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

Authors: Xin Gao; Peiyun Li; Jun Mei; Jing Xie
Journal: Journal of Chemistry
 (2021)

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## 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.


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## Body

## 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 inS. 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 ofS 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. 10μg 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

300μg 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 ×g 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 ×g for 10 min at room temperature in dark. The protein was collected by centrifugation with 10 kDa ultrafiltration centrifuge tube at 12,000 ×g 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 8μL 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, 300μg 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, 75μm × 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).


## 2.1. Bacterial Strain and Growth Conditions

Broth cultures ofS 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. 10μg 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

300μg 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 ×g 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 ×g for 10 min at room temperature in dark. The protein was collected by centrifugation with 10 kDa ultrafiltration centrifuge tube at 12,000 ×g 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 8μL 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, 300μg 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, 75μm × 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 Figure1(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.Figure 1
(a) Histogram of log2 protein intensity for each sample. (b) Box plots of log2 protein (or reporter ion) 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 fromS. 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 P 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).Table 1
Detailed information regarding upregulated and downregulated differentially expressed proteins inS. putrefaciens cultivated at 4°C and 30°C.
Accession1Sequence lengthFC2Gene namesLocusProtein namesAmino acid transport and metabolismA4Y8495050.52Sputcn32_2411L-Lysine 6-monooxygenase, EC 1.14.13.59A4Y3157970.61Sputcn32_0617Bifunctional aspartokinase/homoserine dehydrogenase (includes aspartokinase, EC 2.7.2.4; homoserine dehydrogenase, EC 1.1.1.3)A4Y5504560.70Sputcn32_1355Zinc metalloprotease, EC 3.4.24.-A4Y4D84510.72Sputcn32_1093L-Glutamine synthetase, EC 6.3.1.2A4YC195820.80Sputcn32_3795Urocanate reductase, EC 1.3.99.33A4Y7I316140.82Sputcn32_2195Glutamate dehydrogenase, EC 1.4.1.2A4Y7Q79391.20Sputcn32_22692-Oxoglutarate dehydrogenase E1 component, EC 1.2.4.2A4Y4E54251.22Sputcn32_11004-Aminobutyrate aminotransferase, EC 2.6.1.19A4Y6562341.28Sputcn32_17143-Oxoacid CoA-transferase, A subunit, EC 2.8.3.5A4Y1J06791.31Sputcn32_0087Oligopeptidase A, EC 3.4.24.70A4Y3F63581.32alrSputcn32_0759Alanine racemase, EC 5.1.1.1A4Y6613891.35Sputcn32_1719Isovaleryl-CoA dehydrogenase, EC 1.3.8.4A4Y4D32851.39Sputcn32_10883-Mercaptopyruvate sulfurtransferase, EC 2.8.1.2A4Y6573151.42Sputcn32_1715Hydroxymethylglutaryl-CoA lyase, EC 4.1.3.4A4Y3U21891.43Sputcn32_0896Alkyl hydroperoxide reductase C, EC 1.11.1.15A4Y2I95241.44Sputcn32_0440Histidine ammonia-lyase, EC 4.3.1.3A4Y5402741.45dapDSputcn32_13452,3,4,5-Tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase, EC 2.3.1.117A4Y5422781.45mapSputcn32_1347Methionine aminopeptidase, EC 3.4.11.18A4YBI53771.46Sputcn32_3610Alanine-glyoxylate aminotransferase, EC 2.6.1.44A4Y3424051.46argDSputcn32_0645Acetylornithine aminotransferase, EC 2.6.1.11A4YCC53411.47tdhSputcn32_3902L-Threonine 3-dehydrogenase, EC 1.1.1.103A4Y3C63861.54Sputcn32_0729Methionine gamma-lyase, EC 4.4.1.11A4Y6M43441.55astESputcn32_1884Succinylglutamate desuccinylase, EC 3.5.1.96A4YCC43971.56kblSputcn32_39012-Amino-3-ketobutyrate coenzyme A ligase, EC 2.3.1.29A4Y7N94441.61astBSputcn32_2251N-succinylarginine dihydrolase, EC 3.5.3.23A4Y1I25551.82hutUSputcn32_0079Urocanate hydratase, EC 4.2.1.49A4Y1I04081.85hutISputcn32_0077Imidazolonepropionase, EC 3.5.2.7A4Y1I74511.89Sputcn32_0084NADPH-glutathione reductase, EC 1.8.1.7A4Y5963001.89Sputcn32_14023-Hydroxyisobutyrate dehydrogenase, EC 1.1.1.31A4YAE03641.93gcvTSputcn32_3211Aminomethyltransferase, EC 2.1.2.10A4Y6123462.05Sputcn32_16704-Hydroxyphenylpyruvate dioxygenase, EC 1.13.11.27A4Y6592882.09Sputcn32_1717Methylglutaconyl-CoA hydratase, EC 4.2.1.18A4Y8W21972.18Sputcn32_2676Thiol peroxidase, EC 1.11.1.15A4Y9463702.20aguASputcn32_2760Putative agmatine deiminase, EC 3.5.3.12A4Y3Y41692.21luxSSputcn32_0938S-Ribosylhomocysteine lyase, EC 4.4.1.21A4Y7333962.26Sputcn32_2045Aminotransferase, EC 2.6.1.-A4YAD91292.31gcvHSputcn32_3210Glycine cleavage system H proteinCarbohydrate transport and metabolismA4Y4N47070.66Sputcn32_1189Aldehyde ferredoxin oxidoreductase, EC 1.2.7.5A4Y7R11310.67Sputcn32_2273Succinate dehydrogenase subunit C, EC 1.3.5.1A4Y9V15940.80opgDSputcn32_3021Glucans biosynthesis protein DA4Y4935451.32pgiSputcn32_1048Glucose-6-phosphate isomerase, EC 5.3.1.9A4Y6B41641.41Sputcn32_17724-Hydroxy-4-methyl-2-oxoglutarate aldolase, EC 4.1.1.112A4Y9A64041.43deoBSputcn32_2820Phosphopentomutase, EC 5.4.2.7A4Y5663051.49Sputcn32_1371N-Acetylglucosamine kinase, EC 2.7.1.59A4Y6K92271.53Sputcn32_18692-Keto-3-deoxy-phosphogluconate aldolase, EC 4.1.2.14A4Y5793021.60Sputcn32_1385UTP--glucose-1-phosphate uridylyltransferase, EC 2.7.7.9A4Y7695441.76opgGSputcn32_2081Glucans biosynthesis protein GCell cycle control, cell division, chromosome partitioningA4Y3E03700.73zapESputcn32_0743Cell division protein ZapECell wall/membrane/envelope biogenesisA4Y6J01920.59rnfASputcn32_1850Ion-translocating oxidoreductase complex subunit A, EC 7.