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. 2025 Jun 7;109(1):141. doi: 10.1007/s00253-025-13525-y

Phosphoproteomics analysis of acid stress response of Alicyclobacillus acidoterrestris in response to acid stress

Yingli Liu 1, Kunrun Wu 1, Lingxia Jiao 1,2,✉, Rui Shen 1, Junjian Ran 1, Youzhi Wu 2
PMCID: PMC12145292  PMID: 40483348

Abstract

Abstract

Alicyclobacillus acidoterrestris causes pasteurized acidic juices spoilage, resulting in a significant decline in juice quality and causing economic losses. Exploration of A. acidoterrestris in response to acid stress could help control contamination caused by the bacteria. In this study, the mechanism of A. acidoterrestris in response to acid stress was studied by quantitative phosphoproteomics technique. Results showed that the phosphorylation of 40 proteins in A. acidoterrestris was closely related to the regulation of acid stress. The KEGG pathway enrichment analysis showed that the quorum sensing pathway, which might be involved in the perception of A. acidoterrestris, was mainly enriched. We found that the upregulation of Spo0A and YidC phosphorylationmay resist acid stress by forming spores. The phosphorylation level of pyruvate kinase increased, which may improve bacterial acid stress resistance through the formation of energy supply. The phosphorylation level of ABC transporter permease was significantly upregulated, which may be part of the cell adaptation adjustment and contribute to the survival of A. acidoterrestris under acid stress. In summary, the molecular mechanism of acid stress regulation of A. acidoterrestris was proposed via quantitative phosphoproteomics, which provided a theoretical and experimental basis for further investigation of the acid resistance mechanism of A. acidoterrestris.

Key points

• Spo0 A, Yidc proteins may be the key regulatory proteins for acid stress response.

• ABC transporters are beneficial to the survival of the bacteria under acidic stress.

• A. acidoterrestris may sense and transmit pH signal through quorum sensing pathway.

Keywords: Alicyclobacillus acidoterrestris, Acid stress, Phosphorylated proteomics, Acid tolerance mechanism, Protein modification

Introduction

Alicyclobacillus acidoterrestris (A. acidoterrestris DSM 3922 T) is an acid, heat-resistant, and non-pathogenic gram-positive bacterium, which is widely distributed in acidic fruit juice and is one of the main strains leading to the spoilage of acidic fruit juices (Flauzino et al. 2021; Silva et al. 2021). It exhibits unique physiological and biochemical characteristics such as acidophilia, high-temperature resistance, and spore production (Smit et al. 2011). A. acidoterrestris is an obligate acidophilic bacterium, and its optimal growth pH is 3.5–4.5, and it can survive in an environment with a pH of 2.2. However, it cannot grow and reproduce when the environmental pH exceeds 6.0, demonstrating its remarkable acid stress adaptability. In the field of food science and juices manufacturing, A. acidoterrestris has attracted much attention due to its unique acidophilic and heat-resistant properties. This microorganism can grow and reproduce in an acidic environment, and its spores have strong heat resistance and can resist the traditional pasteurization process. This has led to the possible contamination of juices products even after pasteurization, and the resulting deterioration of juices sensory quality has led to significant economic losses, making the control of A. acidoterrestris pollution a key issue that needs to be solved in the industry (Sourri et al. 2022; Wang et al. 2021).

It is well known that microorganisms can grow and reproduce rapidly in suitable environments, such as rich nutrients, suitable temperature, and pH. Meanwhile, environmental changes can cause stress to microorganisms. Acid stress is a common pressure encountered by bacteria, which inhibits their growth and even results in cell death (Zhao et al. 2022b). The key for bacteria to survive in harsh environments is to perceive changes in the external environment and to adjust the life activities of the cells to adapt to the changing environment so that they can maintain a certain metabolic activity under adverse conditions (Bruggeman et al. 2020). Acid stress response is a cell protection mechanism that rapidly produces various acid stress proteins to resist sudden adverse environments. Protein post-translational modification can be used as a rapid and effective signal transduction mode to participate in the regulation of bacterial metabolism, gene expression, replication, and other life processes (Blanco-Touriñán et al. 2020). Protein phosphorylation is one of the most important reversible post-translational modifications, which can change protein activity, cellular localization, and interaction with other proteins, thereby regulating protein function (Mallick and Das 2023). In recent years, with the rapid development of phosphorylation modification enrichment technology and high-resolution liquid chromatography–mass spectrometry technology, phosphorylated proteomics has been widely used in the study of bacterial stress regulation mechanisms. The researchers performed a comprehensive quantitative phosphoproteomics analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation (Saric et al. 2022). Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. Zhang et al. (2024) carried out a phosphorylation modification omics analysis on foxtail millet under salt stress, and the identified phosphorylation modification proteins related to salt stress were mainly involved in metabolism, transcription, translation, protein processing, and other regulatory processes (Jayaraman et al. 2008; Wu et al. 2016).

