Abstract

   Meat is often contaminated by food-borne pathogens, resulting in
   significant economic losses. Linalool from plant essential oils (EOs)
   has been reported to have excellent antibacterial properties.
   Therefore, this study aims to elucidate the mechanism of linalool
   against Shigella sonnei (S. sonnei) based on proteomic and
   physiological indicators. The results indicated that linalool severely
   perturbed the expression levels of intracellular proteins, of which 208
   were up-regulated and 49 were down-regulated. Moreover, linalool
   exerted its inhibitory effect mainly through the induction of amino
   acid limitation and insufficient energy levels based on the pathways
   involved in differential expressed proteins (DEPs). After 8 h, alkaline
   phosphatase (AKP) leakage increased 20.96 and 21.52-fold in the MIC and
   2MIC groups while protein leakage increased 2.17 and 2.50-fold,
   respectively, which revealed the potential of linalool on cell
   structure damage combined with nucleic acid leakage. In addition, the
   ATP content decreased to 36.92% and 18.84% in the MIC and 2MIC groups,
   respectively when processed for 8 h. In particular, linalool could
   effectively control the quality change of fresh beef by measuring pH,
   total volatile basic nitrogen (TVB-N), total viable counts (TVC) while
   not affecting its sensory acceptability based on the result of sensory
   evaluation. This research provides theoretical insights for the
   development of linalool as a new natural antibacterial agent.

1. Introduction

   Meat including beef is often an ideal carrier for pathogenic bacteria
   due to its high protein, fat and moisture content ([37]Lin, Mao, Sun,
   Rajivgandhi, & Cui, 2019). Shigella, is commonly detected in multiple
   foods including ground beef ([38]Ahmed and Shimamoto, 2015, [39]Kang et
   al., 2018), which not only threatens human health, but also causes
   great economic losses. Shigella, a gram-negative bacteria, consists of
   four species including Shigella dysenteriae, Shigella flexneri,
   Shigella boydii, and Shigella sonnei, which ranked third after
   Salmonella and Campylobacter in the bacteria associated with
   food-related infections ([40]Zhang et al., 2011). As one of the
   Shigella, Shigella sonnei (S. sonnei) is expanding unprecedentedly in
   industrialized regions including Asia, Latin America and the Middle
   East due to the resistance exhibited to multiple antibiotics ([41]Gu et
   al., 2012, [42]Thompson et al., 2015). Despite improvements in drinking
   water and food hygiene, Shigellosis caused by S. sonnei was still
   internationally prevalent, and its multi-drug resistant strains were
   rapidly expanding ([43]Tickell et al., 2017, [44]Ud-Din et al., 2013).
   They can transmit exogenous genes related to mobile genetic components
   including transposons, integrons, R-plasmids and genome islands related
   to bacterial chromosomes ([45]Ud-Din et al., 2013). Once the intestinal
   tract is infected with S. sonnei, abdominal cramping, diarrhea, nausea,
   vomiting, fever, and severe fluid loss may occur ([46]Chai, Lee, Kim, &
   Oh, 2016). Therefore, the risk of S. sonnei infection combined with
   negative perceptions of synthetic preservatives, has forced researchers
   to seek effective and safe natural antibacterial agents.

   In recent years, essential oils (EOs) from natural ingredients have
   attracted the attention of researchers. Reports confirmed that EOs
   including oregano and clove have excellent antibacterial effects
   against a variety of pathogenic bacteria ([47]Cui et al., 2019, [48]Hu
   et al., 2019). However, inconsistencies in composition due to
   geographic origin and harvest time may limit the application of EOs in
   the food industry ([49]Burt, 2004). One of the effective ways to
   overcome this problem is to isolate specific components with
   antibacterial potential from EOs. Linalool is widely found in the EOs
   including green huajiao and basil ([50]Diao et al., 2013, [51]Hollander
   and Yaron, 2021, [52]Pereira et al., 2018), proving to have excellent
   inhibitory effect on multiple pathogenic bacteria. Our previous studies
   also found that linalool has outstanding inhibitory potential against
   spoilage and pathogenic bacteria including Pseudomonas aeruginosa,
   Pseudomonas fluorescens and Listeria monocytogenes, mainly by
   disrupting cell structure and impairing respiratory metabolism ([53]Guo
   et al., 2021, [54]He et al., 2022, [55]Liu et al., 2020). Although the
   mechanisms of linalool inhibition on S. sonnei have been initially
   reported based on the membrane damage and oxidative stress ([56]He et
   al., 2023), the mechanisms involved at the molecular level have not
   been systematically investigated. Furthermore, it was reasonable to
   speculate that components from EOs would disrupt the expression of
   bacterial intracellular protein profiles according to previous report
   ([57]Du, Yin, Yang, He, & Sun, 2021). However, the response mechanism
   of S. sonnei to linalool has not been clearly clarified based on
   physiological indicators combined with proteomic technology.

