Abstract

   In the food industry, food spoilage caused by spores is a pressing
   scientific challenge that needs to be addressed urgently, and spore
   germination is a key approach to solving this problem. Studies have
   shown that peptidoglycan-induced spore germination represents a novel
   mechanism of action, which can bind to the PASTA domain of the
   serine/threonine kinase PrkC. However, the signaling mechanism of
   peptidoglycan-induced spore germination remains unclear. This study
   focuses on Bacillus subtilis, using pull-down experiments to screen for
   proteins interacting with PrkC. There are 80 interaction proteins of
   PrkC that were identified in the spore. GO analysis reveals that
   PrkC-interacting proteins in the spore are mainly involved in metabolic
   processes, cell part and catalysis. KEGG results indicate that
   PrkC-interacting proteins in the spore are mainly involved in RNA
   degradation, quorum sensing, oxidative phosphorylation, etc.
   Additionally, proteins are categorized into six groups by function
   based on events that may be associated with post-germination triggered
   by peptidoglycan-induced activation of the PrkC signaling pathway,
   including “stimulate translation initiation” and “ATP synthesis and
   energy metabolism”. The experimental results provide a theoretical
   basis for further elucidating the signaling mechanism of PrkC,
   revealing the signaling pathway of peptidoglycan-induced spore
   germination, and identifying targeted inducers and repressors of spore
   germination.

   Keywords: Bacillus subtilis, spore germination, PrkC, interaction
   protein

1. Introduction

   The spores of Bacillus, as the resting bodies, exhibit no metabolism
   and possess strong resistance to conditions and substances such as
   heat, radiation, dryness, and toxic chemicals. They can persist in the
   environment for extended periods, even years [[36]1]. However, spores
   sense the environment and respond to favorable conditions by
   germinating and restoring cell growth [[37]2]. Currently, research on
   spores primarily focuses on their germination mechanism, as germinated
   spores lose their resistance and become more susceptible to killing.
   Many Bacillus spores can cause food spoilage, food poisoning, and human
   diseases [[38]3,[39]4]. The dormant spores are non-pathogenic, and only
   synthesize toxins when they germinate and grow into vegetative cells
   [[40]5]. Therefore, inhibiting spore germination or rapidly inducing
   germination of all spores holds significant research importance.

   When the external environment becomes suitable, nutrients or
   non-nutrients can be utilized, and the spore exits dormancy to rapidly
   form a vegetative reproductive body [[41]6]. In this process, spore
   germination is known as the key step. Spore germination is induced by a
   series of nutrient molecules, including amino acids, sugars, and cell
   wall peptides, which bind to receptors on the spore inner membrane and
   interact to activate downstream signaling substances, guiding spore
   resuscitation [[42]7]. In the spore germination pathway, germination is
   induced mainly by nutrients and non-nutrients.

   Previous studies have shown that spores possess a non-nutrient
   germination pathway through the PrkC protein kinase, which located on
   the inner membrane of B. subtilis spores can induce spore germination
   through binding to peptidoglycan (PGN) [[43]8]. This germination
   pathway exists in the genera Bacillus and Clostridium, and is a new
   signal transmission pathway that is relatively independent of
   nutritional germination. Studies have shown that ΔprkC spores still
   responded to the nutrient germinant L-alanine and to the chemical
   germinant Ca^2+-dipicolinic acid (Ca-DPA). Spores with low expression
   levels of germination receptors (GRs), activated by L-alanine and AGFK
   (a mixture of l-asparagine, d-glucose, d-fructose, and potassium ions),
   can germinate via the PrkC pathway [[44]9]. This indicates that during
   the germination process of spores, the PrkC receptor germination
   pathway is independent and can work together with the traditional
   nutritional receptor germination pathway. Although superdormant spores
   cannot be induced to germinate by GRs, they can be induced to germinate
   by PrkC receptors [[45]10]. The PrkC-mediated germination pathway only
   a single identified receptor, represents a promising and tractable
   target for controlling spore outgrowth in food industry applications.
   Therefore, the study of the mechanism of action of the PrkC germination
   pathway is of great significance, which can improve the mechanism of
   spore germination and provide a theoretical basis for more effective
   spore killing in food industry.

