TMT-Based Quantitative Proteomics Analysis of the Fish-Borne Spoiler <i>Shewanella putrefaciens</i> Subjected to Cold Stress Using LC-MS/MS

Gao, Xin; Li, Peiyun; Mei, Jun; Xie, Jing

doi:https://doi.org/10.1155/2021/8876986

Journal of Chemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2021 | Article ID 8876986 | https://doi.org/10.1155/2021/8876986

TMT-Based Quantitative Proteomics Analysis of the Fish-Borne Spoiler Shewanella putrefaciens Subjected to Cold Stress Using LC-MS/MS

Xin Gao,^1,2,3,4Peiyun Li,^1,2,3,4Jun Mei ,^1,2,3,4and Jing Xie^1,2,3,4,5

Academic Editor: Ajaya Kumar Singh

Received29 Aug 2020

Revised04 Nov 2020

Accepted26 Dec 2020

Published08 Jan 2021

Abstract

Shewanella putrefaciens is a specific spoilage bacterium for fish during cold storage. To better understand the molecular mechanisms of cold stress adaptation of S. putrefaciens, tandem mass tag- (TMT-) based quantitative proteomic analysis was performed to detect the effects of cold stress on protein expression profiles in S. putrefaciens which had been cultivated at 4°C and 30°C, respectively. A total of 266670 peptide spectrum matching numbers were quantified proteins after data analysis. Of the 2292 proteins quantitatively analyzed, a total of 274 were found to be differentially expressed (DE) under cold stress compared with the nonstress control. By integrating the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, 9 common KEGG terms were found notable for the cold-responsive proteins. Generally, the DE proteins involved in carbohydrate, amino acid, and fatty acid biosynthesis and metabolism were significantly upregulated, leading to a specific energy conservation survival mode. The DE proteins related to DNA repair, transcription, and translation were upregulated, implicating change of gene expression and more protein biosynthesis needed in response to cold stress.

1. Introduction

Fresh fish is very perishable due to endogenous enzymes and microbial activities, which can result in large economic losses [1]. Indeed, the intrinsic properties of fresh fish are favourable for growth and enzymatic activity of spoilage bacteria, with the consequent off-flavours, off-odours, discoloration, textural changes, and slime formation [2]. Cold storage is widely used to maintain the quality of fish and prolong the shelf life [3, 4]. Shewanella putrefaciens is considered as the common specific spoilage organisms (SSOs) in fish during cold storage [5, 6]. S. putrefaciens has been reported to be able to use electron acceptors, such as TMAO, instead of oxygen to survive under oxygen or hypoxia conditions. It can produce proteolytic and lipolytic enzymes broken down proteins and produce various flavour defects to lower the fish quality [7]. In addition, S. putrefaciens is a cold-adapted bacterium in refrigerated fish and exhibits many special characteristics and molecular mechanisms that allow them to adapt to the cold stress environment [8, 9].

Some bacteria show a variety of physiological adaptation mechanisms, in order to cope with cold stress, survive, and grow in the cold stress environment. The mechanism is as follows: (i)Increased fluidity of cell membranes(ii)The freezing point of the aqueous phase in the cytoplasm decreased(iii)Macromolecules with enhanced stability(iv)Under the effects of cold shock and cold acclimation, the reaction protein of cells to temperature decreases(v)Peroxidase, catalase, redox, and superoxide dismutase protect reactive oxygen species(vi)Whether the catalytic efficiency is maintained under cold stress and under cold stress [10–14].

Proteomics can provide advanced information on microbial metabolism and mechanisms of adaption to the cold stress environment, and this knowledge could be useful to reveal the cold-adaptation mechanisms in S. putrefaciens. Proteomics techniques have been widely used in microbiology, among which two-dimensional electrophoresis and protein identification are commonly used [15]. Due to its high technical reproducibility, improved proteome coverage, and more confident peptide identification and quantification, proteome analysis based on the tandem mass tag (TMT) is suitable for analyzing the abundance of thousands of proteins in complex biological samples [16, 17]. Some research studies have been performed to determine the proteomics changes of bacteria under cold stress. For example, proteomics methods were used to investigate the quantitative proteomics of Edwardsiella tarda in the midexponential growth phase at the optimal temperature of 37^°C for 24 h and then through the hatch at 4°C for two weeks without vibration. Several key proteins related to DNA synthesis and transcription were significantly upregulated [18]. Similar comprehensive studies for S. putrefaciens have yet to be carried out. To provide insight into potential mechanisms underlying the ability of S. putrefaciens to grow at a temperature of 4°C, we investigated the whole proteome response of S. putrefaciens exposed to cold stress using mass spectrometry.

