showed a significant positive correlation according to WGCNA (Supplementary Table 5). A peptidoglycan synthesis pathway was constructed based on these findings (Fig. 4).

To validate the results obtained from transcriptome sequencing, all the DEGs of the peptidoglycan synthesis pathway were subjected to qRT‒PCR tests, which showed consistent results with the transcriptome data (Fig. 5). Although the transcriptome sequencing data indicated that the expression of DEGs for peptidoglycan synthesis increased at low temperature, it should be noted that transcriptome sequencing was performed at only 2 hours and 6 hours. The results showed that most changes occurred at 6 hours rather than 2 hours. To further determine the role of peptidoglycan synthesis in the strain, qRT‒PCR was performed after varying the treatment time. The results showed that the expression of those genes peaked at 8 hours and gradually resumed at 10 hours, whether at high temperature or low temperature (Fig. 5).

In the culture experiment, the viscosity of the experimental bacterial strain was higher than that of other strains. This inference was made because the other strains were completely precipitated when centrifuged at 12,000 rpm for 10 min, while the experimental strain could not be completely precipitated, and orange liquid appeared between the bacteria and the water phase. We speculated that this might have been due to the production of exopolysaccharides by the strain. Exopolysaccharides have been shown to present various properties, such as antioxidant activity²⁵, immune modulation, antitumour activity²⁶, and cold resistance²⁷. The experimental strain could still grow at 5°C, which might indicate cold resistance, probably due to the production of extracellular polysaccharides. Our algal-bacterial symbiosis experiment also supported this hypothesis, as it showed an increased cold tolerance time at normal temperatures. Temperature is a very important environmental factor for all organisms. To adapt to temperature, cold-tolerant bacteria have developed special cell structures and enzyme metabolism mechanisms during their long-term biological evolution. It is well documented that the cold tolerance mechanisms of strains involve a complex regulatory process, which can be explained based on several aspects, such as membrane structure, cold-active enzymes, and protein components. However, there are still many microorganisms that have not been discovered in the deep-sea environment, and their temperature adaptation mechanisms are unknown and deserve further exploration.

Transcriptome sequencing revealed that the expression of differentially expressed genes involved in glycan biosynthesis and metabolism increased under low-temperature conditions but decreased under high-temperature conditions, with notable variation in the expression of peptidoglycan biosynthesis genes. The murC gene of the peptidoglycan synthesis pathway showed high weight values in the brown module of WGCNA. Peptidoglycan is a basic component of all bacteria and plays a role in maintaining bacterial morphology and adjusting osmotic pressure²⁸. Suyal observed increased expression of peptidoglycan synthesis proteins in Rhodococcus qingshengii S10107 under low-temperature nitrogen deficiency²⁹. As early as 1972, temperature-sensitive Escherichia coli strains were constructed by knocking out or replacing genes such as murE³⁰, murA and murB³¹. A plasmid carrying the murB gene can restore the temperature-sensitive growth of six Staphylococcus aureus mutants at a restrictive temperature³². Moreover, studies have shown that temperature sensitivity can be weakened by restoring murA and/or murB in a temperature-sensitive mutant of Corynebacterium glutamicum³³. These findings suggest that our experimental strain has adapted to temperature stress by increasing its peptidoglycan content under cold stress and decreasing it under heat stress, similar to how humans dress to survive in cold weather.

The differential gene expression of the ABC transport system, an important component responsible for the export of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria³⁴, was found to be upregulated under low-temperature conditions but decreased under high-temperature conditions. In contrast, the expression of two-component system genes decreased at low temperature and increased at high temperature. Two-component systems play a role as thermosensors and regulate gene expression in microorganisms³⁵. The phoR and phoP genes, which are involved in the response to phosphate limitation, were primarily upregulated in Ant34-E75 under these conditions³⁶, and they also presented a strong relationship with autophosphorylation at a conserved histidine residue³⁵. Although PhoPR is essential for the normal growth and metabolism of a broad range of species, the regulatory functions and mechanisms of PhoP can vary in different bacteria³⁶. Differential gene expression was also found to be clustered in a two-component system under different pH conditions in Pseudoalteromonas agarivorans³⁷. Both the ABC transport system and the two-component system play important roles in the response to environmental changes, including temperature stress.