Although there have been some reports on A. acidoterrestris response under acidic stress conditions in recent years, for example, Zhao et al. (2022a) studied the acid adaptive response of A. acidoterrestris, which provided an important understanding of A. acidoterrestris to acid stress. Research by Liu et al. (2024) studied the molecular mechanism of A. acidoterrestris exposed to acid stress at sublethal pH. It was found that A. acidoterrestris adapted to the acid stress environment by rapidly regulating protein expression levels, metabolic pathways, and membrane permeability at an acidic pH of 2.5. However, the mechanism of bacterial acid stress response is a complex process. Despite these insights, the mechanism by which A. acidoterrestris specifically senses and transmits acid stress signals and maintains pH homeostasis through specific pathways has not been fully elucidated. In particular, the specific acid-resistant molecular mechanism of Suandicao is still unclear. Particularly, the specific acid-resistant molecular mechanism of A. acidoterrestris is still unclear. Therefore, the study of protein phosphorylation modification is of great significance for analyzing the pathways related to phosphorylation signals in obligate acidophilic bacteria and their signal transduction mechanisms in response to acid stress. In this paper, the quantitative techniques of proteomics and phosphorylated proteomics were used to explore the changes in metabolic pathways of proteins and phosphorylated proteins after acid stress. Bioinformatics analysis of key phosphorylated proteins involved in acid stress regulation laid a theoretical foundation for further revealing the unique stress resistance mechanism of the bacteria.

Materials and methods

Bacterial strain and medium conditions

A. acidoterrestris was purchased from Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). A. acidoterrestris broth medium (AAM broth): glucose 2.0 g, yeast extract powder 2.0 g, CaCl2 0.38 g, KH2PO4 1.2 g, MgSO4•7H2O 1.0 g, MnSO4•H2O 0.38 g, (NH4)SO4 0.4 g, sterile water top up volume to 1 L and titrating pH to 4.0. The medium was autoclaved at 121℃ for 30 min (Jiao et al. 2015; Yamazaki et al. 1996).

Main reagents

Protease inhibitor (Merck Millipore, USA); Trypsin (Promega, USA); urea, dithiothreitol, niacinamide, and trifluoroacetic acid and TFA (all are from Sigma-Aldrich, Germany), phosphotransferase inhibitor (Merck Millipore, Germany), BCA-Kit (Bi yun tian, China), deacetylase inhibitor (MedChemExpress, USA), deubiquitinase inhibitor (Selleck Chemicals, USA), and acetonitrile (Thermo Fisher Scientific, USA).

Culture of A. acidoterrestris and acid stress treatment

The spores of A. acidoterrestris cryopreserved at − 80℃ were activated by inoculating in the AAM medium and cultured at 45℃ to a logarithmic phase (Liang et al. 2023). The activated bacterial suspension was centrifuged and resuspended into pH 2.0, 2.5, 3.0, and 4.0 (control) medium, which were correspondingly cultured in medium for 20 and 60 min, respectively. Scanning electron microscopy was used to monitor the bacterial activity and morphology under different acid stresses.