   Proteomics was widely used as a highly sensitive method to study the
   action mechanisms of different drugs ([58]Yang et al., 2016).
   Therefore, proteomic technology was used to analyze the mechanism of S.
   sonnei response to linalool in this study and its preservation
   potential for fresh beef. In addition, alkaline phosphatase (AKP),
   protein and nucleic acid leakage were also measured to validate the
   insights drawn in proteomics. Particularly, intracellular ATP content
   and SDS-PAGE were also performed as components of the mechanism of
   linalool against S. sonnei. This investigation aims to reveal the
   molecular mechanism of S. sonnei inhibition by linalool using
   proteomics techniques, encouraging the proposal of linalool as a
   natural antibacterial agent in the food industry.

2. Materials and methods

2.1. Bacterial strains and chemicals

   S. sonnei ATCC 25931 was purchased from Guangdong Microbial Culture
   Preservation Center (Guangzhou, China), and cultured at 37 °C with
   nutrient broth medium (Solarbio Science & Technology Co., Ltd, China).
   Linalool (purity > 99%) was provided by Hainan Camphora Biotech Co.,
   Ltd. (Hainan, China). AKP A059-1, Bicinchoninic acid (BCA) A045-4 and
   ATP A095-1-1 assay kits were obtained from Nanjing JianCheng
   Bioengineering Institute (Nanjing, China).

2.2. Determination of cell wall integrity

   Referring to our previous report, the minimum inhibitory concentration
   (MIC) of linalool against S. sonnei used in this study was 1.5 mL/L
   ([59]He et al., 2023). Subsequently, the leakage of AKP was used as an
   indicator to evaluate the cell wall integrity and simply, linalool at
   final concentrations of MIC and 2MIC was added as treatments when the
   bacterial suspension was grown to log phase. The supernatant was
   obtained for the determination of AKP content by centrifugation (6000
   g, 4 °C, 10 min). In particular, the absorption value was measured at a
   wavelength of 520 nm using a microplate reader (SP-Max3500FL, Shanghai
   Flash Spectrum Biotechnology Co., Ltd., Shanghai, China).

2.3. Leakageof extracellular proteins and nucleic acids

   The bacterial suspension was treated with linalool (MIC and 2MIC) after
   incubation to log phase and subsequently, the supernatant was collected
   by centrifugation (6000 g, 4 °C, 10 min) when treated for 0, 2, 4, 6,
   8 h, respectively. Nucleic acid leakage was defined as absorbance at
   260 nm and the content of protein was quantified using BCA protein
   assay kit.

2.4. SDS-PAGE

   Intracellular proteins were analyzed using Sodium dodecyl
   sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) following the
   procedures ([60]Sitohy, Mahgoub, & Osman, 2012). Cells in log phase
   were harvested by centrifugation (6000 g, 4 °C, 10 min) and then
   resuspended after washing with PBS (phosphate buffer solution, 0.01 M,
   pH 7.2). Subsequently, linalool (MIC and 2MIC) was added to the
   bacterial suspension for co-culture and the samples were centrifuged
   again to get the cell pellet at 4 h. The prepared bacteria cells were
   processed with ultrasound (power 300 W, 15 min, interval 5 s) to
   release intracellular proteins. The samples (10 μL) were loaded on
   SDS-PAGE electrophoresis (Bio-Rad Mini-PROTEAN® Tetra, Bio–Rad
   Laboratories, Hercules, USA) at 80 V for 30 min followed by 120 V for
   60 min. Subsequently, the gel was stained using coomassie blue R-250
   stain solution and decolorized in water/methanol/acetic acid (5:4:1,
   v/v). Finally, the eluted protein bands were visualized by
   photographing.