2. Materials and Methods

2.1. Bacterial Strain and Growth Conditions

Broth cultures of S putrefaciens (ATCC 8071) were prepared as follows: 1 mL aliquots of logarithmic phase grown broth cultures were transferred to 250 mL Erlenmeyer flasks containing 100 mL medium. The flasks were incubated aerobically agitating at 200 rpm, at 30 and 0°C, respectively, until an absorbance (OD600) of 0.4 was attained. Six independent replicates were collected for each sample. The cell pellets prepared by centrifugation of the bacterial culture were resuspended and washed three times with phosphate-buffered saline (PBS).

2.2. Protein Extraction and Quantification

The spoilage cells were resuspended in a 600 L lysis buffer and subjected to high-intensity probe ultrasound in a 200 w ice bath (UP-250S sonicator, Scientz, Ningbo, China). The mixtures were centrifuged at 16000 g at 4°C for 5 min. The supernatant was collected, and the protein concentration was quantified using the bicinchoninic acid method. 10 μg protein samples were added to 5X loading buffer at a rate of 5 : 1 (V/V), and then, the mixture was put in the boiling water for 5 min. The purity of proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on the basis of Hou et al. [19].

2.3. Protein Enzymatic Hydrolysis and Peptide Desalting

300 μg of each sample and 0.1 M DTT were mixed together. The mixture was heated in the boiling water for 5 min and then cooled to room temperature. Then, 200 μL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) was added to the mixture, and the protein was collected by centrifugation with 10 kDa ultrafiltration centrifuge tube at 12,000 × for 15 min. This process was repeated twice. Subsequently, the protein was added with 100 μL IAA (50 mM IAA in UA) and oscillated at 600 rpm for 1 min and centrifuged at 12,000 × for 10 min at room temperature in dark. The protein was collected by centrifugation with 10 kDa ultrafiltration centrifuge tube at 12,000 × for 15 min, and this procedure was repeated twice.

2.4. TMT Labelling

Desalted peptides were reconstituted in 0.1% FA, and the concentration of peptide was determined with the total protein assay kit (BCA method, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Peptides were reconstituted in 50 mM 2-hydroxyethyl (pH 8.5) and TMT zero reagent (Thermo Fisher, Waltham, USA) was added from stocks dissolved in 100% anhydrous ACN. The peptide-TMT mixture was incubated for 1 h at 25°C and 400 rpm, and the labelling reaction was stopped by the addition of either 5% hydroxylamine to a final concentration of 0.4% or 8 μL of 1 M Tris, pH 8.0, and incubation for 15 min at 25°C and 400 rpm. Peptide solutions were acidified with 45% (v/v) of 10% FA in 10% ACN prior to drying or directly frozen at 80°C and dried by vacuum centrifugation. For in-depth proteome analyses, peptides derived from Lys-C/trypsin digests of luminal and basal PDX tumors were processed as described in Fang et al. [4] but following the optimized TMT labelling protocol.

Briefly, 300 μg peptides were dissolved in 60 μL of 50 mM HEPES (pH 8.5), and the labelling reaction was started by the addition of 300 μg TMT reagents (15 μL of 56.7 mM (20 μg/μL) TMT stocks). Samples were incubated for 1 h at 25°C and 1,000 rpm, and the labelling reaction was quenched using 5 μL of 5% hydroxylamine (15 min; 25°C; 1,000 rpm). Peptide solutions were pooled, frozen at 80°C, and dried by vacuum centrifugation. Subsequently, TMT-labelled samples were desalted using tC18, RP solid-phase extraction cartridges (Waters Corp.; wash solvent: 0.1% TFA; elution solvent: 0.1% FA in 50% ACN), frozen at −80°C, and dried by vacuum centrifugation. TMT-labelled peptides were fractionated via a high-pH reversed-phase column (PierceTM High-pH Reversed-Phase Peptide Fractionation Kit, Thermo Fisher). Peptides were pooled into 15 fractions. Enrichment was performed using Ni-nitrilotriacetic acid superflow agarose beads (Qiagen) loaded with iron (III) ions. Subsequently, phosphopeptides were desalted using self-packed StageTips (wash solvent: 0.1% FA; elution solvent: 0.1% FA in 50% ACN), frozen at 80°C, and dried by vacuum centrifugation.