The ability of microorganisms to cope with temperature stress is largely dependent on their ability to maintain the activity of proteins required for growth. Studies have shown that the amino acid composition of thermophilic proteins exhibits certain general features, with many of the amino acids showing a significant temperature-dependent difference in frequency^38,39. Our study showed a decrease in differential gene expression related to arginine and proline metabolism at low temperature and an increase at high temperature (Supplementary Table 4). Similarly, studies on other microorganisms, such as Bacillus cereus⁴⁰, Shewanella gelidimarina⁴¹ and Halomonas sp.⁴², have shown decreased arginine and proline contents at low temperature, which was also observed in the insect Bactrocera dorsalis⁴³. This reduction in arginine and proline content is thought to enhance protein conformational flexibility and improve enzyme activity at low temperatures by reducing hydrogen bonds and salt bridges. In contrast, the contents of lysine and glutamine increase under such conditions⁴⁴, as observed in our experiment. The results also showed a consistent trend of increased expression of serine and threonine in addition to the downregulation of arginine and proline⁴¹. At high temperature, the biosynthesis of tyrosine and tryptophan was significantly upregulated, while arginine biosynthesis was downregulated, which may be related to the adjustment of G + C contents, with a higher G + C content being beneficial for the maintenance of protein structure.

Temperature stress generally leads to limited microbial growth and inhibited metabolism. However, our results showed that the nitrogen metabolism of Ant34-E75 was significantly up-regulated at both low temperatures and high temperatures, which could be due to utilization of additional nitrogen sources. Besides, oxidative phosphorylation was significantly up-regulated at high temperature, while nicotinate and nicotinamide metabolism were significantly up-regulated at low temperature, which may play a role in the adaptation of microorganisms to temperature. And last but not least, the WGCNA screened several genes with unknown functions, newly perspective genes, which are most likely related to the temperature adaptation of warrant further investigation.

Strain source and culture medium

The strain investigated in this study, designated Ant34-E75, was isolated from deep-sea sediment collected during the 34th Chinese National Antarctica Expedition. The samples were collected from waters of the Antarctic and the southern end of the Atlantic Ocean in the Scotia Sea at 37°32.471' W, 60°23.008' S and a water depth of 2560 m. Aequorivita viscosa 8-1b^T (= CGMCC 1.11023), which was obtained from the China General Microbiological Culture Collection Center (CGMCC), was employed as a reference strain in this study. The cultivable strain was obtained through a combination of gradient dilution coating and streaking inoculation techniques on marine ZoBell 2216E medium (peptone 5 g; yeast extract 1 g; filtered seawater: ultra-pure water (v/v) = 2: 1).

Strain identification

The identification of strain Ant34-E75 was conducted by evaluating its growth at different temperatures (5°C, 15°C, 27°C, and 37°C), pH levels (pH 2.0–12.0 with intervals of 2 units), and NaCl concentrations (0–10% in 2% increments, w/v). The degradation of gelatine, casein, sodium alginate, urea, starch, Tween 80 and xylan was tested on ZoBell 2216E plates supplemented with appropriate substrates as described previously⁴⁵. Catalase activity was determined by bubble production in 3% (v/v) H₂O₂, and oxidase activity was determined using 1% (w/v) N’N’N’N’-tetramethyl-p-phenylenediamine. Other biochemical tests and enzyme assays were performed using the API 20NE and API ZYM kits (bioMérieux) and GN2 MicroPlates (Biolog) according to the manufacturers’ instructions. Respiratory quinone was measured by using HPLC⁴⁶. Fatty acids were measured by GC/MS⁴⁷. Polar lipids were analysed by two-dimensional TLC⁴⁸.