2.5. Measurement of intracellular ATP concentrations

   Linalool (MIC and 2MIC) was added to the bacterial suspension when the
   bacteria grew to log phase. Cell precipitates were collected by
   centrifugation and resuspended in saline for sonication as described in
   2.4. Next, ATP in the samples was measured to assess the level of
   intracellular energy perturbation under linalool stress with the
   instructions of ATP content assay kit.

2.6. Proteomic analysis

2.6.1. Sample preparation

   The change in protein profile of S. sonnei treated with linalool was
   analyzed to uncover the mechanism of linalool as description of
   [61]Liao et al. (2019). Generally, the final concentration of linalool
   at MIC was selected as the treatment when S. sonnei was cultured to log
   phase. Subsequently, cells were harvested by centrifugation (3000 g,
   4 °C, 10 min) and washed 3 times with PBS when co-cultured for 4 h.
   Cell pellets were quenched with liquid nitrogen immediately for
   proteomic analysis.

2.6.2. Protein extraction

   Lysate (4% SDS, 100 mM Tris-HCl, pH 7.6) was added to the cell pellet
   and then pellets were disrupted using MP Fastprep-24 homogenizer
   (6.0 M/S, 30 s, 2 times). Boiling water bath was performed for 10 min
   after sonication and subsequently, the supernatant was collected by
   centrifugation (14000 g, 15 min). Afterwards, the protein content was
   determined with BCA protein assay kit when the supernatant was filtered
   using 0.22 μm filters. Protein (20 μg) was mixed with loading buffer
   and then treated with boiling water bath for 5 min for SDS-PAGE
   analysis.

2.6.3. FASP (Filter aided sample Preparation) enzymatic hydrolysis and TMT
labeling

   Referring to previous report ([62]Sarengaowa et al., 2019), protein
   solution (150–200 μg) were reduced with dithiothreitol (DTT, 100 mM)
   for 5 min in boiling water. Protein samples were centrifuged under the
   condition (12500 g, 15 min) after mixing with UA buffer (8 M Urea,
   150 mM Tris-HCl, pH 8.5). Subsequently, the protein was alkylated using
   IAA butter (Iodoacetamide, 100 uL) at 600 rpm for 1 min and centrifuged
   at 12,500 g for 15 min before left standing for 30 min at 25 °C. In
   particular, this process was performed twice. After TEAB was mixed with
   the sample for centrifugation (12500 g, 15 min), the solution was
   incubated under agitation (600 rpm, 1 min) when mixing with TEAB
   (0.1 M, 40 μL) buffer containing trypsin (4 μg). Peptides (100 μg) were
   labeled with TMT kit (Thermo Scientific, USA) as directed by the
   instructions due to the high accuracy compared to label-free proteomic.

2.6.4. LC–MS/MS analysis

   The peptide solution was fractionated with Thermo Scientific Easy nLC
   equipped column (Thermo Fisher Scientific, Acclaim PepMap RSLC
   50um X 15 cm, nano viper, P/N164943). A fivestep linear gradient of A/B
   mixture (A: ddH[2]O with 0.1% formic acid, B: ACN with 0.1% formic
   acid) was applied to elute the peptides. The sample was loaded onto the
   analytical column for separation by the autosampler and the flow rate
   was 300 nL/min when the column was equilibrated with 100% A. The
   elution gradient was as follows: 0–5 min, the linear gradient of B was
   3%-5%; 5–75 min, the linear gradient of B was 5%-28%; 75–80 min, the
   linear gradient of B increased from 28% to 38%; 80–84 min, the linear
   gradient of B rose to 99%; 84–90 min, B was maintained at 99%. Samples
   were analyzed using Q Exactive plus (Thermo Fisher Scientific,
   Shanghai, China)-coupled tandem mass spectra after chromatographic
   separation. The detection method was positive ion, and the scanning
   range of precursor ion was 350–1800 m/z. MS1 resolution was 70,000 and
   AGC target was 3e6 with Level 1 Maximum IT of 50 ms. The acquisition
   parameters of the secondary spectrum were as follows: MS2 Activation
   Type was HCD; Isolation window was 2.0 m/z; MS2 resolution was 17,500
   or 35000; AGC target was 2e5; Minimum AGC target was 2e3; Secondary
   Maximum IT was 50 ms. The collected charge was between 2 and 6, the ion
   dynamic exclusion time was 30 s, with the Normalized Collision Energy
   of 30 eV.