   PGN is a crucial cell wall polymer for bacteria, consisting of
   N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNar) sugar
   chains linked by β-1,4 glycosidic bonds and connected by short peptides
   [[46]11]. Depending on the third amino acid in its peptide chain, PGN
   is classified into Lys-type and meso-diaminopimelic acid (DAP)-type.
   The muropeptide fragment of PGN can bind to the PASTA (consisting of 3
   β sheets and an α helix) [[47]12] domain of the serine/threonine kinase
   PrkC, activating the intracellular kinase functional domain and
   inducing spore germination [[48]8,[49]13]. Notably, it is the DAP-type
   muropeptide, rather than the Lys-type muropeptide, that plays a role
   [[50]8]. The recognition process of PrkC for DAP-type peptidoglycan
   fragments is semi-mediated by DAP. DAP-type peptidoglycan can interact
   with an arginine (Arg) residue located at position 500 on the PASTA
   domain of the C-terminal of PrkC. Furthermore, mutation of the arginine
   residue completely inhibits the binding of muropeptide to PrkC
   [[51]13]. However, only a few studies have reported molecules
   downstream of PrkC-mediated spore germination. The elongation factor G
   (EF-G) in B. subtilis possesses ribosomal GTPase activity and can be
   phosphorylated by PrkC, thereby affecting the germination process of
   spores, and this modification is likely necessary for germination in
   response to PG. Elongation Factor Tu (Ef-Tu) also as a substrate of
   PrkC and phosphorylated by PrkC [[52]14,[53]15]. In Bacillus anthracis
   spores, the enolase (Eno) is phosphorylated by the conserved PrkC which
   decreases the catalytic activity of Eno, thereby controlling the
   overall spore germination process [[54]16]. Another study showed that
   phosphoglycerate mutase (Pgm) is also regulated by PrkC
   phosphorylation, which is important for spore germination [[55]17]. The
   specific details of the downstream signaling pathway mediated by PrkC
   are currently unclear. Therefore, this study will use pull-down
   experiments to screen interaction proteins of PrkC kinase in spores,
   and establish the interaction network of PrkC kinase. These results
   could provide reference for revealing the mechanism of PrkC protein in
   inducing the spore germination pathway.

2. Materials and Methods

2.1. Strain and Plasmid

   The Bacillus subtilis 168 and PY79 strains are preserved in our
   laboratory. The prkC gene was amplified from Bacillus subtilis str. 168
   genomic DNA using primers PrkC-F
   (5′-CAGCAAATGGGTCGCCTCGAGGTGCTAATCGGCAAGCGGATCAGC-3′) and PrkC-R
   (5′-GTGGTGGTGGTGGTGGCGGCCGCTTCATCTTTCGGATACTCAATGGT-3′). Both the
   purified PCR product and pGEX-4T-1 vector were digested with NcoI and
   XhoI. Ligate prkC gene and plasmid by Gibson assembly, and validated by
   Sanger sequencing.

   B. subtilis 168 were cultured in LB broth medium until an optical
   density at 600 nm (OD[600]) of 1–2. Spores were grown for around 1 day
   on DSM medium and purified using the Histodenz gradient centrifugation
   method [[56]18]. Spores were kept at −20 °C.

2.2. Induced Expression of PrkC Protein

   The correctly sequenced plasmids were transferred into E. coli
   expression strain BL21, respectively. Positive monoclonal plaques were
   selected and inoculated into 5 mL of LB medium containing the
   corresponding antibiotics. The culture was incubated overnight at 37 °C
   and 220 rpm. The bacterial solution was inoculated into 200 mL of LB
   liquid medium containing the corresponding antibiotics at a volume
   ratio of 1:100, and incubated at 37 °C and 220 rpm until the OD[600]
   reached 0.6–0.8. IPTG was added to the bacterial solution to a final
   concentration of 0.1 mM, and the culture was induced at 22 °C for 6–7
   h. The bacterial solution was transferred to a new centrifuge tube,
   centrifuged for 10 min at 4 °C, and the supernatant was discarded. A
   buffer solution (100 mM Tris-HCl pH 7.5, 150 mM NaCl) was added to wash
   the bacterial cells, which were then centrifuged at 5000× g for 10 min
   at 4 °C, and the supernatant was discarded.

2.3. Purification of PrkC Protein

   The precipitate was resuspended in 3 mL of lysis buffer (100 mM
   Tris-HCl pH 7.5, 150 mM NaCl, 1 mM PMSF). Following complete
   resuspension, ultrasonic disruption was performed at an output power of
   270 W using 6–8 cycles of 20 s pulses interspersed with 40 s cooling
   intervals. The lysate was centrifuged at 12,000× g for 30 min at 4 °C,
   and the supernatant was transferred to a fresh centrifuge tube. For
   His-tagged protein purification, a Chelating Sepharose Fast Flow nickel
   affinity chromatography column was employed. Briefly, 2 mL of resin
   slurry was packed into a column and equilibrated with 10 mL of lysis
   buffer. The clarified supernatant was loaded onto the equilibrated
   column and incubated with the resin at 4 °C for 2 h. The flow-through
   was collected, and the column was sequentially washed with 10 column
   volumes of wash buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 50 mM
   imidazole) to remove nonspecifically bound impurities. Target protein
   elution was achieved using elution buffer (100 mM Tris-HCl pH 7.5, 150
   mM NaCl, 300 mM imidazole), with five 2-mL fractions collected. Protein
   concentrations in the eluted fractions were quantified via Bradford
   assay (Coomassie Brilliant Blue G-250), and purification efficiency was
   assessed by SDS-PAGE analysis (12% resolving gel, Coomassie R-250
   staining).

