ABSTRACT
Cellulose fibers from ramie is a kind of functional biological material with multipurpose in the textile industry, which called as “China grass.” The degumming of ramie fibers performed with Bacillus subtilis were regulated by response regulator CitT from CitS/CitT two-component system through specifically functioning on the degradation of component pectin. In this study, comparative proteomic analysis was executed to obtain insights into the sensitivity by which the metabolic network induced by the absence of CitT protein, and to further explore the regulatory mechanism during bio-degumming process of ramie fibers. Results showed that 29 differentially expressed proteins were detected from original strain and mutant strain, which were mainly involved in transmembrane transport system, two-component system, and amino acid metabolism. This study demonstrated that the lack of CitT protein could result in the down-regulation of enzymes in histidine biosynthesis pathway, and the up-regulation of enzyme in arginine degradation pathway. This study is the first time to reveal comprehensive information about the regulatory function of CitT protein in bio-degumming of ramie fibers, and may provide important scientific and technological basis for targeted constructing engineering strains in degumming of ramie fibers.
摘要
苎麻纤维是一种在纺织工业中具有多种用途的功能性生物材料, 被称为“中国草”。枯草芽孢杆菌CitS/CitT双组分系统中的应答调节蛋白CitT通过特异性调控胶质组分果胶的降解实现对苎麻脱胶过程的调控。本研究通过比较蛋白质组学分析, 深入了解因CitT蛋白缺失而诱导的代谢网络敏感性, 并进一步探究苎麻生物脱胶过程的调控机制。结果表明, 从原始菌株和突变菌株中检测到29个差异表达蛋白(DEPs), 主要参与跨膜转运系统、双组分系统和氨基酸代谢等过程。此外, 缺乏CitT蛋白可引起组氨酸生物合成途径中酶表达量的下调, 精氨酸降解途径中酶表达量的上调。本研究首次全面揭示了CitT蛋白在苎麻生物脱胶中的调控作用, 可为定向构建苎麻脱胶工程菌株提供重要的科学技术依据。
Introduction
Natural fibers are vital components of cellulose, which have been used for a great diversity of purposes since ancient times (Yang, Duan, et al. Citation2022). Natural fibers are promising and hopeful alternatives to current synthetic fibers on account of their numerous advantages, such as environmentally friendly and specific properties and performances desirable for a wide range of technological engineering applications (Nair et al. Citation2013). Ramie (Boehmeria nivea (L) Gaud), which produce one of the strongest and longest single plant bast fibers, could be used to make twines, canvas, and clothing fabrics with wide application potential.
However, raw ramie fibers are usual sticking to each other because of the presence of much non-cellulosic compounds (usually called as gums, almost 20–30 wt%), which have hemicellulose (8–16 wt%), pectin (2–6 wt%), lignin (1–4 wt%), and wax (1–2 wt%) (Qu et al. Citation2020). A treatment process defined as “degumming” is necessarily required to remove the gummy non-cellulosic materials closely adhered on the surface of bast fibers, the aim of this procedure is to eliminate the gummy non-cellulosic compounds with keeping the integrality of chemical structures or physical properties of ramie fibers (Xu et al. Citation2022). Degumming is the vital and critical method to isolate cellulosic fibers and prepare bast fibers in the textile industry (J. H. Liu, Guan, and Li Citation2018; Nair et al. Citation2015). Degumming technology has been extensively explored and widely used in extraction of cellulose fibers from ramie, which could be classified as chemical, physical, and biological techniques (Mishra et al. Citation2021; Wu et al. Citation2022; Yang et al. Citation2021). Usually, it is the traditional chemical degumming methods which have been widely applied in textile industrial application. As compared to biological techniques, the chemical degumming methods are time saving, however, the chemical degumming technique is high-polluting and high energy consuming that has resulted in seriously restriction to the development of ramie bast fibers processing industry. Therefore, it is highly rewarding and pressing to explore new technique with improvement of fibers quality for ramie degumming and less environmental pollution. Biological degumming methods, which are considered as low-carbon and green approach compared with conventional chemical degumming, are gradually become more attracting strategies for ramie fibers degumming (Ren et al. Citation2023). Biological technologies include two approaches, that are enzymatic method and microbial method. Nevertheless, enzymatic method demands for the strong specificity of the enzymes for ramie degumming, and it is difficult to form a comprehensive enzymatic system due to the complicated composition of much gums. Therefore, microbial technique is no doubt the most suitable and feasible technology in the degumming process for ramie fibers.