2.6.5. Proteomics data analysis

   Pearson correlation, PCA and heat map analyses were performed using the
   R language in this study. Difference level >1.2 or <0.83 folds was
   defined as differentially expressed protein (DEPs) combined with p
   value < 0.05 based on previous study ([63]Sarengaowa et al., 2019).
   Then, Blast2GO was carried out for functional annotation and
   classification with DEPs and the Kyoto Encyclopedia of Genes and
   Genomes (KEGG) was employed using KOALA (KEGG Orthology And Links
   Annotation) software to display the functions from the metabolic
   pathways.

2.7. Application in fresh beef

   In the current study, the preservation potential of linalool was also
   investigated by selecting fresh beef as a food model. Specifically,
   fresh beef was cut into pieces (2 cm × 2 cm, 10 g) after sterilization
   with UV irradiation, and all samples were inoculated with S. sonnei
   (10^6 CFU/mL, 10 μL) by spot painting. The samples were left for 30 min
   to ensure that the bacteria were adsorbed and then divided into 4
   groups for processing (linalool at MIC and 2MIC concentrations, 1%
   ethanol and sterile water). Subsequently, the prepared fresh beef
   samples were stored in sterile packaging bags at 4 °C and the quality
   was evaluated by quantitative indicators including pH, total volatile
   basic nitrogen (TVB-N), the total viable counts (TVC) and sensory
   evaluation.

2.7.1. pH

   The blender was used to homogenize the beef samples when mixed with
   sterile saline at a ratio of 1:50 (v/v). After soaking for 30 min, the
   samples were filtered to obtain a filtrate for pH determination with a
   pH meter, previously calibrated with pH 4.00 and pH 7.00 buffers.

2.7.2. Total volatile basic nitrogen (TVB-N)

   TVB-N values of beef samples during storage were also quantified in
   this study. Briefly, the samples were dispersed in deionized water
   (75 mL) and then filtered using a stirrer to obtain the supernatant
   after thorough mixing. Subsequently, TVB-N analyses were performed
   using the automatic Kjeldahl analyzer (K9840, Shandong Haineng
   Scientific Instrument Co., Ltd., China). Finally, hydrochloric acid
   (0.1 mol/L) was used to titrate distillation products and the results
   were expressed as mg of TVB-N/100 g of beef.

2.7.3. Total viable counts (TVC)

   The TVC was determined by plate counting method according to the
   Chinese National Standard (GB/T 4789.2–2016). The churned beef samples
   were mixed with sterile saline and diluted at a ratio of 1:9 (v/v). The
   diluted samples were uniformly coated on the cooled medium and
   incubated for 48 h before observation. The result of microbial count
   was recorded as Log CFU/g of beef sample.

2.7.4. Sensory evaluation

   The effect of linalool on the sensory properties of beef samples was
   evaluated through a panel of 12 semi-trained students in food science
   and engineering. In particular, the sensory evaluation of this study
   consisted of 5 parts including “color”, “odor”, “texture”, and
   “overall-acceptability”, which were evaluated using a 5-point
   descriptive range with reference to previous report ([64]Shokri,
   Parastouei, Taghdir, & Abbaszadeh, 2020). The higher the score of the
   sample, the better the quality of the related index, where 5 means the
   best and 1 means the worst. Samples were randomly assigned to group
   members for evaluation at 1, 3, 5, 7, and 9 days during the evaluation
   process. In addition, samples with scores < 3 were specified as
   unacceptable.