2.4. Pull-Down Experiment

   Fusion-expressed GST empty-vector and GST-PRKC bacteria were obtained,
   treated with PDS solution, vortexed, and briefly centrifuged. After
   removal of the supernatant, the pellet was rinsed, resuspended in lysis
   buffer containing protease inhibitors, and lysed by ultrasonication.
   The lysate was centrifuged, and the supernatant was collected as the
   input control. Pre-equilibrated glutathione resin was incubated with
   different protein mixtures to assess interaction specificity. GST resin
   was incubated with spore lysate as a negative control for nonspecific
   binding, GST-PRKC resin was incubated with lysis buffer only to confirm
   that binding requires spore proteins, and GST-PRKC resin was incubated
   with spore lysate as the experimental group. After incubation at 4 °C,
   spores were lysed separately under similar conditions, and their lysate
   was collected. Following incubation, the resin was washed thoroughly to
   remove unbound proteins, and bound proteins were eluted using 20 mM
   freshly prepared reduced glutathione. The eluate was stored at −80 °C
   as the pull-down product. A portion was mixed with loading buffer,
   heated, and analyzed by Western blot.

2.5. Western Blot Analysis

   The input and pull-down products were mixed with an appropriate volume
   of 4× loading buffer and incubated in a boiling water bath for 10 min.
   The mixture was centrifuged at 12,000× g for 3 min at 4 °C, and the
   supernatant was subjected to Western blot analysis. Electrophoresis was
   performed at a constant current of 16 mA per gel until the bromophenol
   blue tracking dye migrated to the bottom of the gel. Following
   electrophoresis, the gel was carefully removed, and a transfer sandwich
   was assembled for electroblotting. Protein transfer was conducted at a
   constant voltage of 100 V for 1 h in an ice-water-cooled
   electrotransfer tank. Upon completion of transfer, the membrane was
   retrieved and placed in separate antibody incubation chambers. Blocking
   was performed by incubating the membrane with 5% (w/v) skimmed milk
   powder or 2% (w/v) bovine serum albumin (BSA) in 1 × TBST for 1 h on a
   horizontal shaker. After discarding the blocking solution, the membrane
   was briefly rinsed with 1 × TBST and transferred to an antibody
   incubation tray. The GST-specific primary antibody (1:6000 dilution in
   1 × TBST) was applied, and the membrane was incubated overnight at 4
   °C. The primary antibody was removed, and the membrane was washed three
   times with 1 × TBST (10 min per wash). A horseradish peroxidase
   (HRP)-conjugated secondary antibody (1:8000 dilution in 1 × TBST) was
   then added, followed by incubation at room temperature for 2 h. After
   three additional TBST washes (10 min each), residual buffer was
   absorbed using filter paper. The membrane was exposed to X-ray film in
   a light-tight cassette for signal detection.

2.6. LC–MS/MS Analysis

   The collected protein solution was digested with trypsin to generate
   peptide segments, which were then dried. Each dried peptide sample was
   re-dissolved in a sample solution containing 0.1% formic acid and 2%
   acetonitrile and subjected to LC–MS/MS analysis with a 100-min LC
   gradient for protein identification and label-free quantification
   (LFQ). Mass spectrometry (MS) data acquisition was performed using an
   Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher
   Scientific, San Jose, CA, USA) coupled to an Ultimate 3000 RSLCnano
   UHPLC system Peptide trapping was conducted on an Acclaim PepMap 100
   nanoViper column (75 μm × 20 mm) at a 5 µL/min flow rate for 5 min,
   followed by separation on an Acclaim PepMap RSLC nanoViper column (75
   μm × 500 mm, C-18, 2 μm, 100 Å, Thermo Fisher) at 300 nL/min and 40 °C.
   The mobile phases consisted of 0.1% formic acid in water (solvent A)
   and 0.1% formic acid in acetonitrile (solvent B). A multi-step linear
   gradient was applied as follows: 5% B (0–4 min), 5% B (5 min), 50% B
   (45 min), 90% B (50 min), 90% B (55 min), and re-equilibration to 5% B
   (65 min). A data-dependent acquisition (DDA) strategy was employed with
   3 s cycles, utilizing specific MS scan parameters. Full MS scans were
   performed in the Q Exactive analyzer at 70,000 resolving power to
   precisely determine peptide mass-to-charge ratios (m/z), Scan range:
   350–1800 m/z. MS2 analysis targeted precursor ions at 70,000 resolving
   power. Selected peptide ions were isolated via quadrupole (0.7 m/z
   window), fragmented using high-energy collision dissociation (HCD, 27%
   normalized collision energy). Additionally, a dynamic exclusion window
   of 30 s was applied to improve spectral acquisition.

2.7. MS Data Analysis and Peptide Identification

   Acquired raw MS data were analyzed using the Bacillus subtilis UniProt
   database (strain-specific entries) with Sequest HT (Proteome Discoverer
   2.5) and MaxQuant (v2.0.3). Full tryptic peptides were searched with up
   to two missed cleavages allowed, and fixed modifications included
   phosphorylation, ubiquitination, methylation, acetylation, and
   glycosylation. Data were filtered for precursor ions with a
   signal-to-noise (S/N) ratio of at least 1.5, and peptide spectrum
   matches (PSMs) were refined using a delta Cn threshold of 0.05.

   To control the false discovery rate (FDR), q-values were calculated at
   the PSM level using Percolator and subsequently at the peptide level
   using the Qvality algorithm. For protein identification, explicit
   statistical criteria were applied: candidate proteins were validated
   using Sum PEP protein scores and accepted as significant if they met a
   strict FDR threshold of 0.01, with an alternative relaxed threshold of
   0.05 provided in order not to filter proteins.