Scanning electron microscopy to monitor bacterial morphology change

According to the method of Anakha et al. (2023), a 1 mL bacterial liquid of the control group and acid treatment group at different time periods was transferred into a sterile centrifuge tube, spinning down, and pellets were washed 2–3 times with pre-cooled sterile water. The supernatant was discarded, adding 1 mL 2.5% glutaraldehyde (fixing cell form) to suspend the pellet. The suspension was placed in a 4℃ refrigerator for more than 2 h (avoid light). The suspension was further separated by centrifugation, the supernatant was discarded, and the pellet was washed with sterile water before ethanol gradient elution (35%, 50%). After each elution, place the sample into the refrigerator for 10 to 15 min. After centrifugation, 1 mL of ethanol was added with a final concentration of 50%. Each sample was taken as 10 μL onto a clean circular cover slide, air-dried, and then gold-sprayed with an ion spray and imaged by scanning electron microscopy.

Extraction and enzymatic hydrolysis of total bacterial protein

The appropriate amount of A. acidoterrestris bacterial sample was ground to powder in liquid nitrogen. After that, a fourfold volume of lysis buffer (8 mol/L urea, 1% protease inhibitor, 1% phosphatase inhibitor, 50 μmol/L deubiquitinase inhibitor, 3 μmol/L deacetylase inhibitor, 50 mmol/L nicotinamide) was added. The sample was ultrasonically lysed, centrifuged at 12,000 g for 10 min, and the supernatant was kept for further analysis. The total bacterial protein concentration was determined by the BCA method. The working reagent was prepared by mixing the BCA reagent with the copper ion reagent at a volume ratio of 50:1. At the same time, the standard protein solution was prepared. Subsequently, 0.05 to 1 mL of the standard protein solution or sample to be tested was mixed with 1 mL of a working reagent and incubated at 37 °C for 30 min. After incubation, the cells were cooled to room temperature, and the absorbance was measured at a wavelength of 562 nm to determine the protein concentration.

According to the method of FASP reported by Wiśniewski et al. (2009), the appropriate amount of standard protein and lysate were mixed to adjust the concentration of each group of protein. Add 20% trichloroacetic acid, vortex mixing, and precipitate at 4 °C for 2 h. After precipitation, the pellet was separated by centrifugation at 4500 g for 5 min. The supernatant was discarded, and the pellet was washed 2–3 times with pre-cooled acetone. After drying the precipitate, 200 mmol/L tetraethylammonium bromide was added, and the precipitate was dispersed by ultrasound. Trypsin was added at a ratio of 1:50 (protease: protein, m/m) and digested at 37 °C overnight; dithiothreitol was added to make the final concentration of 5 mM and reduced at 56 °C for 30 min. Finally, iodoacetamide was added to a final concentration of 11 mM and incubated in the dark at room temperature for 15 min to obtain polypeptides for subsequent enrichment and phosphorylation.

Enrichment of phosphorylated peptides

The peptides were dissolved in an enrichment buffer containing 50% acetonitrile/6% trifluoroacetic acid. The supernatant was transferred to a clean IMAC material and incubated on an oscillator at room temperature for 30 min, then washed with buffer 50% acetonitrile 6% trifluoroacetic acid and 30% acetonitrile/0.1% trifluoroacetic acid for three times. The modified peptides were eluted with 10% ammonia, and the eluants were collected. After vacuum freezing and drying, the salt was removed according to the instructions of C18 ZipTips; finally, it was stored at − 80 °C for a subsequent LC–MS/MS analysis.

LC–MS/MS analysis of enzymatic hydrolysates

The peptide was dissolved in an aqueous solution of 0.1% (V/V) HCOOH, and the liquid phase gradient separation was performed using an ultra-high performance liquid phase system. The conditions are as follows: mobile phase A: 0.1% HCOOH, 2% C2H3N; mobile phase B: 0.1% HCOOH, 90% C2H3N. The gradient settings of the liquid phase are 0–40 min (3% ~ 19% B), 40–52 min (19% ~ 29% B); 52–56 min (29% ~ 80% B); 56–60 min 80% B), and the flow rate is set to 400 nL/min. The ion source (2.0 kV voltage) was ionized and then analyzed by the QExactive Plus mass spectrometry. The parent ions and secondary fragments were detected and analyzed by the high-resolution Orbitrap. The raw data obtained from the mass spectrometry operation were analyzed according to the MS data analysis and evaluation method reported (Liu et al. 2024; Soggiu et al. 2016).