The 16S rDNA sequence of strain Ant34-E75 was aligned via a BLAST search in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were reconstructed using the neighbour-joining and maximum-likelihood algorithms based on the 16S rDNA sequences using Escherichia coli as an outgroup genus with MEGA X, and the best DNA model was selected by MODELS in MEGA X. The Kimura 2-parameter model was used to produce the maximum likelihood tree of 16S rDNA, with a bootstrap value of 1000 via model K2 + G + I.

Whole-genome sequencing and assembly

The genome of the strain was sequenced using third-generation sequencing at Beijing Novogene Bioinformatics Technology Co., Ltd. (P.R. China). After qualified testing, the DNA samples were randomly interrupted with a Covaris ultrasonicator, and library construction was completed. Then, raw reads were filtered to obtain clean reads, which were assembled using SMRT Link v5.0.1 software. The preliminary assembly results were further evaluated using average nucleotide identity (ANI) analysis via JSpeciesWS (http://jspecies.ribohost.com/jspeciesws/#analyse).

Algae-bacteria symbiosis

The bacterial solution (1 mL) was centrifuged, and the supernatant was discarded. The bacteria were then resuspended in 1 mL of TAP liquid medium. Chlamydomonas reinhardtii (2 mL) grown to the logarithmic phase was centrifuged, and the algae liquid was resuspended in 1 mL of TAP liquid medium. The algae and bacteria were mixed at proportions of 1%, 10% and 20% (ratio by volume) and then cultured in a light incubator at 26℃ (optimum growth temperature of Chlamydomonas reinhardtii) for 1.5 hours. The coculture mixture was treated at -20℃ for different times (10 min, 20 min, 30 min, 40 min, and 50 min). Finally, 6 µL of the treated mixture was inoculated into TAP solid medium and incubated at 26°C for 5 days at a light intensity of 50 ± 10 µEm^− 2s^− 1.

Sequencing for transcriptomic analysis

The strain was grown to the logarithmic growth phase at 27℃ and then treated at 5℃ or 37℃ for 2 hours and 6 hours. To obtain the DEGs of the strain under different temperature conditions, we sent the samples to Sangon Biotech (Shanghai) Co., Ltd. for transcriptome sequencing using the Illumina HiSeq. The raw data were evaluated for quality using FastQC and cut by Trimmomatic. To validate the accuracy of the transcriptome data, the processed data were compared to the reference genome by Bowtie2. Then, the processed data were assembled according to the reference genome using Rockhopper. Finally, differential gene expression analysis was performed using DESeq2.

Weighted gene co-expression network construction

WGCNA analysis was used to construct a coexpression network for the filtered genes⁴⁹. The gene expression matrices of all the DEGs and sample traits, including OD₆₀₀ (bacterial concentration), OD₄₉₀ (polysaccharide content) and temperature, were used for WGCNA. The polysaccharide content was determined by the acetone sulfuric acid method⁵⁰. After sample clustering, scale independence and mean connectivity analysis of modules with different power values was performed to determine the soft threshold for module analysis. The power value was set from 1 to 30, and the soft threshold was determined when the scale independence value reached 0.8⁵¹. The relationship between module and trait was calculated, and genes in significant modules were exported for further analysis.

Quantitative real-time RT‒PCR analysis

The transcriptome data were validated using real-time quantitative PCR. The treatment conditions were the same as those for transcriptome sequencing: the strain was grown to the logarithmic growth phase at 27°C and then treated at 5°C or 37°C. However, a greater number of treatment times at different temperatures were examined, including 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours. The experiment was performed in triplicate. The 16S RNA gene was used as the reference gene for normalization⁵². The primers used (Supplementary Table 6) in the experiments were designed according to the functional gene sequence using Primer6.0. Total RNA was isolated using the TransZol Up Plus RNA Kit (ER501-01-V2) and transcribed to cDNA using a One-Step gDNA Removal kit (AT341-02). The 2^−△△Ct procedure was applied to evaluate the qRT‒PCR data.