-.-.-A4YBG84600.59Sputcn32_3593Membrane fusion protein (MFP) family proteinA4Y5V01780.61palSputcn32_1608Peptidoglycan-associated proteinA4Y2N54030.69ftsWSputcn32_0486Probable peptidoglycan glycosyltransferase FtsW, EC 2.4.1.129A4YB042440.70zapDSputcn32_3427Cell division protein ZapDA4Y3783300.71Sputcn32_0681Mg2+ transporter protein, CorA family proteinA4Y3B51860.75lptCSputcn32_0718Lipopolysaccharide export system protein LptCA4Y8T73950.77bamBSputcn32_2649Outer-membrane protein assembly factor BamBA4Y3B61831.72Sputcn32_07193-Deoxy-D-manno-octulosonate 8-phosphate phosphatase KdsC, EC 3.1.3.45A4YBR91971.78gmhASputcn32_3694Phosphoheptose isomerase, EC 5.3.1.28Energy production and conversionA4Y9203200.58thrBSputcn32_2734Homoserine kinase, EC 2.7.1.39A4Y6S93340.62ruvBSputcn32_1939Holliday junction ATP-dependent DNA helicase RuvB, EC 3.6.4.12A4Y3985450.68groL groELSputcn32_070160 kDa chaperoninA4Y7T76370.68htpGSputcn32_2299Chaperone protein HtpGA4YCH71420.68atpCSputcn32_3955ATP synthase epsilon chain (ATP synthase F1 sector epsilon subunit) (F-ATPase epsilon subunit)A4YCH92860.69atpGSputcn32_3957ATP synthase gamma chainA4Y6J26680.69uvrBSputcn32_1852UvrABC system protein B, protein UvrBA4Y3P64100.69nqrBSputcn32_0850Na (+)-translocating NADH-quinone reductase subunit B, Na (+)-NQR subunit B, Na (+)-translocating NQR subunit B, EC 7.2.1.1A4Y2C04700.70ntrCSputcn32_0371DNA-binding transcriptional regulator NtrCA4Y4K25960.70lepASputcn32_1157Elongation factor 4, EF-4, EC 3.6.5.n1A4YBH46700.73repSputcn32_3599ATP-dependent DNA helicase Rep, EC 3.6.4.12A4YAH66040.73dsbDSputcn32_3247Thiol:disulfide interchange protein DsbD, EC 1.8.1.8A4Y3F54680.74Sputcn32_0758Replicative DNA helicase, EC 3.6.4.12A4YCH84630.75atpDSputcn32_3956ATP synthase subunit beta, EC 7.1.2.2A4YAV27220.75Sputcn32_3373DNA helicase, EC 3.6.4.12A4Y1A44610.76dnaASputcn32_0001Chromosomal replication initiator protein DnaAA4Y9348560.76mutSSputcn32_2748DNA mismatch repair protein MutSA4YCI05130.76atpASputcn32_3958ATP synthase subunit alpha, EC 7.1.2.2A4Y4B74290.76Sputcn32_1072NADH dehydrogenase, EC 1.6.99.3A4Y6757200.77ppkSputcn32_1733Polyphosphate kinase, EC 2.7.4.1A4Y9651490.77nrdRSputcn32_2779Transcriptional repressor NrdRA4YBZ93190.77birASputcn32_3775Bifunctional ligase/repressor BirAA4Y4062740.78hmuVSputcn32_0960Hemin import ATP-binding protein HmuV, EC 7.6.2.-A4Y9Z64940.78Sputcn32_3066NAD(P) transhydrogenase subunit beta, EC 7.1.1.1A4Y3M44640.79mplSputcn32_0827UDP-N-acetylmuramate--L-alanyl-gamma-D-glutamyl-meso-2,6-diaminoheptandioate ligase, EC 6.3.2.45A4Y9A04540.79radASputcn32_2814DNA repair protein RadAA4Y2J96960.80recGSputcn32_0450ATP-dependent DNA helicase RecG, EC 3.6.4.12A4Y4708570.80clpBSputcn32_1024Chaperone protein ClpBA4Y8E41920.80tdkSputcn32_2506Thymidine kinase, EC 2.7.1.21A4Y9N92070.81Sputcn32_2957Corrinoid adenosyltransferase, EC 2.5.1.17A4Y5P96020.81msbASputcn32_1557Lipid A export ATP-binding/permease protein MsbA, EC 7.5.2.6A4Y4273880.82obgSputcn32_0981GTPase Obg, EC 3.6.5.-A4Y4E03780.83potASputcn32_1095Spermidine/putrescine import ATP-binding protein PotA, EC 7.6.2.11A4Y8T35251.20guaASputcn32_2645GMP synthase (glutamine-hydrolyzing), EC 6.3.5.2A4YBZ83161.21coaASputcn32_3774Pantothenate kinase, EC 2.7.1.33A4Y8F65711.22proSSputcn32_2518Proline--tRNA ligase, EC 6.1.1.15A4Y9M110741.22carBSputcn32_2939Carbamoyl-phosphate synthase large chain, EC 6.3.5.5A4Y5H45561.24glnSSputcn32_1482Glutamine--tRNA ligase, EC 6.1.1.18A4Y3346401.25deaDSputcn32_0637ATP-dependent RNA helicase DeaD, EC 3.6.4.13A4YBS33321.25trpSSputcn32_3698Tryptophan--tRNA ligase, EC 6.1.1.2A4Y7N17891.25Sputcn32_2243Phosphoenolpyruvate synthase, EC 2.7.9.2A4Y9M23861.29carASputcn32_2940Carbamoyl-phosphate synthase small chain, EC 6.3.5.5A4Y5T83451.32purMSputcn32_1596Phosphoribosylformylglycinamidine cyclo-ligase, EC 6.3.3.1A4Y6M83601.33nadASputcn32_1888Quinolinate synthase A, EC 2.5.1.72A4Y5452451.34pyrHSputcn32_1350Uridylate kinase, EC 2.7.4.22A4Y2N44391.37murDSputcn32_0485UDP-N-acetylmuramoylalanine--D-glutamate ligase, EC 6.3.2.9A4Y4H33971.37tyrSSputcn32_1128Tyrosine--tRNA ligase, EC 6.1.1.1A4YA385501.37rhlESputcn32_3108ATP-dependent RNA helicase RhlE, EC 3.6.4.13A4Y9289581.38valSSputcn32_2742Valine--tRNA ligase, EC 6.1.1.9A4Y9Z35651.40cysISputcn32_3063Sulfite reductase [NADPH] hemoprotein beta-component, EC 1.8.1.2A4Y9593231.41thiLSputcn32_2773Thiamine-monophosphate kinase, EC 2.7.4.16A4Y6G83271.42pheSSputcn32_1828Phenylalanine--tRNA ligase alpha subunit, EC 6.1.1.20A4Y7E32311.43lolDSputcn32_2155Lipoprotein-releasing system ATP-binding protein LolD, EC 7.6.2.-A4YBL92921.44prpBSputcn32_36442-Methylisocitrate lyase, 2-MIC, EC 4.1.3.30A4Y4A49401.46ileSSputcn32_1059Isoleucine--tRNA ligase, EC 6.1.1.5A4Y5I74011.49fabVSputcn32_1495Enoyl-[acyl-carrier-protein] reductase [NADH], EC 1.3.1.9A4YA913171.51gshBSputcn32_3162Glutathione synthetase, EC 6.3.2.3A4Y7C91431.53ndkSputcn32_2141Nucleoside diphosphate kinase, EC 2.7.4.6A4Y6Z54281.55serSSputcn32_2007Serine--tRNA ligase, EC 6.1.1.11A4Y9Q36391.60dnaKSputcn32_2971Chaperone protein DnaKA4Y2H21981.71azoRSputcn32_0423FMN-dependent NADH-azoreductase, EC 1.7.1.17A4YAY04322.11purDSputcn32_3402Phosphoribosylamine--glycine ligase, EC 6.3.4.13A4Y3S03912.25pgkSputcn32_0874Phosphoglycerate kinase, EC 2.7.2.3A4Y397963.88groS groESSputcn32_070010 kDa chaperoninLipid transport and metabolismA4Y4F42030.54plsYSputcn32_1109G3P acyltransferase, GPAT, EC 2.3.1.275A4Y9G12190.70lipBSputcn32_2875Octanoyltransferase, EC 2.3.1.181A4Y6252380.72fadRSputcn32_1683Fatty acid metabolism regulator proteinA4Y8984360.72fadISputcn32_24603-Ketoacyl-CoA thiolase, EC 2.3.1.16A4Y5533410.75lpxDSputcn32_1358UDP-3-O-acylglucosamine N-acyltransferase, EC 2.3.1.