Bioinformatics analysis

The identified phosphorylation modification sites correspond to the protein that were analyzed using the eggnog – mapper software (v2.0) (http://eggnog-mapper.embl.de) for Gene Ontology annotation. Using the KAAS online annotation tool (https://www.genome.jp/tools/kaas) and KEGG Mapper analysis tool (https://www.genome.jp/kegg/mapper), a molecular regulatory network was constructed based on the corresponding proteins of the previously screened phosphorylation modification sites to obtain a clear and concise KEGG enrichment distribution bubble map. In this book, the MoMo analysis tool (http://meme-suite.org) based on the motif-x algorithm is used to analyze the motif features of modification sites. The parameter requirements are as follows: the number of peptides in the form of characteristic sequences is greater than 20, and the statistical test p-value is less than 0.000001.

Data processing and statistical analysis

Each sample was analyzed by three biological replicates and three technical replicates. The difference between the experimental group and the control group was analyzed by a paired t test in a statistical software SPSS (Version11.0). The significance level was set at a p-value of less than 0.05 or 0.01.

Results

Effects of acid stress on cell morphology of A. acidoterrestris

Effects of acid stress on cell morphology and survival rate of A. acidoterrestris were studied before (Liu et al. 2024). In order to further investigate the survival status of A. acidoterrestris under stress conditions, the control group and the bacterial cells treated for 20 min and 30 min at their corresponding pH 2.0, pH 2.5, and pH 3.0 were monitored and imaged under the scanning electron microscopy. It can be interpreted from (Fig. 1) that the surface of the bacteria in the control group was smooth, rod-shaped, and had no wrinkles. Compared with the control group, there was no significant difference in the morphology of the bacterial cells treated with acid at pH 3.0 for 20 min or 30 min, but most of the cells showed compression and shrinkage when treated with pH 2.0 for 20 min and 30 min and pH 2.5 for 30 min. According to cell survival rate and cell morphological changes, it was suggested that A. acidoterrestris bacterial cells were alive and the protein expression levels in the cells may have changed when the cells were treated with acid at pH 2.5 for 20 min. In view of this, this study selected samples treated at pH 2.5 for 20 min for a subsequent phosphoproteomic analysis.

Fig. 1.

Fig. 1

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Cell morphology of A. acidoterrestris under acid stress. A–B pH 4.0 (control) 20 min, 30 min; C–D pH 2.0 20 min, 30 min; E–F pH 2.5 20 min, 30 min; G–H pH 3.0 20 min, 30 min. Legend: The electron microscope image of this experiment was taken under the condition of SU10000 20 kV 9.6 mm × 5.00kSE (U)

Identification and quantification of phosphorylated proteins under acid stress

The acid-treated A. acidoterrestris was subjected to phosphorylation modification proteomics analysis. A total of 805 phosphorylation modification sites on 380 proteins were identified, and 509 sites of 280 proteins displayed quantitative information. Based on the screening criteria of localization probability > 0.75, 635 phosphorylation modification sites located on 351 proteins were finally identified, and 460 sites of 269 proteins contained quantitative information. The data was used for subsequent bioinformatic analysis.

Screening of acid stress–related phosphorylation differential proteins

The criteria for screening differentially expressed proteins in this paper were as follows: when the P-value < 0.05, the change of differential expression was more than 1.3 times as significantly upregulated and less than 1/1.3 times as significantly downregulated. The phosphorylation levels of 44 sites of 40 proteins in the acid treatment group changed significantly; the modification level of 23 sites (the phosphorylation modification ratio of serine, threonine and tyrosine was 14:8:1) was upregulated; the modification level of 21 sites (the phosphorylation modification ratio of serine, threonine and tyrosine was 12:8:1) was downregulated (Table 1 and Fig. 2). The upregulated proteins were mainly N007_03770 (predictive phosphoglycerate mutase), EF-Tu (elongation factor Tu), N007_14990 (imaginary protein), GatB [aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit B], N007_03016 (stage 0 sporulation protein A homolog), N007_05905 (pyruvate kinase), N007_21270 (predictive ABC transporter permease), N007_06630 (predictive protein translocase subunit YidC), and N007_16190 (cell division protein ZapA). The downregulated proteins were mainly GpmI (2,3-diphosphoglycerate-independent phosphoglycerate mutase), rpsC (30S ribosomal protein S3), AtpA (ATP synthase subunit α), Clp family proteases, N007_02735 (predictive Bacillusthiol biosynthetic deacetylase BshB2), N007_19330 (cysteine synthase), N007_03105 (predictive serine/threonine protein kinase), and N007_11495 (predictive two-component system sporulation sensor kinase A).