2.8. Bioinformatics Analysis

   Identified proteins were annotated using DAVID (v6.8) with Gene
   Ontology (GO) terms (biological process, molecular function, cellular
   component, and only result FDR p < 0.05 and raw p values < 0.05).
   Proteins were functionally annotated and categorized using the
   SubtiWiki database. ([57]https://subtiwiki.uni-goettingen.de/, accessed
   on 15 February 2025). Pathway enrichment analysis was subsequently
   conducted via the KEGG database ([58]https://www.kegg.jp/, accessed 20
   January 2025).

3. Results

3.1. In Vitro Expression and Purification of PrkC Protein

   A prokaryotic expression vector of GST-tagged PrkC protein was
   constructed, and GST-PrkC protein was purified ([59]Figure 1A). The
   total proteins of B. subtilis spores were extracted, hybridized with
   purified GST-PrkC protein, and the proteins bound to PrkC protein were
   collected using GST antibody protein ([60]Figure 1B). Then, the
   collected proteins were analyzed by liquid chromatography-mass
   spectrometry (LC–MS), and the data of these proteins were compared to
   analyze the signaling pathways and biological functions involved in
   them.

Figure 1.

   [61]Figure 1
   [62]Open in a new tab

   Purification of GST-PrkC and GST pull-down assay. (A) The SDS-PAGE of
   GST- PrkC. Lane Marker: Protein molecular weight marker; Lane 2: Input
   control; Lane 3 and 4: Elution once and twice. (B) Proteins were
   expressed in E. coli BL21(DE3) cells, and bacterial extracts were
   applied onto a glutathione resin. Empty plasmid GST+spore protein and
   GST-PrkC+ lysis buffer act as negative controls. Eluted proteins were
   probed with an anti-GST antibody by Western blotting.

3.2. Screening of PrkC-Interacting Proteins in B. subtilis Spores

   The LC–MS data were analyzed using ProteomeDiscoverer 2.4 against a
   target-decoy database of B. subtilis 168 (UniProt taxonomy ID 224308)
   with stringent filtering (1% FDR, ≥2 unique peptides). The data showed
   that in addition to PrkC protein, there were 79 interacting proteins in
   the spore ([63]Table 1). Gene and protein names were annotated in the
   table.

Table 1.