2.8. Statistical analysis

   Graphs were performed using Origin 2018 (Origin Lab Co., Northampton,
   MA, USA) and results were expressed as mean ± standard deviation (SD)
   with three replicates in each experiment. The significance of data was
   conducted by one-way analysis of variance (ANOVA), followed by Duncan’s
   multiple range tests using SPSS version 13.0 (SPSS Inc., Chicago, IL,
   USA). In particular, significance analysis was performed between MIC of
   linalool and blank control in the proteomics data while in MIC of
   linalool, 2MIC of linalool, 1% ethanol and blank control among other
   indicators. A p-value < 0.05 was considered as a significant
   difference.

3. Results

3.1. Cell wall integrity analysis

   In this study, the content of AKP was maintained at a very low level
   and hardly changed significantly in the absence of linalool ([65]Fig.
   1A). However, the AKP activity showed a significant increasing trend
   (p < 0.05) and reached a maximum at 6 h under linalool stress, implying
   that the addition of linalool caused severe damage to the cell wall
   thereby leading to the leakage of the AKP. Similar report also
   supported that linalool can damage the cell wall structure of
   Shewanella putrefaciens ([66]Guo et al., 2021b). A potential
   explanation might be attributed to the interaction between the
   hydrophobic group of linalool and the hydrophobic peptidoglycan
   ([67]Vadillo-Rodriguez, Cavagnola, Perez-Giraldo, & Fernandez-Calderon,
   2021).

Fig. 1.

   [68]Fig. 1
   [69]Open in a new tab

   The leakage of AKP in S. sonnei with or without linalool (A); SDS–PAGE
   profiles of S. sonnei under linalool stress (B); Extracellular protein
   (C) and nucleic acid (D) contents in S. sonnei when exposed to
   linalool; Intracellular ATP content in S. sonnei when treated with
   linalool (E). PCA (F) and Heat map (G) analysis of the protein profiles
   in S. sonnei between control and linalool treatment.

3.2. SDS-PAGE

   The SDS-PAGE electrophoresis analysis was performed in this study and
   the protein bands of samples and Markers were visualized in [70]Fig.
   1B, where Markers ranged from 10 kDa to 180 kDa. The protein bands were
   clear without linalool. The bands were blurred and even some bands were
   barely discernible when exposed to linalool. The current result
   suggested that the loss of soluble protein was extremely significant
   combined with the result of intracellular protein content ([71]Fig.
   S1A). These blurred protein bands revealed the inhibitory effect of
   linalool on protein synthesis of S. sonnei and leakage of proteins,
   which were thought to interfere with physiological functions due to
   proteins responsible for information transmission and cell structure
   ([72]Kohanski, Dwyer, & Collins, 2010). Our previous study found that
   linalool also reduced intracellular protein content of Shewanella
   putrefaciens ([73]Guo et al., 2021b) and speculated that it may be due
   to the following reasons: protein leakage caused by loss of cellular
   integrity; excessive protein consumption in response to stimuli or
   blocked protein synthesis when exposed to linalool.

3.3. Cell membrane integrity assessment

   The content of extracellular macromolecules including proteins and
   nucleic acid was also measured as an indicator to monitor the integrity
   of the cell membrane in the current work. As shown in [74]Fig. 1C and
   1D, both protein and nucleic acid contents were increased with the
   prolongation of linalool acting time (p < 0.05). The amount of nucleic
   acid leakage in the MIC group and the 2MIC group was 2.61 and 2.63
   times that of the control group, respectively, while the amount of
   protein leakage was 3.02 and 3.33 times. Taken together, the current
   evidence revealed that membrane damage may make an important
   contribution to the inhibition of S. sonnei by linalool.

3.4. Intracellular ATP content analysis

   ATP is widely used to assess a variety of cellular functions including
   material transport across cell membranes, which may be a potential
   target for understanding how natural antibacterial agents work
   ([75]Bajpai, Sharma, & Baek, 2013). As illustrated in [76]Fig. 1E,
   intracellular ATP content was rapidly dissipated when linalool was
   selected as the treatment (p < 0.05). ATP content decreased by 63.08%
   and 81.16% in the MIC and 2MIC groups, respectively, revealing the
   phenomenon of energy shortage in the cell. It implied an intracellular
   energy deficit, which was attributed to the excessive consumption of
   ATP caused by excessive apoptosis ([77]Zamaraeva et al., 2005). Another
   important factor in the decrease of intracellular ATP content might be
   the leakage of intracellular ATP due to the loss of cell membrane
   integrity.