-A4Y5S42481.22Sputcn32_15823-Oxoacyl-[acyl-carrier-protein] reductase, EC 1.1.1.100A4Y4D04981.34Sputcn32_1085Aldehyde dehydrogenase (acceptor), EC 1.2.5.2A4Y7Z33541.38fabHSputcn32_23553-Oxoacyl-[acyl-carrier-protein] synthase 3, EC 2.3.1.180A4Y3G12451.49Sputcn32_0764Enoyl-CoA hydratase, EC 4.2.1.17A4Y5S33081.74Sputcn32_1581Malonyl CoA-acyl carrier protein transacylase, EC 2.3.1.39A4Y8834111.83Sputcn32_24453-Oxoacyl-[acyl-carrier-protein] synthase I, EC 2.3.1.41A4Y5X91712.05fabASputcn32_16373-Hydroxydecanoyl-[acyl-carrier-protein] dehydratase, EC 4.2.1.59A4Y5S5772.54acpPSputcn32_1583Acyl carrier protein, ACPNucleotide transport and metabolismA4Y9517350.64Sputcn32_2765(P)ppGpp synthetase I, SpoT/RelA, EC 2.7.6.5A4Y9S53431.32pyrCSputcn32_2994Dihydroorotase, EC 3.5.2.3A4Y5T92081.33uppSputcn32_1597Uracil phosphoribosyltransferase, EC 2.4.2.9A4Y7I23391.37pyrDSputcn32_2194Dihydroorotate dehydrogenase, EC 1.3.5.2A4YAX95431.46purHSputcn32_3401Bifunctional purine biosynthesis protein PurH (includes phosphoribosylaminoimidazolecarboxamide formyltransferase, EC 2.1.2.3 (AICAR transformylase); IMP cyclohydrolase, EC 3.5.4.10 (ATIC))A4Y8X82051.49Sputcn32_2692dITP/XTP pyrophosphatase, EC 3.6.1.66A4Y9A74431.54deoASputcn32_2821Thymidine phosphorylase, EC 2.4.2.4A4Y2U22521.55Sputcn32_0543Uridine phosphorylase, EC 2.4.2.3A4YCD73311.62addSputcn32_3914Adenosine deaminase, EC 3.5.4.4A4Y2C81031.64ppnPSputcn32_0379Pyrimidine/purine nucleoside phosphorylase, EC 2.4.2.2A4Y5R02961.71cddSputcn32_1568Cytidine deaminase, EC 3.5.4.5A4Y7B52451.80kdsBSputcn32_21278-Amino-3,8-dideoxy-manno-octulosonate cytidylyltransferase, EC 2.7.7.90A4Y7K51931.99dcdSputcn32_2217dCTP deaminase, EC 3.5.4.13A4Y7262312.08pyrFSputcn32_2038Orotidine 5′-phosphate decarboxylase, EC 4.1.1.23A4Y4P91452.97Sputcn32_1204Cytosine deaminase, EC 3.5.4.1Replication, recombination, and repairA4Y6I42130.74nthSputcn32_1844Endonuclease III, EC 4.2.99.18A4Y5Y26090.82uvrCSputcn32_1640UvrABC system protein C, protein UvrCA4Y9F13430.82Sputcn32_2865DNA polymerase III, delta subunit, EC 2.7.7.7A4Y6M11851.24seqASputcn32_1881Negative modulator of initiation of replicationA4Y8J72371.24fliASputcn32_2559RNA polymerase sigma factor FliAA4Y729951.43ihfB himDSputcn32_2041Integration host factor subunit beta, IHF-betaA4YBK6921.63rpoZSputcn32_3631DNA-directed RNA polymerase subunit omega, EC 2.7.7.6A4Y9D31581.65greASputcn32_2847Transcription elongation factor GreAA4YB05692.50yacGSputcn32_3428DNA gyrase inhibitor YacGSecondary metabolites biosynthesis, transport, and catabolismA4YAT14930.62ubiDSputcn32_33523-Octaprenyl-4-hydroxybenzoate carboxy-lyase, EC 4.1.1.98A4Y7602220.64Sputcn32_2072HAD-superfamily hydrolase, subfamily IA, variant 1, EC 3.1.3.18A4Y3M61760.69Sputcn32_0829Hypoxanthine phosphoribosyltransferase, EC 2.4.2.8A4Y9304180.71Sputcn32_2744Aspartokinase, EC 2.7.2.4A4YAL82890.73psdSputcn32_3289Phosphatidylserine decarboxylase proenzyme, EC 4.1.1.65 (cleaved into phosphatidylserine decarboxylase alpha chain; phosphatidylserine decarboxylase beta chain)A4Y7Q64000.74Sputcn32_2268Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, EC 2.3.1.61A4Y9A33310.75Sputcn32_2817Phosphoserine phosphatase, EC 3.1.3.3A4Y7C71640.76Sputcn32_2139Acetolactate synthase, small subunit, EC 2.2.1.6A4Y7194370.79Sputcn32_2031Phospholipase D/transphosphatidylase EC 2.7.8.8A4Y9C62770.82Sputcn32_2840Dihydropteroate synthase, EC 2.5.1.15A4Y2M05221.21leuASputcn32_04712-Isopropylmalate synthase, EC 2.3.3.13A4Y9M32701.22dapBSputcn32_29414-Hydroxy-tetrahydrodipicolinate reductase, EC 1.17.1.8A4Y9W81731.27Sputcn32_3038Protoporphyrinogen oxidase, EC 1.3.3.4A4Y2M24741.29leuCSputcn32_04733-Isopropylmalate dehydratase large subunit, EC 4.2.1.33A4Y7E12671.31Sputcn32_2153Inositol-1-monophosphatase, EC 3.1.3.25A4Y4A73181.32ispHSputcn32_10624-Hydroxy-3-methylbut-2-enyl diphosphate reductase, EC 1.17.7.4A4Y4T44401.34Sputcn32_1239Isocitrate lyase, EC 4.1.3.1A4Y3R86641.35Sputcn32_0872Transketolase, EC 2.2.1.1A4YAZ48881.39Sputcn32_3417Pyruvate dehydrogenase E1 component, EC 1.2.4.1A4YBI76191.40ilvDSputcn32_3612Dihydroxy-acid dehydratase, EC 4.2.1.9A4Y2M13641.42leuBSputcn32_04723-Isopropylmalate dehydrogenase, EC 1.1.1.85A4Y4861631.44purESputcn32_1041N5-carboxyaminoimidazole ribonucleotide mutase, N5-CAIR mutase, EC 5.4.99.18A4Y6E85141.48Sputcn32_1807Fumarate hydratase class I, EC 4.2.1.2A4Y4X12931.49Sputcn32_1276Farnesyl-diphosphate synthase, EC 2.5.1.10A4Y5G23941.49Sputcn32_1470Glycerate kinase, EC 2.7.1.31A4Y4243311.51Sputcn32_0978Trans-hexaprenyltranstransferase, EC 2.5.1.30A4Y7N33571.54Sputcn32_2245Phospho-2-dehydro-3-deoxyheptonate aldolase, EC 2.5.1.54A4Y5913961.55Sputcn32_1397Acetyl-CoA acetyltransferase, EC 2.3.1.9A4YAY78651.58Sputcn32_3409Aconitate hydratase B, EC 4.2.1.3A4Y9W42171.60Sputcn32_3034Dihydropteridine reductase, EC 1.5.1.34A4Y6702881.63Sputcn32_1728Acyl-CoA thioesterase II, EC 3.1.2.2A4YAE83111.66mdhSputcn32_3219Malate dehydrogenase, EC 1.1.1.37A4Y4943181.66talSputcn32_1049Transaldolase, EC 