   PrkC interaction protein in B. subtilis spores.
   UniprotKBc Gene Names Protein Names
   [64]P45867 acdA Acyl-CoA dehydrogenase (EC 1.3.99.-)
   [65]O34324 acoL Dihydrolipoyl dehydrogenase (EC 1.8.1.4)
   (Dihydrolipoamide dehydrogenase) (E3 component of acetoin cleaving
   system)
   [66]P42064 appD Oligopeptide transport ATP-binding protein AppD
   [67]O31788 aprX Serine protease AprX (EC 3.4.21.-)
   [68]P35146 aroD 3-dehydroquinate dehydratase (3-dehydroquinase) (EC
   4.2.1.10) (Type I DHQase) (Type I dehydroquinase) (DHQ1)
   [69]P37808 atpA ATP synthase subunit alpha (EC 7.1.2.2) (ATP synthase
   F1 sector subunit alpha) (F-ATPase subunit alpha) (Vegetative protein
   100) (VEG100)
   [70]P07788 cotA Laccase (EC 1.10.3.2) (Bilirubin oxidase) (EC 1.3.3.5)
   (Spore coat protein A)
   [71]O34656 cotI Spore coat protein I
   [72]P17820 dnaK Chaperone protein DnaK (HSP70) (Heat shock 70 kDa
   protein) (Heat shock protein 70)
   [73]P51831 fabG 3-oxoacyl-[acyl-carrier-protein] reductase FabG (EC
   1.1.1.100) (3-ketoacyl-acyl carrier protein reductase)
   (Beta-Ketoacyl-acyl carrier protein reductase) (Beta-ketoacyl-ACP
   reductase)
   [74]O07639 ftsW Probable peptidoglycan glycosyltransferase FtsW (PGT)
   (EC 2.4.99.28) (Cell division protein FtsW) (Cell wall polymerase)
   (Peptidoglycan polymerase) (PG polymerase)
   [75]P54376 gcvPA Probable glycine dehydrogenase (decarboxylating)
   subunit 1 (EC 1.4.4.2) (Glycine cleavage system P-protein subunit 1)
   (Glycine decarboxylase subunit 1) (Glycine dehydrogenase
   (aminomethyl-transferring) subunit 1)
   [76]P12310 gdh Glucose 1-dehydrogenase (EC 1.1.1.47)
   [77]O07621 hemAT Heme-based aerotactic transducer HemAT
   [78]P21248 hemD Uroporphyrinogen-III synthase (UROS) (EC 4.2.1.75)
   (Hydroxymethylbilane hydrolyase [cyclizing]) (Uroporphyrinogen-III
   cosynthase)
   [79]P13714 ldh L-lactate dehydrogenase (L-LDH) (EC 1.1.1.27)
   [80]O34338 mntB Manganese transport system ATP-binding protein MntB
   [81]O31667 mtnX 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate
   phosphatase (HK-MTPenyl-1-P phosphatase) (EC 3.1.3.87)
   [82]P40406 nagZ Beta-hexosaminidase (EC 3.2.1.52)
   (Beta-N-acetylhexosaminidase) (N-acetyl-beta-glucosaminidase)
   (N-acetylglucosaminidase) (ORF1)
   [83]P38050 pbpF Penicillin-binding protein 1F (PBP-1F)
   (Penicillin-binding protein F) (Peptidoglycan TGase);
   Penicillin-sensitive transpeptidase (DD-transpeptidase)]
   [84]O34798 pdaC Peptidoglycan-N-acetylmuramic acid deacetylase PdaC
   (Peptidoglycan MurNAc deacetylase) (EC 3.5.1.-) (Polysaccharide
   deacetylase PdaC)
   [85]O34819 pelB Pectin lyase (PNL) (EC 4.2.2.10)
   [86]O07617 phoE Uncharacterized phosphatase PhoE (EC 3.1.3.-)
   [87]P40806 pksJ Polyketide synthase PksJ (PKS)
   [88]Q05470 pksL Polyketide synthase PksL (PKS)
   [89]P40872 pksM Polyketide synthase PksM
   [90]O34996 polA DNA polymerase I (POL I) (EC 2.7.7.7)
   [91]P39846 ppsB Plipastatin synthase subunit B (EC 2.3.1.-) (Peptide
   synthase 2) [Includes ATP-dependent tyrosine adenylase 1 (TyrA 1)
   (Tyrosine activase 1); ATP-dependent threonine adenylase (ThrA)
   (Threonine activase)]
   [92]O34507 prkC Serine/threonine-protein kinase PrkC (Ser/Thr-protein
   kinase PrkC) (EC 2.7.11.1)
   [93]O31755 proS Proline-tRNA ligase (EC 6.1.1.15) (Prolyl-tRNA
   synthetase) (ProRS)
   [94]P54528 prpB 2-methylisocitrate lyase (2-MIC) (MICL) (EC 4.1.3.-)
   [95]P00497 purF Amidophosphoribosyltransferase (ATase) (EC 2.4.2.14)
   (Glutamine phosphoribosylpyrophosphate amidotransferase) (GPATase)
   [96]P25994 pyrAB Carbamoyl phosphate synthase pyrimidine-specific large
   chain (EC 6.3.4.16) (EC 6.3.5.5) (Carbamoyl phosphate synthetase
   ammonia chain)
   [97]P16971 recA Protein RecA (Recombinase A)
   [98]P17894 recN DNA repair protein RecN (Recombination protein N)
   [99]P35161 resB Cytochrome c biogenesis protein ResB
   [100]Q03222 rho Transcription termination factor Rho (EC 3.6.4.-)
   (ATP-dependent helicase Rho)
   [101]O31466 rtpA Tryptophan RNA-binding attenuator protein inhibitory
   protein (Anti-TRAP protein) (AT)
   [102]O06714 sbcC Nuclease SbcCD subunit C
   [103]P21458 spoIIIE DNA translocase SpoIIIE
   [104]P17867 spoIVCA Putative DNA recombinase (Stage IV sporulation
   protein CA)
   [105]P40869 spoVAD Stage V sporulation protein AD
   [106]P39627 spsG Spore coat polysaccharide biosynthesis protein SpsG
   [107]P27206 srfA1 Surfactin synthase subunit 1
   [108]P40401 ssuC Putative aliphatic sulfonates transport permease
   protein SsuC
   [109]O32165 sufD Iron-sulfur cluster assembly protein SufD
   [110]P33166 tuf Elongation factor Tu (EF-Tu) (P-40)
   [111]P54334 xkdO Phage-like element PBSX protein XkdO
   [112]P37557 yabO RQC P-site tRNA stabilizing factor (RqcP) (Hsp15)
   (Ribosome-associated protein quality control protein P)
   [113]O34772 ycdC Uncharacterized protein YcdC
   [114]P96605 ydbJ Uncharacterized ABC transporter ATP-binding protein
   YdbJ
   [115]O31557 yfjB Uncharacterized protein YfjB
   [116]P54601 yhcQ Spore coat protein F-like protein YhcQ
   [117]O07529 yhdA FMN-dependent NADPH-azoreductase (EC 1.7.-.-)
   (Azobenzene reductase)
   [118]C0SP94 yhfQ Putative ABC transporter substrate-binding lipoprotein
   YhfQ
   [119]O31607 yjbI Group 2 truncated hemoglobin YjbI (Truncated Hb)
   (trHbO) (Hemoglobin-like protein YjbI) (Truncated BHb)
   [120]O31649 yjdH Uncharacterized protein YjdH
   [121]O34798 yjeA Peptidoglycan-N-acetylmuramic acid deacetylase PdaC
   (Peptidoglycan MurNAc deacetylase) (EC 3.5.1.-) (Polysaccharide
   deacetylase PdaC)
   [122]O07638 ylaN UPF0358 protein YlaN
   [123]O34549 ylbO Uncharacterized protein YlbO
   [124]O34569 yoaA Uncharacterized N-acetyltransferase YoaA (EC 2.3.1.-)
   [125]O34748 yocI ATP-dependent DNA helicase RecQ (DNA 3′-5′ helicase
   RecQ)
   [126]O31993 yolB SPbeta prophage-derived uncharacterized protein YolB
   [127]P50830 yprA Uncharacterized ATP-dependent helicase YprA (EC
   3.6.4.-)
   [128]P45931 yqbO Uncharacterized protein YqbO
   [129]P45942 yqcG Toxin YqcG (DNase YqcG)
   [130]P54463 yqeW Uncharacterized protein YqeW
   [131]P54516 yqhR Uncharacterized protein YqhR
   [132]O05389 yrbE Uncharacterized oxidoreductase YrbE (EC 1.-.-.-)
   [133]O34758 yrrL Endolytic murein transglycosylase (EC 4.2.2.29)
   (Peptidoglycan lytic transglycosylase) (Peptidoglycan polymerization
   terminase)
   [134]O34496 ytpQ UPF0354 protein YtpQ
   [135]C0SPA7 yukB ESX secretion system protein YukB
   [136]O05267 yumB NADH dehydrogenase-like protein YumB (EC 1.6.-.-)
   [137]O34817 yvoA HTH-type transcriptional repressor NagR
   (N-acetylglucosamine utilization regulator)
   [138]P96726 ywqN Putative NAD(P)H-dependent FMN-containing
   oxidoreductase YwqN (EC 1.-.-.-)
   [139]P42296 yxiD Toxin YxiD (DNase YxiD)
   [140]P42299 yxiG Uncharacterized protein YxiG
   [141]P55183 yxjM Sensor histidine kinase YxjM (EC 2.7.13.3)
   [142]Q07835 yxxF Uncharacterized transporter YxxF
   [143]C0SP91 yycJ Exodeoxyribonuclease YycJ (EC 3.1.11.-)
   [144]P45867 acdA Acyl-CoA dehydrogenase (EC 1.3.99.-)
   [145]O34324 acoL Dihydrolipoyl dehydrogenase (EC 1.8.1.4)
   (Dihydrolipoamide dehydrogenase) (E3 component of acetoin cleaving
   system)
   [146]P42064 appD Oligopeptide transport ATP-binding protein AppD
   [147]Open in a new tab