Table 1.

Differentially protein information

Gene name Protein description A/C ratio Regulated type
N007_03770 Phosphoglycerate mutase (predicted) 6.355 Up
tuf Elongation factor Tu 3.079 Up
N007_14990 Uncharacterized protein 2.434 Up
gatB Aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit B 2.367 Up
N007_10430 Superoxide dismutase 2.182 Up
N007_03610 Stage 0 sporulation protein A homolog 2.12 Up
N007_01705 6-phosphogluconate dehydrogenase 1.894 Up
rplI 50S ribosomal protein L9 1.87 Up
pyrE Orotate phosphoribosyltransferase 1.838 Up
N007_14500 Glutamate dehydrogenase 1.788 Up
sat Sulfate adenylyltransferase 1.757 Up
N007_14500 Glutamate dehydrogenase 1.749 Up
N007_11120 Peptidase U32 1.681 Up
N007_05905 Pyruvate kinase 1.642 Up
N007_21270 ABC transporter permease (predicted) 1.575 Up
N007_06630 Protein translocase subunit yidC(predicted) 1.564 Up
rpsJ 30S ribosomal protein S10 1.473 Up
rplK 50S ribosomal protein L11 1.457 Up
N007_00440 SBP_bac_5 domain-containing protein 1.452 Up
N007_16190 Cell division protein ZapA 1.448 Up
N007_06920 Riboflavin biosynthesis protein 1.362 Up
N007_09400 HTH gntR-type domain-containing protein 1.338 Up
N007_07035 Chemotaxis protein CheA (predicted) 1.333 Up
N007_03105 Serine/threonine protein kinase (predicted) 0.752 Down
N007_09400 HTH gntR-type domain-containing protein 0.742 Down
N007_11495 Two-component system sporulation sensor kinase A (predicted) 0.738 Down
N007_00095 S1 motif domain-containing protein 0.722 Down
N007_12235 Uncharacterized protein 0.71 Down
N007_03105 Serine/threonine protein kinase (predicted) 0.693 Down
clpX ATP-dependent Clp protease ATP-binding subunit ClpX 0.67 Down
N007_07615 ATP-dependent Clp protease ATP-binding protein 0.651 Down
glmU Bifunctional protein GlmU 0.635 Down
ndk Nucleoside diphosphate kinase 0.626 Down
N007_02680 HPr domain-containing protein 0.606 Down
rplN 50S ribosomal protein L14 0.595 Down
N007_04395 Short-chain dehydrogenase 0.562 Down
tuf Elongation factor Tu 0.529 Down
N007_02680 HPr domain-containing protein 0.485 Down
tuf Elongation factor Tu 0.475 Down
N007_19330 Cysteine synthase 0.401 Down
N007_02735 Bacillithiol biosynthesis deacetylase BshB2 (predicted) 0.389 Down
atpA ATP synthase subunit alpha 0.385 Down
rpsC 30S ribosomal protein S3 0.382 Down
gpmI 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 0.345 Down
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Fig. 2.

Fig. 2

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Acid stress differential phosphorylation protein volcano plot

Bioinformatics analysis

Differential phosphorylation sites corresponding to GO secondary annotation classification of proteins

GO can be used to annotate various properties of genes and gene products. In this study, the distribution of proteins corresponding to differential phosphorylation sites in GO secondary annotation was statistically analyzed. The results showed that those differential phosphorylated proteins are belonging to different biological processes. The differential proteins are mainly involved in cellular processes (13.39%), metabolic processes (12.60%), growth (4.72%), and stimulation responses (3.94%). Some other proteins corresponding to cell composition are mainly related to cells (11.81%), intracellular (10.24%), and macromolecular complex related (6.30%); additionally, there are still some differential phosphorylated proteins classified by molecular function that mainly include catalytic activity (11.81%), combination (7.09%), and structural molecular activity (3.93%) (Fig. 3).

Fig. 3.