3.5. Global analysis of proteomic responses

   Microbial responses to linalool can be analyzed with advanced
   information provided by proteomics ([78]Hu et al., 2017). A
   quantitative proteomic approach based on TMT label was employed to
   obtain the global protein profile of S. sonnei after exposure to
   linalool. Pearson’s correlation coefficient and principal component
   analysis (PCA) were performed to assess the reproducibility of protein
   quantification ([79]Fig. S1B and 1F). These data supported that there
   were significant changes in the protein profiles of the
   linalool-treated and control group (p < 0.05), which was consistent
   with the normalized heatmap results ([80]Fig. 1G). Subsequently, the
   DEPs were plotted in [81]Fig. S1C in the form of volcano map. A total
   of 257 DEPs were identified, of which 208 were overexpressed and 49
   were downregulated ([82]Fig. S1D). In order to more intuitively display
   the differences in protein expression between different groups, the
   contents of the top 9 proteins with significant expression were
   displayed in the form of scatter plots ([83]Fig. S2).

3.6. Gene ontology (GO) analysis

   The obtained DEPs were further categorized according to GO annotations
   ([84]Fig. 2A), which were classified based on cellular components (CC),
   biological process (BP) and molecular functions (MF). In BP, most of
   DEPs mainly involved in cellular process, metabolism process and
   biological regulation. The CC analysis indicated that these proteins
   were mainly located in cell part, protein-containing complex and
   membrane part. Binding, catalytic activity and structural molecular
   activity contained a large number of DEPs known from MF. Significant
   enrichment analysis of GO functional annotations was conducted to
   observe whether DEPs has significant enrichment trends in certain
   functional types. The top 10 enriched classification results of the
   three categories were displayed in the form of bubble charts ([85]Fig.
   S3A). Among the three categories, the most enriched were transport (in
   BP), out membrane-bounded periplasmic space (in CC) and structural
   constituent of ribosomes (in MF).

Fig. 2.

   [86]Fig. 2
   [87]Open in a new tab

   Gene Ontology (GO) functional annotation of differentially expressed
   proteins (DEPs) (A). KEGG pathway annotation according to DEPs (C).

Fig. 3.

   [88]Fig. 3
   [89]Open in a new tab

   Enrichment analysis of domains in the form of a circle plot (A);
   Enrichment analysis of domains in histogram format (B). Application of
   linalool in fresh beef preservation, pH (C); TVB-N (D) and TVC (E).

3.7. KEGG pathway analysis

   KEGG analysis was performed to discover the metabolic or signaling
   pathways that DEPs involved in. [90]Fig. 2B showed that the top 20 KEGG
   pathways were sorted according to the number of DEPs, and the pathways
   with more DEPs mainly included metabolic pathways, ribosomes, ABC
   transport, microbial metabolism in diverse environments and
   biosynthesis of secondary metabolites. These data supported that
   intracellular metabolic processes were disrupted in the presence of
   linalool based on the highest number of DEPs involved. To identify
   significantly affected metabolic and signal transduction pathways, KEGG
   pathway enrichment analysis was also carried out through fisher's exact
   test ([91]Fig. S3B). Pathways that were highly enriched and possibly
   related to potential antibacterial mechanisms mainly involved
   ribosomes, cellular chemotaxis, phosphotransferase system (PTS) and ABC
   transporters.

3.8. Domain annotation and enrichment analysis

   It is important to study the domain of proteins for understanding the
   biological functions of proteins and their evolution. The domain
   enrichment results were summarized in [92]Fig. 3A and [93]Fig. 3B after
   statistical analysis. Pathways with higher enrichment involved
   Solute-binding protein family 3, conserved site, Solute-binding protein
   family 3/N-terminal domain of MltF, and Cold-shock conserved site,
   containing 5, 6, and 4 DEPs, respectively. Moreover, except for the
   Globin-like superfamily, the proteins involved in the other domains all
   showed a tendency to be overexpressed (p < 0.05).