3.3. GO Analysis of PrkC-Interacting Proteins in B. subtilis Spores

   To determine what biological characteristics these interacting proteins
   have, we performed GO analysis on the identified interacting proteins
   in the spores. The results showed ([148]Figure 2) that the
   PrkC-interacting proteins in the spores were involved in numerous
   molecular pathways. The GO functional annotation mainly includes
   biological processes (BP), molecular functions (MF), and cellular
   components (CC). In biological processes, the interacting proteins in
   spore are involved in metabolic processes, stress responses,
   developmental processes, cellular processes, biological regulation,
   localization, signaling pathways, multi-tissue processes and movement.
   Among them, there are more PrkC-interacting proteins involved in the
   metabolic process in the spore, including energy metabolism, nucleotide
   metabolism, and biosynthesis (categorized via SubitiWiki), which may be
   related to the restoration of physiological activity after germination.
   In cellular component, enriched in cell part, membrane and membrane
   part. PrkC localizes to the spore inner membrane and have a
   membrane-spanning sequence. Membrane proteins are more likely to
   interact with PrkC under dormant conditions. Molecular function
   analysis indicated that catalytic activity and binding described the
   majority of proteins in all four comparisons. The detailed functional
   classification analysis is systematically elaborated in the Discussion
   below.

Figure 2.

   [149]Figure 2
   [150]Open in a new tab

   GO analysis of PrkC interaction proteins in B. subtilis spores.

3.4. KEGG Pathway Analysis and Protein–Protein Interaction Analysis of
PrkC-Interacting Proteins in B. subtilis Spores

   In order to deeply analyze the mechanism of action of PrkC-interacting
   proteins, KEGG pathway analysis was carried out. The results showed
   ([151]Figure 3) that in spores, interaction proteins of PrkC kinase are
   mainly involved in RNA degradation, population effect, oxidative
   phosphorylation, etc. More pathways are directed to metabolic processes
   such as amino acid metabolism and biotin metabolism. Consistent with
   GO’s analysis, suggesting that metabolism is important in this process.
   Association analysis of PrkC-interacting proteins in spores showed that
   prkC had direct interactions with ftsW, sbcC, pksJ and dnaK,
   respectively ([152]Figure 4A). These interactions may occur more in
   vegetative cells or in the process of sporulation. Through optimizing
   the protein interaction network by removing non-central proteins and
   those functionally unrelated to dormant spore recovery activity, we
   identified recA and dnaK interacting with prkC. And these two also had
   some direct interactions, and both of them could interact with Tuf
   protein, while RecA protein could also interact with SrfA1 protein
   ([153]Figure 4B). The functions of RecA and DnaK proteins have been
   extensively studied in Bacillus subtilis; however, their roles during
   spore germination remain poorly characterized. Both DNA repair and
   chaperone-mediated folding of newly synthesized proteins are important
   processes for restoring physiological activity following spore
   germination. These proteins may regulate the process of spore
   germination and thus deserve further investigation.

Figure 3.

   [154]Figure 3
   [155]Open in a new tab

   KEGG analysis of PrkC interaction proteins in B. subtilis spores.

Figure 4.