Data availability

The original contributions presented in the study are publicly available. The whole genome sequence data were available in the National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA954161. The transcriptome sequencing data were also deposited in GSA under the accession number: CRA011001. The strain Ant34-E75 (= CCTCC AB 2023079 = KCTC 92993) was deposited into the general collection of microorganism of the China Center for Type Culture Collection (CCTCC) and the Korean Collection for Type Cultures (KCTC).

Acknowledgements

This work was financially supported by the GEF-FAO Project: Demonstration of Estuarine Biodiversity Conservation, Restoration and Protected Area Networking in China (GCP/CPR/045/GFF) and the Chinese National Natural Science Foundation (Grant No: 41006102).

Author contributions

Authors Wenqi Liu and Bailin Cong conceived and designed the study, contributed equally to this work. Jing Lin helped to measure the biochemical characteristics of the strains. Shenghao Liu helped to performe the transcriptome analysis. Aifang Deng provided materials and opinions for the experiment of algae-bacteria symbiosis. Linlin Zhao coordinated the whole genome sequencing. Wenqi Liu wrote most of the manuscript with major review and editing by Bailin Cong and Jing Lin. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Jin, M., Gai, Y., Guo, X., Hou, Y. & Zeng, R. Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review. Mar Drugs 17, 656(2019).
Sogin, M.L. et al. Microbial diversity in the deep sea and the underexplored "rare biosphere". Proceedings of The National Academy of Sciences of The United States of America 103, 12115-12120 (2006).
Zain ul Arifeen, M., Ma, Y.-N., Xue, Y.-R. & Liu, C.-H. Deep-Sea Fungi Could Be the New Arsenal for Bioactive Molecules. Marine Drugs 18, 9 (2020).
Martínez-Espinosa, R.M. Microorganisms and Their Metabolic Capabilities in the Context of the Biogeochemical Nitrogen Cycle at Extreme Environments. International Journal of Molecular Sciences 21, 4228 (2020).
Furhan, J. Adaptation, production, and biotechnological potential of cold-adapted proteases from psychrophiles and psychrotrophs: recent overview. Journal, Genetic Engineering & Biotechnology 18, 36 (2020).
Xu, Z. et al. Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature. Chemosphere 191, 946-953 (2018).
Saide, A., Lauritano, C. & Ianora, A. A Treasure of Bioactive Compounds from the Deep Sea. Biomedicines 9, 1556 (2021).
Tortorella, E. et al. Antibiotics from Deep-Sea Microorganisms: Current Discoveries and Perspectives. Mar Drugs 16, 355(2018).
Glöckner, F.O., Fuchs, B.M. & Amann, R. Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization. Applied and Environmental Microbiology 65, 3721-3726 (1999).
Bowman, J.P. & Nichols, D.S. Aequorivita gen. nov., a member of the family Flavobacteriaceae isolated from terrestrial and marine Antarctic habitats. International Journal of Systematic And Evolutionary Microbiology 52, 1533-1541 (2002).
Park, S.C. et al. Aequorivita capsosiphonis sp. nov., isolated from the green alga Capsosiphon fulvescens, and emended description of the genus Aequorivita. International Journal of Systematic And Evolutionary Microbiology 59, 724-728 (2009).
Hahnke, R.L. et al. Genome-Based Taxonomic Classification of Bacteroidetes. Frontiers in Microbiology 7, 2003 (2016).