Fig. 3

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Statistical distribution of proteins corresponding to different modification sites in GO secondary classification

KEGG enrichment analysis of proteins corresponding to differential phosphorylation modification sites

KEGG is a knowledge base for systematic analysis of gene function, which can link genomic information with high-order functional information. In this paper, through the enrichment analysis of metabolic pathways of proteins corresponding to differential phosphorylation modification sites, one pathway was significantly enriched, namely, the quorum sensing pathway (Fig. 4). The differential phosphorylated proteins were mainly concentrated in the quorum sensing pathways (Table 2). Among them, three differential phosphorylated proteins were related to the quorum sensing pathway. The three upregulated proteins were N007_00440 (SBP_bac_5 domain-containing protein), N007_03610 (stage 0 sporulation protein A homolog), and N007_06630 (protein translocase subunit YidC]; there were no downregulated proteins.

Fig. 4.

Fig. 4

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KEGG enrichment distribution of proteins corresponding to different phosphorylation sites

Table 2.

KEGG pathway enrichment distribution of proteins corresponding to different phosphorylation sites

KEGG pathway Mapping  − log10, (P value) Related proteins
Quorum sensing (map02024) 3 1.31 N007_00440, N007_03610, N007_06630
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Protein modification motif (Motif) analysis

Protein motif analysis is a statistical analysis of the amino acid sequence before and after all the phosphorylation sites, and summarizes the trend of the amino acid sequence in the region where the phosphorylation site occurs. The sequence characteristics of the modified sites were found, and then the enzymes related to the modification were interpreted or determined. Motif-X was used to perform motif enrichment analysis on the identified data, and one serine enrichment motif sequence was found (Table 3). The serine-centered Motif-X is mainly MS (x position represents irregular amino acids), while MS does not find the corresponding kinase, which is likely to be a new motif sequence (Fig. 5).

Table 3.

Motif analysis of proteins corresponding to phosphorylation modification sites

Motif Motif score Foreground Background Fold increase
Matches Size Matches Size
xxxxxM_S_xxxxxx 16.00 43 348 1613 64948 5.0
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Fig. 5.

Fig. 5

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Motif analysis of phosphorylation sites

Discussion

The response of A. acidoterrestris to external acid stress is a complex mechanism (Bostock and Quatrano 1992; Guan and Liu 2020; Zhang et al. 2012). It is still unknown how A. acidoterrestris senses and transmits pH signals under acid stress, and the mechanism by which intracellular pH homeostasis is maintained. Protein phosphorylation is the central link in almost all signal transduction pathways in cells. Here, our study applied quantitative phosphoproteomics towards A. acidoterrestris and tried to find a link of differential phosphoproteomics and acid stress response, which could help to unravel the response mechanism of A. acidoterrestris to acid stress.

Our result showed the three phosphorylation sites (Ser 34, Thr 229, Thr 29) of elongation factor (EF-Tu) of A. acidoterrestris were changed under acid stress. The phosphorylation level of Thr 229 was upregulated, and the phosphorylation levels of Ser 34 and Thr 29 were downregulated. This study revealed a differential phosphorylation pattern, wherein certain sites on the same protein exhibited an increase in phosphorylation levels, while other sites exhibited a concomitant decrease in phosphorylation level. Therefore, the diversification of phosphorylation sites of EF-Tu plays a different role in the acid stress of A. acidoterrestris. Studies have found that the EF-Tu has a similar function as a molecular chaperone in the process of protein folding, which can improve the accuracy of protein translation (Hartl et al. 2011). This function allows the EF-Tu to protect the protein structure and maintain its function under environmental stress. When facing acid stress, the upregulation of EF-Tu expression can ensure the correct folding or assembly of polypeptides. In our study, the EF-Tu may respond to acid stress signals through differential phosphorylation. The regulation of the phosphorylation site of the EF-Tu helps A. acidoterrestris to complete protein synthesis under an acid stress environment. It hints that significant protein expression changes of EF-Tu in the acid stress condition indicate that it might play an important role in the acid stress response.