3.9. Analysis of metabolic pathways

   The data obtained revealed that changes in protein profiles might be
   one of the important targets for linalool to exert its bacteriostatic
   effect. Subsequently, the screened DEPs were introduced into KEGG for
   metabolic network analysis to gain an insight into the action of
   linalool against S. sonnei. The result showed that highlighted pathways
   were related to amino acid and protein metabolism, DNA replication and
   repair, energy metabolism and environmental stimulus response. Other
   important pathways including PTS, amino sugar and nucleotide sugar
   metabolism, and peptidoglycan biosynthesis were also enriched.

3.9.1. Amino acid and protein metabolism

   Bacterial cells consume about 50% of their energy to produce protein,
   while 20–40% of protein synthesis is directed to ribosomes and other
   translation factors ([94]Russell & Cook, 1995). Therefore, the
   ribosome, as a site for protein synthesis, is critical for bacterial
   survival. However, the ribosome-associated proteins were all
   overexpressed except for ribosome large subunit L19 based on the
   results in [95]Fig. 4. These evidences suggested that protein
   starvation might have occurred in the cell, causing the upregulation of
   ribosomal proteins when exposed to linalool, which was also supported
   by results for SDS-PAGE and intracellular soluble proteins. Similar
   study considered that natural bacteriostatic agents might achieve
   bacteriostatic properties by overconsumption of amino acids ([96]Liu et
   al., 2020a), which acted as a negative feedback regulation signal to
   stimulate ribosome expression. In the biosynthesis of various amino
   acids, the protein involved in valine, leucine and isoleucine
   biosynthesis (LeuD) was overexpressed. However, the expression of
   protein responsible for arginine biosynthesis (aspc), which was also
   involved in the biosynthesis of phenylalanine, tyrosine and tryptophan,
   was inhibited. A potential explanation for the unbalanced expression of
   proteins involved in the synthesis of these amino acids might be the
   depletion and accumulation of specific amino acids within the cell.
   Previous study has also shown that under stress conditions, bacterial
   cells usually activated some amino acid biosynthesis pathways to
   survive ([97]Horinouchi et al., 2010). Unlike previous report that
   amino acid restriction downregulated genes controlling amino acid
   metabolism ([98]Liu et al., 2020a), we found that some amino acid
   metabolisms were down-regulated, while some were up-regulated. The
   pathways of amino acid metabolism up-regulation included d-amino acid
   metabolism (alr and dadA), degradation of valine, leucine and
   isoleucine (fadA). Meanwhile, arginine and proline metabolism (aspC and
   adiA), tyrosine metabolism (aspC) were inhibited. Some amino acid
   metabolisms were expressed unbalanced, such as the down-regulation of
   aspC, and the overexpression of pyrB and pyrI, which involved in
   alanine, aspartate, and glutamate metabolism. In cysteine and
   methionine metabolism, aspC was inhibited while mrsC was significantly
   expressed. Proteins involved in phenylalanine metabolism were also
   expressed unbalanced, with aspC repressed and dadA activated. Overall,
   the addition of linalool might have caused amino acid restriction to
   activate most of the proteins participating in ribosomal in an attempt
   to alleviate this stress. Upregulation of specific amino acid synthesis
   pathways (valine, leucine and isoleucine biosynthesis) and inhibition
   of certain amino acid metabolism (tyrosine metabolism, arginine and
   proline metabolism) may also serve this purpose (Liu et al., 2020).
   However, more research was still needed to elucidate the mechanism of
   amino acid disorder in the next step.

Fig. 4.

   [99]Fig. 4
   [100]Open in a new tab

   Pathway analysis of DEPs related to amino acid and protein metabolism.
   The colors used in the ribosomal pathway (top left) include blue, pink
   and yellow to distinguish between different proteins The red box
   reprents up-regulated proteins and blue indicates down-regulated
   proteins in the mapped metabolic network pathways. (For interpretation
   of the references to color in this figure legend, the reader is