Zhang, S., Zhou, H., Sun, C., Hu, Z. & Wang, H. Aequorivita lutea sp. nov., a novel bacterium isolated from the estuarine sediment of the Pearl River in China, and transfer of Vitellibacter todarodis and Vitellibacter aquimaris to the genus Aequorivita as Aequorivita todarodis comb. nov. and Aequorivita aquimaris comb. nov. Int J Syst Evol Microbiol 70, 3117-3122 (2020).
Wang, Q., Cai, S.D., Liu, J. & Zhang, D.C. Aequorivita sinensis sp. nov., isolated from sediment of the East China Sea, and reclassification of Vitellibacter todarodis as Aequorivita todarodis comb. nov. and Vitellibacter aquimaris as Aequorivita aquimaris comb. nov. International Journal of Systematic And Evolutionary Microbiology 70, 3323-3327 (2020).
Liu, J.J. et al. Aequorivita viscosa sp. nov., isolated from an intertidal zone, and emended descriptions of Aequorivita antarctica and Aequorivita capsosiphonis. International Journal of Systematic And Evolutionary Microbiology 63, 3192-3196 (2013).
Palma Esposito, F. et al. Isolation by Miniaturized Culture Chip of an Antarctic bacterium Aequorivita sp. with antimicrobial and anthelmintic activity. Biotechnology reports (Amsterdam, Netherlands) 20, 281 (2018).
Chianese, G. et al. Linear Aminolipids with Moderate Antimicrobial Activity from the Antarctic Gram-Negative Bacterium Aequorivita sp. Marine Drugs 16, 187 (2018).
Morita, R.Y. Psychrophilic bacteria. Bacteriological Reviews 39, 144-167 (1975).
Gounot, A.M. Psychrophilic and psychrotrophic microorganisms. Experientia 42, 1192-1197 (1986).
Russell, N.J. Cold adaptation of microorganisms. Royal Society. Philosophical Transactions. Biological Sciences 326, 595-611 (1990).
Merchant, S.S. et al. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science 318, 245-250 (2007).
Grossman, A.R. et al. Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation. Photosynth Res 106, 3-17 (2010).
Salomé, P.A. & Merchant, S.S. A Series of Fortunate Events: Introducing Chlamydomonas as a Reference Organism. The Plant Cell 31, 1682-1707 (2019).
Richter, M. & Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proceedings of The National Academy of Sciences of The United States of America 106, 19126-19131 (2009).
Li, W. et al. Production of exopolysaccharides by Lactobacillus helveticus MB2-1 and its functional characteristics in vitro. LWT - Food Science and Technology 59, 732-739 (2014).
Ibrahim, H.A.H., Abou Elhassayeb, H.E. & El-Sayed, W.M.M. Potential functions and applications of diverse microbial exopolysaccharides in marine environments. Journal, Genetic Engineering & Biotechnology 20, 151 (2022).
Nwodo, U.U., Green, E. & Okoh, A.I. Bacterial exopolysaccharides: functionality and prospects. International Journal of Molecular Sciences 13, 14002-14015 (2012).
Pazos, M. & Peters, K. Peptidoglycan. Sub-cellular biochemistry 92, 127-168 (2019).
Suyal, D.C., Joshi, D., Kumar, S., Soni, R. & Goel, R. Differential protein profiling of soil diazotroph Rhodococcus qingshengii S10107 towards low-temperature and nitrogen deficiency. Scientific Reports 9, 20378 (2019).
Lugtenberg, E.J. & v Schijndel-van Dam, A. Temperature-sensitive mutants of Escherichia coli K-12 with low activity of the diaminopimelic acid adding enzyme. Journal of Bacteriology 110, 41-46 (1972).
Miyakawa, T., Matsuzawa, H., Matsuhashi, M. & Sugino, Y. Cell wall peptidoglycan mutants of Escherichia coli K-12: existence of two clusters of genes, mra and mrb, for cell wall peptidoglycan biosynthesis. Journal of Bacteriology 112, 950-958 (1972).
Matsuo, M. et al. Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus. FEMS Microbiology Letters 222, 107-113 (2003).
Shi, T. et al. Double deletion of murA and murB induced temperature sensitivity in Corynebacterium glutamicum. Bioengineered 10, 561-573 (2019).