2.2.1.2A4YAY43541.68hemESputcn32_3406Uroporphyrinogen decarboxylase, EC 4.1.1.37A4Y5N52031.74ribASputcn32_1543GTP cyclohydrolase-2, EC 3.5.4.25A4Y1D33021.75hemFSputcn32_0030Oxygen-dependent coproporphyrinogen-III oxidase, EC 1.3.3.3A4YBS42261.75gphSputcn32_3699Phosphoglycolate phosphatase, EC 3.1.3.18A4Y4G44301.77hemLSputcn32_1119Glutamate-1-semialdehyde 2,1-aminomutase, EC 5.4.3.8A4Y6442941.96dapASputcn32_17024-Hydroxy-tetrahydrodipicolinate synthase, EC 4.3.3.7A4Y8462782.00trpASputcn32_2408Tryptophan synthase alpha chain, EC 4.2.1.20A4YBG23102.04hemCSputcn32_3587Porphobilinogen deaminase, EC 2.5.1.61A4Y7T52142.14adkSputcn32_2297Adenylate kinase, EC 2.7.4.3A4Y1E35142.14gpmISputcn32_00402,3-Bisphosphoglycerate-independent phosphoglycerate mutase, EC 5.4.2.12A4Y8813382.14asdSputcn32_2443Aspartate-semialdehyde dehydrogenase, EC 1.2.1.11A4Y9L32202.15rpiASputcn32_2931Ribose-5-phosphate isomerase A, EC 5.3.1.6A4Y9664172.21glyASputcn32_2780Serine hydroxymethyltransferase, SHMT, EC 2.1.2.1A4Y3V15972.22Sputcn32_0905Fumarate reductase flavoprotein subunit, EC 1.3.5.4A4Y4522922.27Sputcn32_1006Cysteine synthase, EC 2.5.1.47A4Y9434312.31enoSputcn32_2757Enolase, EC 4.2.1.11A4Y7M63442.41Sputcn32_2238Leucine dehydrogenase, EC 1.4.1.9TranscriptionA4Y9B83180.63truBSputcn32_2832tRNA pseudouridine synthase B, EC 5.4.99.25A4Y9L43960.79rlmISputcn32_2932Ribosomal RNA large subunit methyltransferase I, EC 2.1.1.191A4Y9C14991.27nusASputcn32_2835Transcription termination/antitermination protein NusAA4YB211011.31fisSputcn32_3444DNA-binding protein FisA4Y2K52371.31rphSputcn32_0456Ribonuclease PH, EC 2.7.7.56A4Y9601341.66nusBSputcn32_2774Transcription antitermination protein NusBTranslation, ribosomal structure, and biogenesisA4Y5U62280.67tolQSputcn32_1604Tol-Pal system protein TolQA4Y7L61020.70clpSSputcn32_2228ATP-dependent Clp protease adapter protein ClpSA4YBZ61230.71secESputcn32_3772Protein translocase subunit SecEA4YA836970.73fusASputcn32_3154Elongation factor G, EF-GA4Y1K12810.73rlmJSputcn32_0098Ribosomal RNA large subunit methyltransferase J, EC 2.1.1.266A4YBY22010.73rplDSputcn32_375850S ribosomal protein L4A4Y8362910.75Sputcn32_2398Pseudouridine synthase, EC 5.4.99.-A4YCI21560.76atpFSputcn32_3960ATP synthase subunit bA4YBX72300.78rpsCSputcn32_375330S ribosomal protein S3A4YCI42730.78atpBSputcn32_3962ATP synthase subunit aA4Y8B93150.79secFSputcn32_2481Protein-export membrane protein SecFA4Y8C11100.79yajCSputcn32_2483Sec translocon accessory complex subunit YajCA4Y4K53380.80eraSputcn32_1160GTPase EraA4YBY41031.26rpsJSputcn32_376030S ribosomal protein S10A4Y4S71741.31bamESputcn32_1232Outer-membrane protein assembly factor BamEA4YBV92061.32rpsDSputcn32_373530S ribosomal protein S4A4Y3E72461.33rlmBSputcn32_075023S rRNA (guanosine-2′-O-)-methyltransferase RlmB, EC 2.1.1.185A4YBW11181.41rpsMSputcn32_373730S ribosomal protein S13A4Y3E11421.42rplMSputcn32_074450S ribosomal protein L13A4Y9C21511.42rimPSputcn32_2836Ribosome maturation factor RimPA4YBW41441.44rplOSputcn32_374050S ribosomal protein L15A4Y5442831.45tsfSputcn32_1349Elongation factor Ts, EF-TsA4YBW01301.47rpsKSputcn32_373630S ribosomal protein S11A4Y3E21301.49rpsISputcn32_074530S ribosomal protein S9A4Y7051431.56infCSputcn32_2017Translation initiation factor IF-3A4Y6A5851.57minESputcn32_1763Cell division topological specificity factorA4Y3F31501.61rplISputcn32_075650S ribosomal protein L9A4YBX9921.67rpsSSputcn32_375530S ribosomal protein S19A4YBY81241.71rpsLSputcn32_376430S ribosomal protein S12A4YBV71311.72rplQSputcn32_373350S ribosomal protein L17A4YBW5601.72rpmDSputcn32_374150S ribosomal protein L30A4Y5S0561.75rpmFSputcn32_157850S ribosomal protein L32A4Y9F31091.77rsfSSputcn32_2867Ribosomal silencing factor RsfSA4Y9B7891.84rpsOSputcn32_283130S ribosomal protein S15A4YBY71561.86rpsGSputcn32_376330S ribosomal protein S7A4Y704641.87rpmISputcn32_201650S ribosomal protein L35A4Y7L4721.90infASputcn32_2226Translation initiation factor IF-1A4Y426841.90rpmASputcn32_098050S ribosomal protein L27A4Y4L3831.90rpsPSputcn32_116830S ribosomal protein S16A4YBM9731.92zapBSputcn32_3654Cell division protein ZapBA4YBX61361.93rplPSputcn32_375250S ribosomal protein L16A4Y6L51861.96efpSputcn32_1875Elongation factor P, EF-PA4Y3F2752.05rpsRSputcn32_075530S ribosomal protein S18A4Y4A1882.09rpsTSputcn32_105630S ribosomal protein S20A4Y5V12492.09cpoBSputcn32_1609Cell division coordinator CpoBA4Y6Z82082.14lolASputcn32_2010Outer-membrane lipoprotein carrier proteinA4YB741542.17trmLSputcn32_3498tRNA (cytidine (34)-2′-O)-methyltransferase, EC 2.1.1.207A4YBX81102.21rplVSputcn32_375450S ribosomal protein L22A4YBX21042.22rplXSputcn32_374850S ribosomal protein L24A4Y2V2792.28rpmESputcn32_055350S ribosomal protein L31A4Y5461852.28frrSputcn32_1351Ribosome-recycling factor, RRFA4YBW71162.44rplRSputcn32_374350S ribosomal protein L18A4YBX01012.61rpsNSputcn32_374630S ribosomal protein S14A4Y7031182.62rplTSputcn32_201550S ribosomal protein L20A4Y2L3573.30rpmGSputcn32_046450S ribosomal protein L33A4Y5X8583.61rmfSputcn32_1636Ribosome modulation factor, RMFA4YCM5453.83rpmHSputcn32_400550S ribosomal protein L34A4YBW2374.44rpmJSputcn32_373850S ribosomal protein L36Not assignedA4Y6552181.41Sputcn32_1713Butyryl-CoA:acetate CoA transferase, EC 2.8.3.81UniProt and NCBI database accession numbers. 2Average ratio from three replicates by TMT experiment. A protein species was considered differentially accumulated if it exhibited a fold change >1.5-fold (cultivated at 4°C/cultivated at 30°C) with a P value of <0.05.