The microbial technology is a metabolic pathway to degrade gummy non-cellulose materials (e.g. pectin, hemicelluloses, and lignin) during the cultivation of various microorganisms (Yang et al. Citation2019). Microbial degumming for ramie fibers, which is a crucial technological way to facilitate the sustainable and green progress of the textile industry for ramie fibers. However, a technical and troublesome bottleneck is existed in this environmentally friendly technology, that the ramie fibers degummed by microbial method usually are subjected to the higher residual gum content. Therefore, it is rather a challenging task to research on the search and construction for efficient engineering strains for such an application. Among the non-cellulose components in ramie bast, pectin can bind other polysaccharide components, the efficient degradation of pectin is not only the guarantee to promote the co-degradation of hemicellulose subsequently, but also the imperative premise to reduce fiber residual gum content and realize industrial application of microbial degumming technology (Li et al. Citation2021). Bacillus subtilis is the abundant and dominant species resource of microorganisms for degumming of ramie fibers, preliminary studies showed that response regulator CitT from CitS/CitT two-component system specifically regulates the pectin degradation pathway during ramie fibers degumming process which is treated with B. subtilis, but the molecular mechanism of CitT to regulate the pectin degradation pathways during degumming of ramie bast fibers is not clear now (Yang, Cheng, et al. Citation2022).
In this study, the regulatory mechanism of the two-component system response regulator CitT on pectin degradation during ramie degumming was explored by proteomic analysis. The research findings of this study will elucidate the regulatory molecular mechanism of CitT protein in pectin degradation during the degumming process of ramie fibers, to support a significant scientific and technological basis for targeted constructing engineering strains with higher efficient pectin degradation ability, and to facilitate the usage of microbial technique in processing industry for degumming of ramie bast fibers.
Materials and methods
Bio-degumming of ramie fibers
For routine use, ramie species used in this study was Zhongzhu No.1. Ramie bast was stripped from the core manually, and dried completely without mildewing. B. subtilis subsp. subtilis strain 168 ΔeglS, a eglS-deletion strain applied in degumming of ramie fibers without cellulase producing during degumming process, was defined as original strain in this study (Yang et al. Citation2019). The citT-deletion strain was constructed on the basis of the original strain in our lab, and was defined as mutant strain in this study (Yang, Cheng, et al. Citation2022). The culturing procedure of these two strains and the degumming process of ramie fibers were executed as previous description (Yang, Cheng, et al. Citation2022). In the present study, a total of 200 g ramie bast were treated by original strain and mutant strain respectively for following analysis. Cells and fermentation broth obtained from the bio-degumming system at the late logarithmic phase (5 h) were selected for proteomic analysis and high performance liquid chromatography (HPLC) determination, respectively; the longitudinal surface morphologies of the ramie fibers after degumming (25 h) were analyzed by scanning electron microscope (SEM, Hitachi SU-3500), and the untreated ramie bast (non-degummed ramie fibers) were taken as the control.
HPLC analysis of fermentation broth
The samples collected from original strain or mutant strain cultured in ramie fibers degumming systems at the late logarithmic phase (after degumming for 5 h) were used for detecting the compositions and contents of monosaccharides by HPLC analysis. The supernatants were collected by centrifuging at 6500 rpm for 10 min and further filtered by 0.22 μm Millipore filters. The standard of monosaccharides were constituted with ribose, mannose, glucuronic acid, rhamnose, N-acetyl-glucosamine, galacturonic acid, N-acetyl-galactose amino, glucose, xylose, galactose, arabinose, and fucose, they were mixed and dissolved in deionized water to make the concentration was 50 µg/ml. Then samples and standard were derived as below: 250 µL samples or standard were transferred into 5 mL tube, then added 250 µL NaOH (0.6 mol/L) and 500 µL PMP-methanol (0.4 mol/L). This system was reacted at 70°C for 1 h, and cooled in cold water for 10 min after reaction. Added 500 µL HCl (0.3 mol/L) for neutralization, further added 1 mL chloroform, vortex oscillation for 1 min, centrifuged at 3000 r/min for 10 min, and carefully collected supernatant. Then repeated extraction for three times, and the supernatants were waited for HPLC analysis.