2.5. LC-MS/MS Measurements

Tryptic peptides for one-shot analyses were analyzed on an EASY-nLC 1200 (Thermo Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). After reconstitution in 0.1% FA, an amount corresponding to 500 ng peptides was injected. Peptides were separated on an analytical column (EASY column, 75 μm × 45 cm, Thermo Fisher Scientific) applying a flow rate of 300 nL/min and following elution program: 0–2 min: from 5 to 8% solvent B (0.1% formic acid + 98% acetonitrile); 2–42 min: from 8 to 23% solvent B; 42–50 min: from 23 to 40% solvent B; 50–52 min: from 40 to 100% solvent B; and 52–60 min: 100% solvent B, respectively. Mass spectrometers were operated in data-dependent and positive ionization mode. On the Q Exactive Plus, MS1 spectra were recorded at a resolution of 70 k using an automatic gain control (AGC) target value of 1e6 charges and maximum injection time (maxIT) of 50 ms. After peptide fragmentation via higher energy collisional dissociation, MS2 spectra of up to 10 precursors were acquired at 17.5 k resolution using an AGC target value of 1e5 and a maxIT of 50.

2.6. Database Searching

MaxQuant: for peptide and TMT titration experiments, peptide identification and quantification were performed using MaxQuant (version 1.6.0.16) with its built-in search engine, Andromeda (15, 16). Tandem mass spectra were searched against UniProt-S. putrefaciens-3949-20190409. fasta (3949 entries, downloaded on April 9, 2019).

3. Results and Discussion

3.1. Identification of Proteins by Quantitative Proteomics Analysis

The results of spectrometry in the present research included protein identification, peptide identification, protein quantification, and differential protein classification analysis. A total of 266670 peptide spectrum matching (PSM) numbers, 19483 unique peptides, and 2292 quantified proteins were obtained after data analysis.

The intensity histogram for each sample is shown in Figure 1(a). Figure 1(b) was the box plot of normalized density and represented the box plots of log 2 protein intensity average for each sample.

(a)

(b)

3.2. Identification of Proteins and Their Total and Differential Abundances

TMT-based quantitative proteomic analysis was developed to identify, quantify, and statistically verify quantitative differences in protein abundance from S. putrefaciens grown at 4°C versus 30°C. The use of this approach ensured that the quantitative differences recorded were reliable, irrespective of the magnitude of the quantitative differences, and provided a robust means of interpreting the biological relevance of the data. A total of six experiments were analyzed by LC-MS/MS. Of these proteins, 274 were significant DE proteins with abundances that changed >1.5-fold (cultivated at 30°C/cultivated at 4°C) and values of <0.05. A total of 189 proteins were upregulated and 85 proteins were downregulated (red and green background colors, respectively, in Table 1).

3.3. Bioinformatics Analysis of DE Proteins Identified by TMT

The upregulated and downregulated DE proteins were annotated by Gene Ontology (GO) with Fisher’s exact test to better understand the roles that these proteins may play in cold adaptation. The significantly upregulated and downregulated DE proteins were classified into three categories using GO terms: biological process (BP), cell component (CC), and molecular function (MF). The downregulated DE proteins were clustered into 50 BP terms (the most representative term was “organic substance metabolic process”), 20 CC terms (the most representative term was “cell”), and 18 MF terms (the most representative term was “binding”). Each of the first ten terms in BP, CC, and MF determined based on values is listed in Figure 2.