### 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 onP values is listed in Figure 2.Figure 2
Gene Ontology (GO) enrichment analysis of differentially expressed proteins (S. putrefaciens cultivated at 4°C and 30°C, respectively. X-axis represented each GO term; Y-axis represented the number of differentially expressed proteins in each category).


### 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.Figure 3
Schematic representation of the differentially expressed proteins involved in the ribosome pathway. The red boxes indicate the unregulated proteins, and the green boxes indicate the downregulated proteins.


### 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 ofS. 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, P<0.05), 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, P<0.05). 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.Figure 4
The metabolism pathway of the main energy sources. Different colors indicate the ranges of the fold change (cultivated at 4°C/cultivated at 30°C) values.


### 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.Figure 5
PPI network: significantly upregulated differentially expressed proteins ofS. putrefaciens cultivated at 4°C and 30°C, respectively. The proteins upregulated in S. putrefaciens are colored in a gradient color from white to red, 1.52≤ FC ≤6.36.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 thatS. 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 (Table1), 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 thatS. 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].


## 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 Figure1(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.Figure 1
(a) Histogram of log2 protein intensity for each sample. (b) Box plots of log2 protein (or reporter ion) 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 fromS. 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 P 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).Table 1
Detailed information regarding upregulated and downregulated differentially expressed proteins inS. putrefaciens cultivated at 4°C and 30°C.
Accession1Sequence lengthFC2Gene namesLocusProtein namesAmino acid transport and metabolismA4Y8495050.52Sputcn32_2411L-Lysine 6-monooxygenase, EC 1.14.13.59A4Y3157970.61Sputcn32_0617Bifunctional aspartokinase/homoserine dehydrogenase (includes aspartokinase, EC 2.7.2.4; homoserine dehydrogenase, EC 1.1.1.3)A4Y5504560.70Sputcn32_1355Zinc metalloprotease, EC 3.4.24.-A4Y4D84510.72Sputcn32_1093L-Glutamine synthetase, EC 6.3.1.2A4YC195820.80Sputcn32_3795Urocanate reductase, EC 1.3.99.33A4Y7I316140.82Sputcn32_2195Glutamate dehydrogenase, EC 1.4.1.2A4Y7Q79391.20Sputcn32_22692-Oxoglutarate dehydrogenase E1 component, EC 1.2.4.2A4Y4E54251.22Sputcn32_11004-Aminobutyrate aminotransferase, EC 2.6.1.19A4Y6562341.28Sputcn32_17143-Oxoacid CoA-transferase, A subunit, EC 2.8.3.5A4Y1J06791.31Sputcn32_0087Oligopeptidase A, EC 3.4.24.70A4Y3F63581.32alrSputcn32_0759Alanine racemase, EC 5.1.1.1A4Y6613891.35Sputcn32_1719Isovaleryl-CoA dehydrogenase, EC 1.3.8.4A4Y4D32851.39Sputcn32_10883-Mercaptopyruvate sulfurtransferase, EC 2.8.1.2A4Y6573151.42Sputcn32_1715Hydroxymethylglutaryl-CoA lyase, EC 4.1.3.4A4Y3U21891.43Sputcn32_0896Alkyl hydroperoxide reductase C, EC 1.11.1.15A4Y2I95241.44Sputcn32_0440Histidine ammonia-lyase, EC 4.3.1.3A4Y5402741.45dapDSputcn32_13452,3,4,5-Tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase, EC 2.3.1.117A4Y5422781.45mapSputcn32_1347Methionine aminopeptidase, EC 3.4.11.18A4YBI53771.46Sputcn32_3610Alanine-glyoxylate aminotransferase, EC 2.6.1.44A4Y3424051.46argDSputcn32_0645Acetylornithine aminotransferase, EC 2.6.1.11A4YCC53411.47tdhSputcn32_3902L-Threonine 3-dehydrogenase, EC 1.1.1.103A4Y3C63861.54Sputcn32_0729Methionine gamma-lyase, EC 4.4.1.11A4Y6M43441.55astESputcn32_1884Succinylglutamate desuccinylase, EC 3.5.1.96A4YCC43971.56kblSputcn32_39012-Amino-3-ketobutyrate coenzyme A ligase, EC 2.3.1.29A4Y7N94441.61astBSputcn32_2251N-succinylarginine dihydrolase, EC 3.5.3.23A4Y1I25551.82hutUSputcn32_0079Urocanate hydratase, EC 4.2.1.49A4Y1I04081.85hutISputcn32_0077Imidazolonepropionase, EC 3.5.2.7A4Y1I74511.89Sputcn32_0084NADPH-glutathione reductase, EC 1.8.1.7A4Y5963001.89Sputcn32_14023-Hydroxyisobutyrate dehydrogenase, EC 1.1.1.31A4YAE03641.93gcvTSputcn32_3211Aminomethyltransferase, EC 2.1.2.10A4Y6123462.05Sputcn32_16704-Hydroxyphenylpyruvate dioxygenase, EC 1.13.11.27A4Y6592882.09Sputcn32_1717Methylglutaconyl-CoA hydratase, EC 4.2.1.18A4Y8W21972.18Sputcn32_2676Thiol peroxidase, EC 1.11.1.15A4Y9463702.20aguASputcn32_2760Putative agmatine deiminase, EC 3.5.3.12A4Y3Y41692.21luxSSputcn32_0938S-Ribosylhomocysteine lyase, EC 4.4.1.21A4Y7333962.26Sputcn32_2045Aminotransferase, EC 2.6.1.-A4YAD91292.31gcvHSputcn32_3210Glycine cleavage system H proteinCarbohydrate transport and metabolismA4Y4N47070.66Sputcn32_1189Aldehyde ferredoxin oxidoreductase, EC 1.2.7.5A4Y7R11310.67Sputcn32_2273Succinate dehydrogenase subunit C, EC 1.3.5.1A4Y9V15940.80opgDSputcn32_3021Glucans biosynthesis protein DA4Y4935451.32pgiSputcn32_1048Glucose-6-phosphate isomerase, EC 5.3.1.9A4Y6B41641.41Sputcn32_17724-Hydroxy-4-methyl-2-oxoglutarate aldolase, EC 4.1.1.112A4Y9A64041.43deoBSputcn32_2820Phosphopentomutase, EC 5.4.2.7A4Y5663051.49Sputcn32_1371N-Acetylglucosamine kinase, EC 2.7.1.59A4Y6K92271.53Sputcn32_18692-Keto-3-deoxy-phosphogluconate aldolase, EC 4.1.2.14A4Y5793021.60Sputcn32_1385UTP--glucose-1-phosphate uridylyltransferase, EC 2.7.7.9A4Y7695441.76opgGSputcn32_2081Glucans biosynthesis protein GCell cycle control, cell division, chromosome partitioningA4Y3E03700.73zapESputcn32_0743Cell division protein ZapECell wall/membrane/envelope biogenesisA4Y6J01920.59rnfASputcn32_1850Ion-translocating oxidoreductase complex subunit A, EC 7.-.-.