The HPLC analysis system was performed by Shimadzu LC-20AD liquid chromatograph equipped with SPD-20A UV-VIS detector. The separation was executed on C18 column (Xtimate C18 4.6 × 200 mm 5 µm) which was attached with loop 20 mul. Elution was performed at a 30°C constant temperature with a mobile phase consisting of 0.05 mol/L pH 6.70 KH2PO4 : acetonitrile (ACN) (83 : 17 v/v) at a flow rate of 1 mL/min and detected at a wavelength of 250 nm. The injection volume was 20 µL.
Preparation of whole cell proteins
Cells were harvested by centrifugation under 6000 rpm and 4°C for 10 min, washed three times by pre-chilled PBS (10 mM, pH 7.8). Then, cells were frozen with liquid nitrogen for 10 min. For extraction of proteins, strains were ground into powder by milling with a pre-chilled mortar and pestle. Frozen powders were transferred into 2-mL Eppendorf tubes. The 800-µL extraction buffer (2.4 g sucrose, 0.058 g NaCl, 0.146 g EDTA·2Na, 0.02 g dithiothreitol (DTT), 2.5 mL 0.5 M pH 6.8 Tris-HCl and 2.5 mL 1.5 M pH 8.8 Tris-HCl dissolved in H2O to a constant volume with 10 mL) was added to each sample, then added phosphatase inhibitor and protease inhibitor to make the final concentration 1 mM. The samples were then supplemented with equal volumes of phenol-Tris-HCl (pH 7.8) buffer and mixed for 40 min at 4°C. Further more, the mixtures were centrifuged at 7000 rpm and 4°C for 10 min to obtain supernatants. Five times of 0.1 M ammonium acetate-methanol buffer (pre-chilled at 4°C) were added into the samples, further precipitated at −40°C for more than 12 h. Then the samples were centrifuged at 12,000 g for 10 min to collect precipitation. The precipitation were washed by five times of methanol (pre-chilled at 4°C), centrifuged at 12,000 g and 4°C for 10 min to collect precipitation, then repeated once. The methanol contamination was removed by replacing with acetone and repeated washing step twice. Furthermore, samples were centrifuged at 12,000 g and 4°C for 10 min to collect precipitation, then dried the precipitation at room temperature for 5 min, further dissolved the precipitation in SDS lysis buffer (Beyotime Biotechnology). At last, the supernatants were collected by centrifuging at 12,000 g for 10 min. Protein concentration was tested by BCA assay (ThermoScientific) and stored at −80°C.
Preparation of tryptic peptides and tandem mass tags (TMT) labeling
Protein samples were collected from three biological replicates, 50-μg proteins from cell lysates of each sample were added into lysis buffer to make the concentration reached at 2.5 μg/μL. Added 25 mM DTT to reach a concentration of 5 mM, then mixed and incubated at 55°C for 30 min. After cooling down to room temperature, iodoacetamide were added to make the final concentration reached at 10 mM, mixed and put at the dark environment for 15 min. Then proteins were precipitated by adding six times of precooled acetone, and placed the precipitation at −20°C for more than 12 h. Proteins were collected by centrifuging at 8000 g and 4°C for 10 min. Proteins were redissolved in 100-μL 200-mM triethylammonium bicarbonate (TEAB) buffer, incubated for more than 12 h with Trypsin-TPCK in a Trypsin-TPCK/Protein ratio of 1:50 (w/w) at 37°C (in a water bath). Samples were lyophilized after enzymolysis and stored at −80°C.
For TMT labeling, the lyophilized samples were resuspended in 50-μL 100-mM TEAB buffer. Eighty-eight-microliter ACN were added to TMT reagent (90066, ThermoFisher) at room temperature. Proteins were labeled by adding 41 μL of the TMT label reagent to each sample, then placed the labeled samples at room temperature for 60 min. Then the reaction was terminated by adding 8 µL of 5% hydroxylamine and incubating for 15 min. These labeling peptides were lyophilized and stored at −80°C.
Reversed phase liquid chromatography (RPLC) separation and mass spectrometry (MS) detection
RPLC separation was executed on an Agilent 1100 HPLC System with an Agilent Zorbax Extend-C18 column (5 µm, 150 mm × 2.1 mm). The mobile phases A was added 2% ACN in HPLC water, then adjusted pH to 10 with ammonia; the mobile phases B was added 90% ACN in HPLC water, then adjusted pH to 10 with ammonia. The solvent gradient for reversed phase (RP) separation was set as follows: 0–8 min, 2% B; 8–8.01 min, 2%-5% B; 8.01–48 min, 5%-25% B; 48–60 min, 25%-40% B; 60–60.01 min, 40–90% B; 60.01–70 min, 90% B; 70–70.01 min, 90%-2% B; 70.01–75 min, 2% B. The flow rate was set as 300 μL/min, and separation process was monitored at 210 nm. The tryptic peptides were collected from 8 min to 60 min, then the samples were further lyophilized for MS detection.