Cuthbertson, L., Kimber, M.S. & Whitfield, C. Substrate binding by a bacterial ABC transporter involved in polysaccharide export. Proceedings of the National Academy of Sciences 104, 19529-19534 (2007).
Najnin, T. et al. Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii. Scientific Reports 6, 24278 (2016).
Zhang, P. et al. Deletion of the Response Regulator PhoP Accelerates the Formation of Aerial Mycelium and Spores in Actinosynnema pretiosum. Frontiers in Microbiology 13, 845620 (2022).
Ju, Y. et al. Effect of Different Initial Fermentation pH on Exopolysaccharides Produced by Pseudoalteromonas agarivorans Hao 2018 and Identification of Key Genes Involved in Exopolysaccharide Synthesis via Transcriptome Analysis. in Marine Drugs 20, 89 (2022).
Sælensminde, G., Halskau, Ø., Helland, R., Willassen, N.-P. & Jonassen, I. Structure-dependent relationships between growth temperature of prokaryotes and the amino acid frequency in their proteins. Extremophiles 11, 585-596 (2007).
Zhou, X.X., Wang, Y.B., Pan, Y.J. & Li, W.F. Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins. Amino Acids 34, 25-33 (2008).
Park, K.M., Kim, H.J., Kim, M.S. & Koo, M. Morphological Features and Cold-Response Gene Expression in Mesophilic Bacillus cereus Group and Psychrotolerant Bacillus cereus Group under Low Temperature. Microorganisms 9, 1225(2021).
Simpson, P.J.L. & Codd, R. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina. Biochemical and Biophysical Research Communications 414, 783-788 (2011).
Hou, Y. et al. Cold-Adapted Glutathione S-Transferases from Antarctic Psychrophilic Bacterium Halomonas sp. ANT108: Heterologous Expression, Characterization, and Oxidative Resistance. in Marine Drugs 17, 147 (2019).
Raza, M.F. et al. Gut microbiota promotes host resistance to low-temperature stress by stimulating its arginine and proline metabolism pathway in adult Bactrocera dorsalis. Plos Pathogens 16, 1008441 (2020).
Siddiqui, K.S. et al. Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis. Proteins 64, 486-501 (2006).
Margesin, R., Gander, S., Zacke, G., Gounot, A.M. & Schinner, F. Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 7, 451-458 (2003).
Tindall, B.J., Wray, V., Huber, R. & Collins, M.D. A Novel, Fully Saturated Cyclic Menaquinone in the Archaebacterium Pyrobaculum organotrophum. Systematic and Applied Microbiology 14, 218-221 (1991).
Nichols, D.S., Nichols, P.D. & McMeekin, T.A. Polyunsaturated fatty acids in Antarctic bacteria. Antarctic Science 5, 149-160 (1993).
Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry & Physiology 37, 911-917 (1959).
Chen, T., Liu, Y.-X. & Huang, L. ImageGP: An easy-to-use data visualization web server for scientific researchers. iMeta 1, 5 (2022).
Karadeniz, D.G. et al. Microbial exopolysaccharide production of Streptococcus thermophilus and its antiquorum sensing activity. Archives of Microbiology 203, 3331-3339 (2021).
Peng, X., Wu, B., Zhang, S., Li, M. & Jiang, X. Transcriptome Dynamics Underlying Chlamydospore Formation in Trichoderma virens GV29-8. Frontiers in Microbiology 12, 654855(2021).
Yan, J., Yang, Z. & Xie, J. Comparative Transcriptome Analysis of Shewanella putrefaciens WS13 Biofilms Under Cold Stress. Frontiers in Cellular and Infection Microbiology 12, 851521 (2022).
Zhang, H. et al. Aequorivita iocasae sp. nov., a halophilic bacterium isolated from sediment collected at a cold seep field in the South China Sea. International Journal of Systematic and Evolutionary Microbiology 72, 5199 (2022).