-A4YBG84600.59Sputcn32_3593Membrane fusion protein (MFP) family proteinA4Y5V01780.61palSputcn32_1608Peptidoglycan-associated proteinA4Y2N54030.69ftsWSputcn32_0486Probable peptidoglycan glycosyltransferase FtsW, EC 2.4.1.129A4YB042440.70zapDSputcn32_3427Cell division protein ZapDA4Y3783300.71Sputcn32_0681Mg2+ transporter protein, CorA family proteinA4Y3B51860.75lptCSputcn32_0718Lipopolysaccharide export system protein LptCA4Y8T73950.77bamBSputcn32_2649Outer-membrane protein assembly factor BamBA4Y3B61831.72Sputcn32_07193-Deoxy-D-manno-octulosonate 8-phosphate phosphatase KdsC, EC 3.1.3.45A4YBR91971.78gmhASputcn32_3694Phosphoheptose isomerase, EC 5.3.1.28Energy production and conversionA4Y9203200.58thrBSputcn32_2734Homoserine kinase, EC 2.7.1.39A4Y6S93340.62ruvBSputcn32_1939Holliday junction ATP-dependent DNA helicase RuvB, EC 3.6.4.12A4Y3985450.68groL groELSputcn32_070160 kDa chaperoninA4Y7T76370.68htpGSputcn32_2299Chaperone protein HtpGA4YCH71420.68atpCSputcn32_3955ATP synthase epsilon chain (ATP synthase F1 sector epsilon subunit) (F-ATPase epsilon subunit)A4YCH92860.69atpGSputcn32_3957ATP synthase gamma chainA4Y6J26680.69uvrBSputcn32_1852UvrABC system protein B, protein UvrBA4Y3P64100.69nqrBSputcn32_0850Na (+)-translocating NADH-quinone reductase subunit B, Na (+)-NQR subunit B, Na (+)-translocating NQR subunit B, EC 7.2.1.1A4Y2C04700.70ntrCSputcn32_0371DNA-binding transcriptional regulator NtrCA4Y4K25960.70lepASputcn32_1157Elongation factor 4, EF-4, EC 3.6.5.n1A4YBH46700.73repSputcn32_3599ATP-dependent DNA helicase Rep, EC 3.6.4.12A4YAH66040.73dsbDSputcn32_3247Thiol:disulfide interchange protein DsbD, EC 1.8.1.8A4Y3F54680.74Sputcn32_0758Replicative DNA helicase, EC 3.6.4.12A4YCH84630.75atpDSputcn32_3956ATP synthase subunit beta, EC 7.1.2.2A4YAV27220.75Sputcn32_3373DNA helicase, EC 3.6.4.12A4Y1A44610.76dnaASputcn32_0001Chromosomal replication initiator protein DnaAA4Y9348560.76mutSSputcn32_2748DNA mismatch repair protein MutSA4YCI05130.76atpASputcn32_3958ATP synthase subunit alpha, EC 7.1.2.2A4Y4B74290.76Sputcn32_1072NADH dehydrogenase, EC 1.6.99.3A4Y6757200.77ppkSputcn32_1733Polyphosphate kinase, EC 2.7.4.1A4Y9651490.77nrdRSputcn32_2779Transcriptional repressor NrdRA4YBZ93190.77birASputcn32_3775Bifunctional ligase/repressor BirAA4Y4062740.78hmuVSputcn32_0960Hemin import ATP-binding protein HmuV, EC 7.6.2.-A4Y9Z64940.78Sputcn32_3066NAD(P) transhydrogenase subunit beta, EC 7.1.1.1A4Y3M44640.79mplSputcn32_0827UDP-N-acetylmuramate--L-alanyl-gamma-D-glutamyl-meso-2,6-diaminoheptandioate ligase, EC 6.3.2.45A4Y9A04540.79radASputcn32_2814DNA repair protein RadAA4Y2J96960.80recGSputcn32_0450ATP-dependent DNA helicase RecG, EC 3.6.4.12A4Y4708570.80clpBSputcn32_1024Chaperone protein ClpBA4Y8E41920.80tdkSputcn32_2506Thymidine kinase, EC 2.7.1.21A4Y9N92070.81Sputcn32_2957Corrinoid adenosyltransferase, EC 2.5.1.17A4Y5P96020.81msbASputcn32_1557Lipid A export ATP-binding/permease protein MsbA, EC 7.5.2.6A4Y4273880.82obgSputcn32_0981GTPase Obg, EC 3.6.5.-A4Y4E03780.83potASputcn32_1095Spermidine/putrescine import ATP-binding protein PotA, EC 7.6.2.11A4Y8T35251.20guaASputcn32_2645GMP synthase (glutamine-hydrolyzing), EC 6.3.5.2A4YBZ83161.21coaASputcn32_3774Pantothenate kinase, EC 2.7.1.33A4Y8F65711.22proSSputcn32_2518Proline--tRNA ligase, EC 6.1.1.15A4Y9M110741.22carBSputcn32_2939Carbamoyl-phosphate synthase large chain, EC 6.3.5.5A4Y5H45561.24glnSSputcn32_1482Glutamine--tRNA ligase, EC 6.1.1.18A4Y3346401.25deaDSputcn32_0637ATP-dependent RNA helicase DeaD, EC 3.6.4.13A4YBS33321.25trpSSputcn32_3698Tryptophan--tRNA ligase, EC 6.1.1.2A4Y7N17891.25Sputcn32_2243Phosphoenolpyruvate synthase, EC 2.7.9.2A4Y9M23861.29carASputcn32_2940Carbamoyl-phosphate synthase small chain, EC 6.3.5.5A4Y5T83451.32purMSputcn32_1596Phosphoribosylformylglycinamidine cyclo-ligase, EC 6.3.3.1A4Y6M83601.33nadASputcn32_1888Quinolinate synthase A, EC 2.5.1.72A4Y5452451.34pyrHSputcn32_1350Uridylate kinase, EC 2.7.4.22A4Y2N44391.37murDSputcn32_0485UDP-N-acetylmuramoylalanine--D-glutamate ligase, EC 6.3.2.9A4Y4H33971.37tyrSSputcn32_1128Tyrosine--tRNA ligase, EC 6.1.1.1A4YA385501.37rhlESputcn32_3108ATP-dependent RNA helicase RhlE, EC 3.6.4.13A4Y9289581.38valSSputcn32_2742Valine--tRNA ligase, EC 6.1.1.9A4Y9Z35651.40cysISputcn32_3063Sulfite reductase [NADPH] hemoprotein beta-component, EC 1.8.1.2A4Y9593231.41thiLSputcn32_2773Thiamine-monophosphate kinase, EC 2.7.4.16A4Y6G83271.42pheSSputcn32_1828Phenylalanine--tRNA ligase alpha subunit, EC 6.1.1.20A4Y7E32311.43lolDSputcn32_2155Lipoprotein-releasing system ATP-binding protein LolD, EC 7.6.2.-A4YBL92921.44prpBSputcn32_36442-Methylisocitrate lyase, 2-MIC, EC 4.1.3.30A4Y4A49401.46ileSSputcn32_1059Isoleucine--tRNA ligase, EC 6.1.1.5A4Y5I74011.49fabVSputcn32_1495Enoyl-[acyl-carrier-protein] reductase [NADH], EC 1.3.1.9A4YA913171.51gshBSputcn32_3162Glutathione synthetase, EC 6.3.2.3A4Y7C91431.53ndkSputcn32_2141Nucleoside diphosphate kinase, EC 2.7.4.6A4Y6Z54281.55serSSputcn32_2007Serine--tRNA ligase, EC 6.1.1.11A4Y9Q36391.60dnaKSputcn32_2971Chaperone protein DnaKA4Y2H21981.71azoRSputcn32_0423FMN-dependent NADH-azoreductase, EC 1.7.1.17A4YAY04322.11purDSputcn32_3402Phosphoribosylamine--glycine ligase, EC 6.3.4.13A4Y3S03912.25pgkSputcn32_0874Phosphoglycerate kinase, EC 2.7.2.3A4Y397963.88groS groESSputcn32_070010 kDa chaperoninLipid transport and metabolismA4Y4F42030.54plsYSputcn32_1109G3P acyltransferase, GPAT, EC 2.3.1.275A4Y9G12190.70lipBSputcn32_2875Octanoyltransferase, EC 2.3.1.181A4Y6252380.72fadRSputcn32_1683Fatty acid metabolism regulator proteinA4Y8984360.72fadISputcn32_24603-Ketoacyl-CoA thiolase, EC 2.3.1.16A4Y5533410.75lpxDSputcn32_1358UDP-3-O-acylglucosamine N-acyltransferase, EC 2.3.1.