In addition, the expression analysis results of differentially phosphorylated proteins after acid stress of A. acidoterrestris showed that the expression of a phosphorylation site (Ser 271) of Spo0 A was significantly upregulated by 2.12 times. The KEGG enrichment analysis showed that it was significantly enriched in the Quorum sensing pathway. It is well-known that Spo0 A is a key transcriptional regulator in the process of bacterial spore formation. By controlling the expression of a series of genes related to spore formation, Spo0 A regulates the transition of bacteria from growth state to spore formation state and helps bacteria to resist harsh environments by forming spores. Our work found the upregulation of Spo0 A, which is consistent with its role that helps bacteria survive under stress conditions. There was a report found that bacteria will form spores with strong stress resistance when they encounter environmental stresses such as high temperature, low pH, and low temperature (Errington 2003).

Recent studies have demonstrated that the transcriptional regulation of Spo0 A in Bacillus subtilis is intricately linked to the quorum sensing (QS) mechanism (Mirouze et al. 2011). Spo0 A protein has been shown to sense fluctuations in cell density via the QS pathway, thereby modulating a variety of physiological processes. This regulatory interplay has been extensively investigated within the Rap-Phr system and the double-layer quorum sensing system in B. subtilis (Cui et al. 2019). The Spo0 A protein could sense the change of external pH through the quorum sensing pathway and form spores, which plays an important role in the signal transduction process of A. acidoterrestris in response to acid stress. In our study, the analysis of differential phosphorylated protein expression after A. acidoterrestris acid stress showed that the phosphorylation level of a two-component system sporulation sensor kinase A protein was downregulated and the phosphorylation level of YidC protein was upregulated. Studies have found that two-component system sporulation sensor kinase A can sense the changes of the external environment and regulate the formation of spores (Sarwar and Garza 2016). It can further regulate the phosphorylation level of Spo0 A and increase its phosphorylation level, which helps bacteria to produce spores to resist acid stress. It is speculated that the two-component system sporulation sensor kinase A and the quorum sensing pathway may be closely related to the perception of extracellular pH level in A. acidoterrestris. The two-component system sporulation sensor kinase A may act as a signal molecule. When a two-component system sporulation sensor kinase A is dephosphorylated, it can activate or promote the phosphorylation of Spo0 A protein, thereby affecting its role on the spore production. Moreover, the interaction between the two-component system sporulation sensor kinase A and Spo0 A may contribute to the dynamic balance of intracellular signaling pathways, ensure that cells respond appropriately to external stimuli, help maintain the intracellular pH homeostasis of A. acidoterrestris, as well as maintain ion homeostasis to resist acid stress, and thus help A. acidoterrestris survive under acid stress. Additionally, YidC protein is located in the bacterial inner membrane and is responsible for inserting the protein into the inner membrane, which is essential for plasma membrane biogenesis in the bacterial plasma membrane. It has been found that yidC gene product (SpoIIIJ) was necessary for the transport of sporulation-related proteins in B. subtilis (Shiota et al. 2023). Therefore, bacterial spores are not only a product of resisting harsh environments, but also spore formation-related proteins that are likely to be closely related to regulate spore-forming microorganisms to better adapt to external stress environments. Based on our results, Spo0 A and YidC proteins could be closely related to the spore formation of A. acidoterrestris under acid stress, and the secreted spore formation–related proteins may play an important role in the signal transduction and quorum sensing of A. acidoterrestris in response to acid stress.

Pyruvate kinase (PK) is one of the key enzymes in the glycolysis pathway. Pyruvate kinase can transfer the phosphate group of phosphoenolpyruvate (PEP) to ADP, thereby producing ATP. The activity of pyruvate kinase can be affected by a variety of regulatory mechanisms, including phosphorylation and dephosphorylation. Bacterial resistance to acid stress is an energy-intensive process involving multiple levels of physiological and molecular mechanisms. Energy consumption is to protect the integrity of cell structure, maintain metabolic activity, and ensure that bacteria can survive and grow in an acidic environment. The phosphorylation level of a phosphorylation site (Thr 537) of pyruvate kinase was upregulated (phosphorylated) in A. acidoterrestris under acid stress. Chen and co-workers found that higher intracellular ATP was maintained in an acid stress environment, which assisted L. lactis to resist acid stress by providing energy for cells (Chen et al. 2017). It was reported that maintaining high cell ATP in an acid stress environment is to provide energy for cells to help bacteria resist acid stress (Ryssel et al. 2014; Xie et al. 2004). Therefore, according to our research results, we speculate that when the energy demand of A. acidoterrestris increases under acid stress conditions, the pyruvate kinase may be activated by phosphorylation to accelerate the glycolysis process and produce more ATP. The increase of the pyruvate kinase phosphorylation level could increase the intracellular ATP of bacteria to improve the acid stress resistance of A. acidoterrestris cells in the form of energy supply, which is essential for cells to maintain pH homeostasis under acid stress. It may also be through the purine metabolic pathway to resist acid stress, which provides a new layer for further study of the purine metabolic pathway to improve the acid resistance of A. acidoterrestris.