   [156]Figure 4
   [157]Open in a new tab

   The role network diagram of PrkC and its interaction proteins in B.
   subtilis spores. (A) A protein–protein interaction (PPI) network was
   reconstructed using the STRING database. (B) Proteins unrelated to
   germination were pruned. The refined network revealed interaction
   between PrkC and two proteins.

4. Discussion

   Previous studies showed that PrkC mutant spores could not germinate
   under peptidoglycan-induced conditions, while GRs mutant spores could
   germinate, indicating that PrkC and GRs receptors were relatively
   independent in the spore germination pathway. In food sterilization,
   the two can interact with each other, which may produce a better
   sporicidal effect, and this result has been confirmed by the previous
   study of our group [[158]19].Therefore study the downstream mechanism
   of PrkC-mediated germination is important for the guidance of food
   sterilization of spores. In this study, pull-down assay was used to
   screen the interaction protein of PrkC protein in the spores of B.
   subtilis.

   The identification and mapping of protein interactions are critical for
   elucidation of their role in unraveling signaling pathways, metabolic
   networks, and regulatory cascades [[159]20]. There are two main
   approaches for detecting interacting proteins, one measuring direct
   physical interactions and the second measuring interactions among
   groups of proteins. Co-immunoprecipitation (Co-IP) and pull-down assays
   are commonly employed to investigate proteomic interactions [[160]21].
   However, in the absence of specific protein antibodies, pull-down
   assays can serve as in vitro screening tools to initially identify
   unknown protein–protein interactions.

   In spore germination, interaction proteins of PrkC kinase can be
   involved. Previous studies have shown that peptidoglycan can bind to
   the PASTA region of PrkC protein and activate downstream signaling
   molecules, so that the spore germination signal can be transmitted and
   promote spore germination. Functional annotation of the detected
   proteins reveals that the events that may be associated with
   post-germination triggered by peptidoglycan-induced activation of the
   PrkC signaling pathway can be categorized into the following
   categories. (i) “stimulate translation initiation”. Previous studies
   have found that the ribosomal GTPase EF-G is phosphorylated by PrkC,
   which stimulates translation of dormant spores and initiates spore
   germination. EF-G catalyzes the ribosome translocation of the mRNA–tRNA
   complex from the A and P sites to the P and E sites. Before that,
   aminoacyl-tRNA (aa-tRNA) is transported to the A site of the ribosome
   by the GTPase EF-Tu [[161]22]. EF-Tu and EF-G exhibit sequential and
   essential functional coordination during ribosomal cycling. Moreover,
   in vitro PrkC specifically phosphorylates EF-Tu on T384 was previously
   reported [[162]15]. The identification of the interaction of tuf
   (EF-Tu) with PrkC in our data suggests that PrkC-stimulated ribosomal
   activation is critical for spore germination. Several studies have
   found that dormant cells contain large amounts of mRNA and ribosomes
   [[163]23,[164]24]. Our data support that translation stimulation might
   be a fundamental mechanism of exit from dormancy [[165]24]. (ii)
   “peptidoglycan metabolism”. Peptidoglycan cortex-degrading enzymes are
   typically released during the early phase of spore germination.
   Notably, we detected the presence of peptidoglycan synthesis-related
   proteins (e.g., PbpF, PdaC, and YrrL) [[166]25,[167]26] that may be
   activated by PrkC. A large PrkC-dependent effect of the padC gene has
   been demonstrated in vegetative cells [[168]27]. We also detected a
   protein involved in the process of peptidoglycan peptide recovery, NagZ
   [[169]28]. But no proteins associated with peptidoglycan degradation.
   Spore cortex-lytic enzymes essential for germination, such as SleB,
   CwlJ have been alternately demonstrated localized to the outer cortex
   (B) or the spore coat (J). They cannot directly interact with PrkC
   localized on the inner membrane. Thus, the signaling molecules for
   peptidoglycan degradation in the cortex after germination may not
   interact directly with PrkC. The mechanism inducing cortex degradation
   during the second stage of germination may be similar to
   nutrient-induced germination. (iii) “ATP synthesis and energy
   metabolism”. The NADH formation, oxygen uptake, and RNA synthesis were
   initiated immediately after germination [[170]29]. We detected several
   proteins associated with energy metabolism (e.g., Ldh, AcoL, PrpB,
   YvoA, and Gdh) [[171]30,[172]31] and ATP synthase AtpA. PrkC has been
   shown to phosphorylate several metabolic enzymes in vitro [[173]32],
   and initiation of metabolic processes with scarce resources in the
   spore is a prerequisite for spore germination. With the progress of
   germination, the spore requires substantial metabolic supplements.
   Multiple substrates may be involved in energy metabolism. Notably, Gdh
   is a glucose dehydrogenase that is directly involved in glucose
   metabolism and has the possibility of providing direct energy during
   the early metabolism of germination. Although studies have shown that
   neither of the two major glucose catabolic pathways is required for
   triggering Bacillus subtilis spore germination [[174]30], Gdh may be
   important for energy provision. (iv) “nucleotide metabolism”.
   Accompanied by nucleotide metabolism in the early stages of germination
   [[175]33]. We similarly detected a number of proteins involved in
   nucleotide metabolism (e.g., PurF and PyrAB). (v) “DNA repair”.
   Oxidative stress due to full hydration of the spore core during
   germination and activation of metabolism in spore outgrowth can lead to
   the production of reactive oxygen species (ROS) [[176]34]. So DNA
   repair proteins in the spore may be extremely important in spore
   germination. We detected some DNA repair functional proteins including
   PolA, RecA, YprA (MrfA), YycJ, and SbcC. (vi) “ABC transporter”. During
   the early stages of spore germination, the involvement of transporter
   proteins is typically limited due to energy constraints. However, in
   the later stages of germination, numerous transporter proteins are
   recruited to facilitate the restoration of metabolic activity
   [[177]33,[178]35]. We detected ABC transporter AppD, YdbJ, and MntB.
   AppD, an oligopeptide transporter, may be activated by PrkC and
   potentially involved in protein transport processes during the late
   stages of spore germination.