-A4Y5S42481.22Sputcn32_15823-Oxoacyl-[acyl-carrier-protein] reductase, EC 1.1.1.100A4Y4D04981.34Sputcn32_1085Aldehyde dehydrogenase (acceptor), EC 1.2.5.2A4Y7Z33541.38fabHSputcn32_23553-Oxoacyl-[acyl-carrier-protein] synthase 3, EC 2.3.1.180A4Y3G12451.49Sputcn32_0764Enoyl-CoA hydratase, EC 4.2.1.17A4Y5S33081.74Sputcn32_1581Malonyl CoA-acyl carrier protein transacylase, EC 2.3.1.39A4Y8834111.83Sputcn32_24453-Oxoacyl-[acyl-carrier-protein] synthase I, EC 2.3.1.41A4Y5X91712.05fabASputcn32_16373-Hydroxydecanoyl-[acyl-carrier-protein] dehydratase, EC 4.2.1.59A4Y5S5772.54acpPSputcn32_1583Acyl carrier protein, ACPNucleotide transport and metabolismA4Y9517350.64Sputcn32_2765(P)ppGpp synthetase I, SpoT/RelA, EC 2.7.6.5A4Y9S53431.32pyrCSputcn32_2994Dihydroorotase, EC 3.5.2.3A4Y5T92081.33uppSputcn32_1597Uracil phosphoribosyltransferase, EC 2.4.2.9A4Y7I23391.37pyrDSputcn32_2194Dihydroorotate dehydrogenase, EC 1.3.5.2A4YAX95431.46purHSputcn32_3401Bifunctional purine biosynthesis protein PurH (includes phosphoribosylaminoimidazolecarboxamide formyltransferase, EC 2.1.2.3 (AICAR transformylase); IMP cyclohydrolase, EC 3.5.4.10 (ATIC))A4Y8X82051.49Sputcn32_2692dITP/XTP pyrophosphatase, EC 3.6.1.66A4Y9A74431.54deoASputcn32_2821Thymidine phosphorylase, EC 2.4.2.4A4Y2U22521.55Sputcn32_0543Uridine phosphorylase, EC 2.4.2.3A4YCD73311.62addSputcn32_3914Adenosine deaminase, EC 3.5.4.4A4Y2C81031.64ppnPSputcn32_0379Pyrimidine/purine nucleoside phosphorylase, EC 2.4.2.2A4Y5R02961.71cddSputcn32_1568Cytidine deaminase, EC 3.5.4.5A4Y7B52451.80kdsBSputcn32_21278-Amino-3,8-dideoxy-manno-octulosonate cytidylyltransferase, EC 2.7.7.90A4Y7K51931.99dcdSputcn32_2217dCTP deaminase, EC 3.5.4.13A4Y7262312.08pyrFSputcn32_2038Orotidine 5′-phosphate decarboxylase, EC 4.1.1.23A4Y4P91452.97Sputcn32_1204Cytosine deaminase, EC 3.5.4.1Replication, recombination, and repairA4Y6I42130.74nthSputcn32_1844Endonuclease III, EC 4.2.99.18A4Y5Y26090.82uvrCSputcn32_1640UvrABC system protein C, protein UvrCA4Y9F13430.82Sputcn32_2865DNA polymerase III, delta subunit, EC 2.7.7.7A4Y6M11851.24seqASputcn32_1881Negative modulator of initiation of replicationA4Y8J72371.24fliASputcn32_2559RNA polymerase sigma factor FliAA4Y729951.43ihfB himDSputcn32_2041Integration host factor subunit beta, IHF-betaA4YBK6921.63rpoZSputcn32_3631DNA-directed RNA polymerase subunit omega, EC 2.7.7.6A4Y9D31581.65greASputcn32_2847Transcription elongation factor GreAA4YB05692.50yacGSputcn32_3428DNA gyrase inhibitor YacGSecondary metabolites biosynthesis, transport, and catabolismA4YAT14930.62ubiDSputcn32_33523-Octaprenyl-4-hydroxybenzoate carboxy-lyase, EC 4.1.1.98A4Y7602220.64Sputcn32_2072HAD-superfamily hydrolase, subfamily IA, variant 1, EC 3.1.3.18A4Y3M61760.69Sputcn32_0829Hypoxanthine phosphoribosyltransferase, EC 2.4.2.8A4Y9304180.71Sputcn32_2744Aspartokinase, EC 2.7.2.4A4YAL82890.73psdSputcn32_3289Phosphatidylserine decarboxylase proenzyme, EC 4.1.1.65 (cleaved into phosphatidylserine decarboxylase alpha chain; phosphatidylserine decarboxylase beta chain)A4Y7Q64000.74Sputcn32_2268Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, EC 2.3.1.61A4Y9A33310.75Sputcn32_2817Phosphoserine phosphatase, EC 3.1.3.3A4Y7C71640.76Sputcn32_2139Acetolactate synthase, small subunit, EC 2.2.1.6A4Y7194370.79Sputcn32_2031Phospholipase D/transphosphatidylase EC 2.7.8.8A4Y9C62770.82Sputcn32_2840Dihydropteroate synthase, EC 2.5.1.15A4Y2M05221.21leuASputcn32_04712-Isopropylmalate synthase, EC 2.3.3.13A4Y9M32701.22dapBSputcn32_29414-Hydroxy-tetrahydrodipicolinate reductase, EC 1.17.1.8A4Y9W81731.27Sputcn32_3038Protoporphyrinogen oxidase, EC 1.3.3.4A4Y2M24741.29leuCSputcn32_04733-Isopropylmalate dehydratase large subunit, EC 4.2.1.33A4Y7E12671.31Sputcn32_2153Inositol-1-monophosphatase, EC 3.1.3.25A4Y4A73181.32ispHSputcn32_10624-Hydroxy-3-methylbut-2-enyl diphosphate reductase, EC 1.17.7.4A4Y4T44401.34Sputcn32_1239Isocitrate lyase, EC 4.1.3.1A4Y3R86641.35Sputcn32_0872Transketolase, EC 2.2.1.1A4YAZ48881.39Sputcn32_3417Pyruvate dehydrogenase E1 component, EC 1.2.4.1A4YBI76191.40ilvDSputcn32_3612Dihydroxy-acid dehydratase, EC 4.2.1.9A4Y2M13641.42leuBSputcn32_04723-Isopropylmalate dehydrogenase, EC 1.1.1.85A4Y4861631.44purESputcn32_1041N5-carboxyaminoimidazole ribonucleotide mutase, N5-CAIR mutase, EC 5.4.99.18A4Y6E85141.48Sputcn32_1807Fumarate hydratase class I, EC 4.2.1.2A4Y4X12931.49Sputcn32_1276Farnesyl-diphosphate synthase, EC 2.5.1.10A4Y5G23941.49Sputcn32_1470Glycerate kinase, EC 2.7.1.31A4Y4243311.51Sputcn32_0978Trans-hexaprenyltranstransferase, EC 2.5.1.30A4Y7N33571.54Sputcn32_2245Phospho-2-dehydro-3-deoxyheptonate aldolase, EC 2.5.1.54A4Y5913961.55Sputcn32_1397Acetyl-CoA acetyltransferase, EC 2.3.1.9A4YAY78651.58Sputcn32_3409Aconitate hydratase B, EC 4.2.1.3A4Y9W42171.60Sputcn32_3034Dihydropteridine reductase, EC 1.5.1.34A4Y6702881.63Sputcn32_1728Acyl-CoA thioesterase II, EC 3.1.2.2A4YAE83111.66mdhSputcn32_3219Malate dehydrogenase, EC 1.1.1.37A4Y4943181.66talSputcn32_1049Transaldolase, EC 2.2.1.2A4YAY43541.68hemESputcn32_3406Uroporphyrinogen decarboxylase, EC 4.1.1.37A4Y5N52031.74ribASputcn32_1543GTP cyclohydrolase-2, EC 3.5.4.25A4Y1D33021.75hemFSputcn32_0030Oxygen-dependent coproporphyrinogen-III oxidase, EC 1.3.3.3A4YBS42261.75gphSputcn32_3699Phosphoglycolate phosphatase, EC 3.1.3.18A4Y4G44301.77hemLSputcn32_1119Glutamate-1-semialdehyde 2,1-aminomutase, EC 5.4.3.8A4Y6442941.96dapASputcn32_17024-Hydroxy-tetrahydrodipicolinate synthase, EC 4.3.3.7A4Y8462782.00trpASputcn32_2408Tryptophan synthase alpha chain, EC 4.2.1.20A4YBG23102.04hemCSputcn32_3587Porphobilinogen deaminase, EC 2.5.1.61A4Y7T52142.14adkSputcn32_2297Adenylate kinase, EC 2.7.4.3A4Y1E35142.14gpmISputcn32_00402,3-Bisphosphoglycerate-independent phosphoglycerate mutase, EC 5.4.2.12A4Y8813382.14asdSputcn32_2443Aspartate-semialdehyde dehydrogenase, EC 1.2.1.11A4Y9L32202.15rpiASputcn32_2931Ribose-5-phosphate isomerase A, EC 5.3.1.6A4Y9664172.21glyASputcn32_2780Serine hydroxymethyltransferase, SHMT, EC 2.1.2.1A4Y3V15972.22Sputcn32_0905Fumarate reductase flavoprotein subunit, EC 1.3.5.4A4Y4522922.27Sputcn32_1006Cysteine synthase, EC 2.5.1.47A4Y9434312.31enoSputcn32_2757Enolase, EC 4.2.1.11A4Y7M63442.41Sputcn32_2238Leucine dehydrogenase, EC 1.4.1.9TranscriptionA4Y9B83180.63truBSputcn32_2832tRNA pseudouridine synthase B, EC 5.4.99.25A4Y9L43960.79rlmISputcn32_2932Ribosomal RNA large subunit methyltransferase I, EC 2.1.1.191A4Y9C14991.27nusASputcn32_2835Transcription termination/antitermination protein NusAA4YB211011.31fisSputcn32_3444DNA-binding protein FisA4Y2K52371.31rphSputcn32_0456Ribonuclease PH, EC 2.7.7.56A4Y9601341.66nusBSputcn32_2774Transcription antitermination protein NusBTranslation, ribosomal structure, and biogenesisA4Y5U62280.67tolQSputcn32_1604Tol-Pal system protein TolQA4Y7L61020.70clpSSputcn32_2228ATP-dependent Clp protease adapter protein ClpSA4YBZ61230.71secESputcn32_3772Protein translocase subunit SecEA4YA836970.73fusASputcn32_3154Elongation factor G, EF-GA4Y1K12810.73rlmJSputcn32_0098Ribosomal RNA large subunit methyltransferase J, EC 2.1.1.266A4YBY22010.73rplDSputcn32_375850S ribosomal protein L4A4Y8362910.75Sputcn32_2398Pseudouridine synthase, EC 5.4.99.