3.4. Categorization of Differentially Downregulated Ribosomal Proteins (RPs)

Ribosomes are thought to act as sensors for the heat and cold shock response networks in bacteria and are involved as signals linking environmental stimulus (temperature) with the increased heat shock gene expression [20]. Numerous studies have shown that RPs have a strong functional role, especially in regulating protein synthesis and maintaining the stability of ribosomal complexes [21]. Among the quantified proteins, 44 RPs were identified including 23 30S RPs and 21 50S RPs. Of the 183 significant DE proteins, 31 RPs were upregulated (14 30S and 17 50S, Figure 3) and 21 RPs were downregulated (6 30S and 15 50S). This result is consistent with the fact that RPs may be important for the correct assembly of rRNA under cold stress. All of these RPs had a significant score based on FC, and the very large number indicates that more RPs were likely downregulated in the cold stress environment.

3.5. Main Energy Source Metabolism Network Analysis

The metabolic network of the main energy sources was established, including the citrate cycle, glycolysis/gluconeogenesis, fatty acid degradation, and main amino acid (valine, leucine, and isoleucine) degradation, to reveal the energy change profiles of S. putrefaciens in cold temperature (Figure 4). Some of these energy change profiles contained several proteins, and the preferred upregulated proteins are shown. Overall, 13 proteins were identified and quantified in the constructed energy metabolism network. In total, 6 proteins (46.15%) showed a downregulated trend (FC <1), including 2 tricarboxylic acid cycle, 3 fatty acid degradation, and 3 main amino acid. Fumarate hydratase class I (EC 4.2.1.2) and isovaleryl-CoA dehydrogenase (EC 1.3.8.4) were slightly upregulated (1.2< FC <1.5, ), and phosphoglycerate kinase (EC 2.7.2.3), enolase (EC 4.2.1.11), malate dehydrogenase (EC 1.1.1.37), aconitate hydratase B (EC 4.2.1.3), and acetyl-CoA acetyltransferase (EC 2.3.1.9) were significantly upregulated (FC ≥1.5, ). In addition, these two enzymes are involved in other metabolic processes, such as fatty acid degradation and degradation of valine, leucine, and isoleucine. In addition, retinal dehydrogenase 1 and 3-ketoacyl coenzyme A thiolase are also involved in the degradation of fatty acids and metabolic processes such as valine, leucine, and isoleucine [22]. In summary, amino acid and fatty acid degradation were decreased while glycolysis/gluconeogenesis was activated in S. putrefaciens under cold stress.

3.6. Interaction Network of Upregulated DE Proteins

Studies have shown that proteins in living cells do not exist as a single entity, but rather as functional associations within the cell [23]. It was of great significance to reveal the qualitative characteristics of proteins through the interaction between the formations of the network [24]. By using Cytoscape software against the S. oneidensis database, we explored protein interaction networks altered in the cold stress of S. putrefaciens by extracting a putative PPI network. According to the filter criteria of score >400, 23 proteins in PPIs showed significantly differential abundance between S. putrefaciens cultivated at 4°C and 30°C (Figure 5). The proteins were mainly associated with the cellular metabolic process, organonitrogen compound biosynthetic process, plasma membrane ATP synthesis coupled proton transport, protein metabolic process, ATP synthesis coupled proton transport, and ATP biosynthetic process. Among these proteins, 30S and 50S RPs were upregulated, including those encoded by rpl and rps. Proteins linked to ATP synthase, such as atpA and atpG, were downregulated significantly. We speculated that these proteins had a pivotal role in the network.

Bacteria may encounter a variety of physiological threats under low temperature stress, such as less and less membrane fluidity, less and less enzyme activity, irregular protein folding, lower and lower ice formation and transcription rate in cells, nutrient transport, translation, cell changes, and even division [25, 26]. To overcome these challenges, bacteria employ several cold-adaptation strategies such as ensuring nutrient uptake, maintaining the integrity of membrane structure, retaining ribosome functionality, and facing inefficient, slowing protein folding, decreasing the ability in DNA replication and transcription and in RNA translation [27, 28]. Moreover, cold stress affects the membrane fluidity, thus leading the cells to counteract by crafting their membrane lipid composition [26]. Furthermore, bacteria under cold stress protect their DNA, and superoxide dismutase (SOD) and catalase (CATALase) are produced in response to oxidative stress in lipids and proteins due to damage at low temperatures [29].