   The IM channel for Ca-DPA, composed of multiple SpoVA proteins (A, C,
   D, Eb, Ea, and F), opens to trigger a rapid release of Ca-DPA following
   spore germination [[179]36]. Previous studies have suggested that
   SpoVAD acts as a similar cytoplasmic plug in the DPA transport complex
   [[180]37]. During nutrient-induced germination, germinant receptor (GR)
   activation initiates cation efflux, which subsequently activates Ca-DPA
   channel opening [[181]38]. SpoVAC acts as a mechanosensitive channel
   and allows the release of Ca-DPA and amino acids [[182]39]. However,
   the mechanism by which muropeptide and PrkC binding leads to downstream
   Ca-DPA release remains unclear. We detected SpoVAD protein interacting
   with PrkC, though phosphorylation of SpoVAD has not been reported.
   Moreover, we detected YqhR (recently designated FigP), which
   oligomerizes with SpoVAF to form ion channels and serves as an
   essential cofactor for 5AF activity [[183]40]. Protein phosphorylation,
   a critical signal transduction process, can induce conformational
   changes and plays a significant role in regulating spore germination
   [[184]41,[185]42]. Further studies are required to determine whether
   PrkC directly phosphorylates DPA channel components.

   We also identified several spore-specific proteins, including SpoIIIE,
   YhcQ, CotI, and CotA. SpoIIIE is known to mediate chromosome
   translocation during sporulation [[186]43], and its role in germination
   has not been reported. YhcQ, CotI and CotA are located on the spore
   coat. Notably, CotI is known as a bacterial spore kinase (BSK). They
   may be able to interact with specific substrates [[187]44]. The tight
   association between some BSKs and glycosyl transferases and predicted
   nucleotide sugar metabolizing enzymes suggests that they may bind or
   phosphorylate one of these reactants [[188]44]. Furthermore, as the
   earliest protein to contact with the signaling molecule, spore coat
   proteins could play a role in the inward transmission of the signaling
   molecule. However, this interaction should not occur on germination
   because of differences in localization in the spore. Additionally, we
   detected several proteins of particular interest in bacterial spores,
   such as the heat shock protein and molecular chaperone DnaK. Chaperones
   have been shown to participate in protein folding during early
   germination stages [[189]45]. And DnaK is phosphorylated and
   dephosphorylated by PtkA and PtpZ in vegetative cells, respectively
   [[190]46]. DnaK may also have a function in regulating protein quality
   in spore formation and germination.

   The mechanism by which muropeptide binding to PrkC activates downstream
   germination events remains largely enigmatic, and our study offers
   critical clues to unravel this process.

5. Conclusions

   In summary, this study successfully identified key proteins interacting
   with PrkC in Bacillus subtilis spores through pull-down assays, mass
   spectrometry, and comprehensive bioinformatics analyses. The findings
   revealed that these interacting proteins are involved in critical
   biological processes, such as metabolic process and are also enriched
   in many metabolic pathways. They were further categorized into six
   functional groups associated with post-germination events triggered by
   the peptidoglycan-induced PrkC signaling pathway, including “stimulate
   translation initiation”, “peptidoglycan metabolism”, “ATP synthesis and
   energy metabolism”, “nucleotide metabolism”, “DNA repair”, and “ABC
   transporter”. In addition, the discussion covers signal mechanisms that
   may induce Ca-DPA release and several proteins that, while crucial in
   vegetative cells, have been less extensively studied in the context of
   spore reactivation. These results enhance the understanding of the
   mechanisms underlying PrkC-mediated germination and provide a
   foundation for future investigations into specific signaling pathways.

Author Contributions

   Conceptualization, K.M., L.D. and X.H.; methodology, K.M. and T.C;
   software, K.M. and T.C.; validation, K.M. and Z.Z.; formal analysis,
   K.M.; investigation, Y.S.; resources, C.F.; writing—original draft,
   K.M., L.D. and X.H.; writing—review and editing, L.D. and X.H.;
   visualization, K.M.; supervision, C.F.; project administration, L.D.
   and X.H.; funding acquisition, C.F. and X.H. All authors have read and
   agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The original contributions presented in this study are included in this
   article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This work was supported by the National Key Research and Development
   Program of China (2022YFD2100404) and National Natural Science
   Foundation of China (31601563).

Footnotes

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References