-A4YCI21560.76atpFSputcn32_3960ATP synthase subunit bA4YBX72300.78rpsCSputcn32_375330S ribosomal protein S3A4YCI42730.78atpBSputcn32_3962ATP synthase subunit aA4Y8B93150.79secFSputcn32_2481Protein-export membrane protein SecFA4Y8C11100.79yajCSputcn32_2483Sec translocon accessory complex subunit YajCA4Y4K53380.80eraSputcn32_1160GTPase EraA4YBY41031.26rpsJSputcn32_376030S ribosomal protein S10A4Y4S71741.31bamESputcn32_1232Outer-membrane protein assembly factor BamEA4YBV92061.32rpsDSputcn32_373530S ribosomal protein S4A4Y3E72461.33rlmBSputcn32_075023S rRNA (guanosine-2′-O-)-methyltransferase RlmB, EC 2.1.1.185A4YBW11181.41rpsMSputcn32_373730S ribosomal protein S13A4Y3E11421.42rplMSputcn32_074450S ribosomal protein L13A4Y9C21511.42rimPSputcn32_2836Ribosome maturation factor RimPA4YBW41441.44rplOSputcn32_374050S ribosomal protein L15A4Y5442831.45tsfSputcn32_1349Elongation factor Ts, EF-TsA4YBW01301.47rpsKSputcn32_373630S ribosomal protein S11A4Y3E21301.49rpsISputcn32_074530S ribosomal protein S9A4Y7051431.56infCSputcn32_2017Translation initiation factor IF-3A4Y6A5851.57minESputcn32_1763Cell division topological specificity factorA4Y3F31501.61rplISputcn32_075650S ribosomal protein L9A4YBX9921.67rpsSSputcn32_375530S ribosomal protein S19A4YBY81241.71rpsLSputcn32_376430S ribosomal protein S12A4YBV71311.72rplQSputcn32_373350S ribosomal protein L17A4YBW5601.72rpmDSputcn32_374150S ribosomal protein L30A4Y5S0561.75rpmFSputcn32_157850S ribosomal protein L32A4Y9F31091.77rsfSSputcn32_2867Ribosomal silencing factor RsfSA4Y9B7891.84rpsOSputcn32_283130S ribosomal protein S15A4YBY71561.86rpsGSputcn32_376330S ribosomal protein S7A4Y704641.87rpmISputcn32_201650S ribosomal protein L35A4Y7L4721.90infASputcn32_2226Translation initiation factor IF-1A4Y426841.90rpmASputcn32_098050S ribosomal protein L27A4Y4L3831.90rpsPSputcn32_116830S ribosomal protein S16A4YBM9731.92zapBSputcn32_3654Cell division protein ZapBA4YBX61361.93rplPSputcn32_375250S ribosomal protein L16A4Y6L51861.96efpSputcn32_1875Elongation factor P, EF-PA4Y3F2752.05rpsRSputcn32_075530S ribosomal protein S18A4Y4A1882.09rpsTSputcn32_105630S ribosomal protein S20A4Y5V12492.09cpoBSputcn32_1609Cell division coordinator CpoBA4Y6Z82082.14lolASputcn32_2010Outer-membrane lipoprotein carrier proteinA4YB741542.17trmLSputcn32_3498tRNA (cytidine (34)-2′-O)-methyltransferase, EC 2.1.1.207A4YBX81102.21rplVSputcn32_375450S ribosomal protein L22A4YBX21042.22rplXSputcn32_374850S ribosomal protein L24A4Y2V2792.28rpmESputcn32_055350S ribosomal protein L31A4Y5461852.28frrSputcn32_1351Ribosome-recycling factor, RRFA4YBW71162.44rplRSputcn32_374350S ribosomal protein L18A4YBX01012.61rpsNSputcn32_374630S ribosomal protein S14A4Y7031182.62rplTSputcn32_201550S ribosomal protein L20A4Y2L3573.30rpmGSputcn32_046450S ribosomal protein L33A4Y5X8583.61rmfSputcn32_1636Ribosome modulation factor, RMFA4YCM5453.83rpmHSputcn32_400550S ribosomal protein L34A4YBW2374.44rpmJSputcn32_373850S ribosomal protein L36Not assignedA4Y6552181.41Sputcn32_1713Butyryl-CoA:acetate CoA transferase, EC 2.8.3.81UniProt and NCBI database accession numbers. 2Average ratio from three replicates by TMT experiment. A protein species was considered differentially accumulated if it exhibited a fold change >1.5-fold (cultivated at 4°C/cultivated at 30°C) with a P value of <0.05.


## 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 onP values is listed in Figure 2.Figure 2
Gene Ontology (GO) enrichment analysis of differentially expressed proteins (S. putrefaciens cultivated at 4°C and 30°C, respectively. X-axis represented each GO term; Y-axis represented the number of differentially expressed proteins in each category).


## 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.Figure 3
Schematic representation of the differentially expressed proteins involved in the ribosome pathway. The red boxes indicate the unregulated proteins, and the green boxes indicate the downregulated proteins.


## 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 ofS. 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, P<0.05), 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, P<0.05). 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.Figure 4
The metabolism pathway of the main energy sources. Different colors indicate the ranges of the fold change (cultivated at 4°C/cultivated at 30°C) values.


## 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.Figure 5
PPI network: significantly upregulated differentially expressed proteins ofS. putrefaciens cultivated at 4°C and 30°C, respectively. The proteins upregulated in S. putrefaciens are colored in a gradient color from white to red, 1.52≤ FC ≤6.36.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 thatS. 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 (Table1), 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 thatS. 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 ofS. 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.



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*Source: 8876986-2021-01-08.xml*