It is noticed that ABC transporter permease is a part of ATP-binding cassette transporter (ABC transporter). The function of ABC transporter permease is mainly to use the energy of ATP to transport substances across the membrane. Our work found that under acid stress, one phosphorylation site (Thr 594) of ABC transporter permease was upregulated by 1.575 times in A. acidoterrestris. Zhu et al. (2019) found that overexpression of specific ABC transporter genes can enhance the tolerance of Lactococcus lactis to acid stress. To maintain the necessary equilibrium conditions for cell survival under acid stress, the transport of various substrates (including sugars, peptides, amino acids, ions, and vitamins) is required, which is performed by transporters on the cell membrane. These transporters provide power for the transmembrane transport of various substrates through ATP binding and hydrolysis. Our results showed that under acid stress, the cells of A. acidoterrestris may activate ABC transporter permease through specific kinases, thereby increasing its phosphorylation level and enhancing its transport capacity. Under acid stress, cells may need to adjust their metabolic pathways and energy distribution to maintain the stability of the intracellular environment. The increase of phosphorylation level of ABC transporter permease may be part of the adaptive adjustment of cells, which is helpful for the survival of A. acidoterrestris in an acid stress environment.

In summary, we speculate that the up-regulation of pyruvate kinase protein phosphorylation level might help to increase the intracellular ATP in the regulation of acid stress resistance mechanism of A. acidoterrestris; the upregulation of ABC transporter permease protein phosphorylation level should be the use of ATP energy transmembrane transport substances, which is conducive to the survival of A. acidoterrestris under acid stress. There may be a regulatory relationship between this pyruvate kinase and ABC transporter permease, but more experimental data are needed to determine whether there is a synergistic relationship between the increase of ABC transporter permease and pyruvate kinase protein phosphorylation levels.

The regulatory mechanisms and pathways involving phosphorylated proteins in A. acidoterrestris were systematically analyzed using bioinformatics approaches, elucidating the molecular mechanisms underlying acid stress regulation at the level of post-translational protein modifications. The response of A. acidoterrestris to external acid stress is a complex process. EF-Tu, two-component system sporulation sensor kinase A, Spo0 A, Yidc, pyruvate kinase, and ABC transporter permease protein were identified as key regulatory proteins implicated in acid stress response. These proteins are involved in the bacterial stress response to external acidic environments through the quorum sensing pathway and the purine metabolic pathways, playing crucial roles in this process. However, the intricate coordination of these acid stress response phosphorylated proteins with the quorum sensing and purine metabolic pathways to maintain pH homeostasis within bacterial cells warrants further investigation. This line of inquiry provides a theoretical and experimental clues for further unraveling the mechanisms underpinning the remarkable acid resistance of A. acidoterrestris.

Author contributions

LY: data curation, methodology, writing—original draft. WK: investigation. JL:funding acquisition, methodology, writing—review and editing. SR: methodology, investigation. RJ: investigation, methodology, validation. WY: data curation, projection administration.

Funding

This work was supported by the National Natural Science Foundation of China (Grant numbers 31771949, awarded to LJ) and Program for Science and Technology Innovation Talents in Universities of Henan Province (Grant numbers 17HASTIT037, awarded to LJ).

Data availability

Website of protein data: https://www.ebi.ac.uk/pride/archive/projects/PXD058649/private

Accessing number: PXD058649.

Declarations

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare no competing interests.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Website of protein data: https://www.ebi.ac.uk/pride/archive/projects/PXD058649/private

Accessing number: PXD058649.

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