Table 1

Comparison of the characteristics of strains belonging to the same genus. Strains: 1, *Aequorivita scotiaensis* Ant34-E75; 2, *Aequorivita viscosa* 8-1b^T; 3, *Aequorivita lutea* q18^T13; 4, *Aequorivita sinensis* S1-10^T14; 5, *Aequorivita iocasae* KX20305^T53.
Characteristic	1	2	3	4	5
NaCl (%, w/v)	0–8, 4–5	0.5-8, 1–3	0–8, 5	0.5-8, 1	1–10, 3–6
pH	5–9, 6	6.0-9.5, 7.0-8.5	5–8, 7	7–10, 9	6–11, 6–8
Temperature (°C)	5–37, 27	4–39, 28–32	10–40, 30	4–42, 30–35	4–41, 25–30
Catalase	+	+	+	+	+
Oxidase	-	-	+	-	+
Gelatine	+	+		+	+
Sodium alginate	-	-		-
Casein	-	-		+	+
Starch	-	-	-	-	-
Tween 80	-	+	-	-	-
Tween 20	-	+			+
Urea	-	+		-	-
Xylan	-	-
Respiratory quinone	MK-6	MK-6	MK-6	MK-6	MK-6
Polar lipids*	PE, L1-3, AL1-3	PE, AL1-3, GL1-4, L1-5	PE, L1-2, GL1-2, PGL	PE, L1-2, AL1-2, GL, PGL1-3	PE, AL1-2, L1-2
G + C (mol%)	38.41	36.6	42.8	34.6	38.71

*PE, phosphatidylethanolamine; L, unidentified polar lipid; AL, aminolipid; GL, glycolipid; PGL, phosphoglycolipid.

Table 2

Comparison of fatty acids of strains belonging to the same genus. The values are percentages of total fatty acids. Strains: 1, *Aequorivita scotiaensis* Ant34-E75; 2, *Aequorivita viscosa* 8-1b^T; 3, *Aequorivita* lutea q18^T13; 4, *Aequorivita sinensis* S1-10^T14; 5, *Aequorivita iocasae* KX20305^T53.
The fatty acids	1	2	3	4	5
iso-C_15 : 1G	22.59	6.4	4.8	6.0	1.4
iso-C_15 : 0	21.66	34.4	40.3	43.2	32.8
iso-C_17 : 0 3-OH	12.05	18.3	14.6		19.0
anteiso-C_15 : 0	7.26	5.0	7.3	21.3	2.4
Summed Feature 3	4.17	3.5	3.2	2.4	9.9
iso-C_16 : 0	4.1		5.6	1.9	3.1
C_17 : 02-OH	3.53			4.5
Summed Feature 9	3.07		8.4		16.8
iso-C_15 : 0 3-OH	2.95	3.5	2.2	2.1	3.5
anteiso-C_17:1w9c	2.53
iso-C_16:03-OH	1.95	1.5	2.8	2.7	2.9
anteiso-C_15:1A	1.81
C_16:00	1.78		2.0		1.7
C_15:0 2-OH	1.53			1.1

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Taxonomic identification and temperature stress tolerance mechanisms of Aequorivita scotiaensis sp. nov.

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Abstract

Figures

Introduction

Results

Polyphasic taxonomy of the strain

Algal-bacterial symbiosis

Transcriptome sequencing and differentially expressed genes (DEGs) analysis

Weighted gene co-expression network

Pathway prediction and qRT-PCR

Discussion

Methods

Strain source and culture medium

Strain identification

Whole-genome sequencing and assembly

Algae-bacteria symbiosis

Sequencing for transcriptomic analysis

Weighted gene co-expression network construction

Quantitative real-time RT‒PCR analysis

Declarations

References

Tables

Additional Declarations

Supplementary Files

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