3.7. Energy Metabolism

We detected 6 downregulated proteins and 7 upregulated proteins related to energy metabolism. A significantly higher level of enzymes involved in glycolysis, which may suggest that S. putrefaciens under implicates the production of high energy intermediates, such as pyruvate (Figure 3). Upregulation of the key enzymes phosphoglycerate kinase and enolase showed similar results in glycolysis. Phosphoglycerate kinase is a housekeeping gene referred to carbon metabolism and energy production [26]. There are eight DE proteins participating in phosphoglycerate kinase (PGK, EC 2.7.2.3), glycolysis, and enolase, which were more abundant in S. putrefaciens cultivated at 4°C than that at 30°C (S. putrefaciens cultivated at 4°C/S. putrefaciens cultivated at 30°C >1.5) (Figure 3). Glycolysis was a more active energy-producing pathway in S. putrefaciens cultivated at 4°C under nonfavorable temperature, which was also demonstrated by Sun et al. [30]. PGK is found in every domain and in almost all living organisms [31]. It is coded by pgk and PGK activity relates to the conversion of ADP + 3-phospho-d-glyceryl phosphate to ATP + 3-phospho-d-glycerate. PGK is one of the oldest “housekeeping” enzymes, which is found in the most ubiquitous three-carbon portion of the best studied and probably the most ancient metabolic pathway—glycolysis, the Embden–Meyerhof–Parnas cycle (fermentation). PGK activity is also present in several other biochemical processes (e.g., Calvin–Benson–Bassham CO₂ fixation cycle = “CBB”) and is often characterized as metabolically essential [32]. Enolase is a rich expression of cytoplasmic protein in many organisms. It plays a very important role, especially at the end of the catabolic glycolytic pathway, it is a key glycolytic enzyme, and its main role is to catalyze the dehydration of 2-phosphate-d-glycerol (2-PGA) to produce phosphoenolpyruvate (PEP). In the presence of magnesium ions, it provides energy for organisms, so it is an important energy obtaining way for cells [33]. The upregulation of PGK and enolase was accompanied by the increase of glycolysis rate, which indicates the accumulation of pyruvate.

3.8. RPs

As mentioned above, RPs are one of the most abundant proteins. The effect of cold stress significantly increased their expression (Table 1), indicating their important role in the response to cold stress. Cold stress made the structure of ribosome subunits [34] incomplete, which stalled the translation process and reduced the number of polymers, but this reduction was temporary, accompanied by an increase in the number of a single 70 ribosome and 50 and 30 subunits [15]. Consistent with this, quantities of RPs increased when S. putrefaciens was cultivated at 4°C in the current research. For L. monocytogenes strains, the strongest and most active genomes are associated with ribosomal genes under cold stress [35]. We observed that nearly all of 30S and 50S RPs were upregulated, including those encoded by rpl, rps, and rpm. The RPs themself adapt to the new stress conditions [36]. In vitro experiments showed that the E. coli translation apparatus in the cold environment can preferentially act on mRNAs from cold-induced genes [37]. The drop in temperature reduces cell membrane fluidity at the cellular level, and the active transport and secretion of proteins are also affected. The role of RNA helicase in different desiccants was studied. Helicase helps to unlock the secondary structure of RNA so that efficient transcription and translation processes can be achieved under cold stress [38]. Some studies on cold-loving and mesothermal prokaryotes have shown that external ribonuclease and helicase (dead box protein) have this effect; chaperonin proteins have important roles in RNA degradation, nucleotide excision repair pathways, and cold adaptation [39]. There was evidence that RNA degradation under cold stress helps the cells to adapt its RNA metabolism for subsequent growth under cold stress [40]. With the stability of the secondary structure of DNA and RNA, in the case of reduced transcription and translation efficiency and low protein folding efficiency, RPs need to adapt to cold stress in order to function normally [41]. RPs are responsible for ribosome biogenesis and protein translation and play important roles in controlling cell growth, division, and development [42]. In the current research, 47 PRs were identified as being upregulated. The kinetic properties of RPs are different at 4°C than at 30°C, and a large increase in ribosome proteins may lead to the improvement of translation efficiency. Upregulated RPs under cold stress could enhance the appropriate translation or function of ribosome assembly in response to growth requirements. Under this circumstance, ribosomes that are more active under cold stress might be very important for S. putrefaciens under cold stress.

3.9. Translation

In the current study, several factors are upregulated under low temperature stress, such as proteins involved in translation (such as initiation factors IF-2 and extension factors), chaperones involved in protein folding, and proteins involved in transcription (such as DNA-directed RNA polymerase) found in Streptococcus putrefaciens. This shows that S. putrefaciens entered a stabilized phase, which is distinct from the state upon cold shock [15]. In the case of elongation factors, GreA, EF-P, and EF-Ts were upregulated. All were produced at a high level in S. putrefaciens under cold stress, indicating that protein synthesis was maintained to live under cold stress. The transcript cleavage factor, GreA, interacts with the RNAP secondary channel and stimulates the intrinsic transcript cleavage activity of RNAP for the removal of the aberrant RNA 3′ ends. Therefore, polymerization activity can be restarted from the end of a cleaved RNA allowing transcription to resume [43]. GreA is essential for the survival of bacteria under stress [44] and it can facilitate RecBCD-mediated resection and inhibits RecA, which plays an important role in impeding DNA break repair in E. coli [45], indicating the role of GreA in adapting to the stressful environments. The eukaryotic and archaea extension factor 5A (E/AEF-5A) and its prokaryotic bacterial translation extension factor P (EF-P) would slow the ribosome stagnation. The L-type EF-P is also composed of three bucket domains. Ef-p binds to the polyproline-stalled ribosomes between the peptide-TRNA binding site (P site) and the exiting tRNA (E site) and stimulates the formation of peptide bonds by stabilizing the CCA end of prolyl-TRNA at P site. During translation elongation, the ribosome, with the help of translation elongation factors EF-G, EF-TU, and EF-TS, binds the corresponding amino acid of each codon to the growing polypeptide chain and drives along the coding sequence of the mRNA. Ef-ts are involved in protein synthesis and translation extension, and their expression is increased [46–49].

3.10. Lipid Transport and Metabolism

The effect of temperature on bacterial membrane lipids has been extensively studied [50, 51]. Bacteria adapt their membranes to lower the phase-transition temperature below which their membrane changes from a “fluid” (liquid-crystalline) to a “rigid” phase to maintain sufficient membrane fluidity under cold stress [52, 53]. In the current research, 13 proteins (5 downregulated and 8 upregulated) were associated with lipid transport and metabolism in S. putrefaciens under cold stress. PlsY, lipB, fadR, fadI, and lpxD related to the lipid transport and metabolism were downregulated in S. putrefaciens under cold stress. FadR regulatory protein acts as a regulator controlling bacterial lipid metabolism by inhibiting the fatty acid degradation (fad) system and activating the synthesis of unsaturated fatty acids [54]. In E. coli, FadR is a key gene for the synthesis of unsaturated fatty acids and positively regulates fabA and fabB. However, the FadR in S. putrefaciens under cold stress only regulates fabA fatty acid synthesis gene and the fabB protein did not change significantly, which was similar to the result of Yang et al. [54]. Fatty acid degradation occurs through the well-characterized β-oxidation cycle and produces acetyl-CoA, which is further metabolized for energy and precursors of cellular biosynthesis [55]. Acyl-CoA dehydrogenases could catalyze the initial steps in fatty acid β-oxidation. The long-chain fatty acyl-CoA ligase could activate free fatty acids to acyl-CoA thioesters and DE proteins related to lipid synthesis including ketoacyl-ACP synthase III (FabH), which was upregulated under cold stress, which shows the protein expression in S. putrefaciens involved in fatty acid elongation and plays a critical role in maintaining the fluidity of membrane [56]. In some research studies, the FabH enzyme of L. monocytogenes prefers 2-methylbutyryl-CoA as the precursor of odd-numbered anteiso fatty acids under cold stress and increases the synthesis of anteiso fatty acids [57].

4. Conclusions

The ability to canvass a high proportion of the expressed proteome and define quantitatively large or small changes in protein abundance with strict statistical rigour has provided a strong view of S. putrefaciens under cold stress. The proteomics provided functional evidence supporting the importance of specific functional classes of genes that were identified as genomic markers of cold adaptation (e.g., energy metabolism). The quantitative analyses particularly showed the factor in S. putrefaciens under cold stress and the study identified specific pathways that were linked to the cold stress environment. The study provides a platform for comparative analyses of cold adaptation of other bacteria.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (grant numbers 31972142 and 31571914).

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Copyright © 2021 Xin Gao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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