MS detection was executed on a Q-Exactive mass spectrometer with a Nanospray Flex source (ThermoFisher, USA). The tryptic peptides were loaded to a RP-C18 precolumn (Acclaim PepMap100 100 μm × 2 cm, ThermoFisher, USA) and separated by a RP-C18 column (Acclaim PepMap RSLC, 75 μm × 50 cm, ThermoFisher, USA) on an EASY-nLC 1200 system (Thermo, USA). The flow rate was set as 300 μL/min, and the linear gradient was set as 75 min: 0–50 min, 2–28% B; 50–60 min, 28–42% B; 60–65 min, 42–90% B; 65–75 min, 90% B. The mobile phase A was 0.1% formic acid (FA) in water and the mobile phase B was 80% ACN/0.1% FA in water. The full MS scans were acquired in the mass range of 300–1500 m/z with a mass resolution of 45,000, the automatic gain control (AGC) target value was 3e6, the max injection time was set as 50 ms. The 20 most intense peaks in MS were further fragmented with higher-energy collisional dissociation (HCD) under normalized collision energy (NCE) of 32. MS/MS spectra were acquired with the resolution of 15,000 with a max injection time of 80 ms and an AGC target of 2e5. The Q-E dynamic exclusion was run under positive mode and set as 30.0 s.
Proteomic data searching
The Q-Exactive raw data was analyzed by software Proteome Discoverer V.2.4 (Thermofisher) thoroughly against an in-house protein database of B. subtilis strain 168, which was downloaded from the Uniprot website (https://www.uniprot.org/). Database search was executed under trypsin digestion specificity. In the database searching, the static modifications were set as TMT (N-term, K) and Carbamidomethyl (C), while the dynamic modifications were set to Oxidation (M) and Acetyl (N-term). The instrument was set to Q Exactive HF, MS1 tolerance was 10 ppm, MS2 tolerance was 0.02 Da. The missed cleavages was set to 2. The global false discovery rate (FDR) was considered as 0.01.
Bioinformatics analysis
Differentially expressed proteins (DEPs) in mutant strain (while compared to original strain) were controlled based on two criteria: (i) fold change < 0.83 or fold change > 1.2; (ii) p < .05 (Luo et al. Citation2021). DEPs were further analyzed on their protein–protein interactions and/or their functional association by searching against the STRING database (http://string-db.org/), the confidence score was set to 0.150, and all DEPs were presented in the map. Metabolic pathways and functional categories of B. subtilis were referenced to the information on the KEGG website (http://www.kegg.jp/). All information of DEPs was listed in Table S1.
Quantitative real-time polymerase chain reaction (qRT-PCR) detection
The qRT-PCR detection were experimented on a StepOne Plus Real-Time PCR System (ABI, Foster, CA, USA) with 2X SG Fast qPCR Master Mix (High Rox, B639273, BBI). Eight genes (yfmC, liaS, liaH, nagP, pelA, dctA, bglP, and citT) were selected and analyzed by qRT-PCR detection to reveal their transcriptional levels. Transcript obtained from the 16S rRNA gene was chose for normalization during qRT-PCR detection process (Park et al. Citation2012). All the primers used in qRT-PCR detection were designed with software Primer version 5.00 and showed in Table S2.
Results and discussion
Morphology of degummed ramie fibers
The surfaces and appearance morphology of ramie fibers were analyzed by the SEM observation after degumming by original strain or mutant strain. The non-degummed ramie fibers were tightly coated with gum; and as compared to ramie fibers degummed by mutant strain, the ramie fibers experiencing original strain degumming were more dispersive, the surface of these fibers was smoother (). Besides, the single fiber breaking strength value of ramie fibers treated with original strain was 24.17 cN (Yang et al. Citation2019). And the gum components in the ramie bast without degumming were mainly consisting of cellulose (78.52%), hemicellulose (12.55%), pectin (4.08%), lignin (2.39%), and water soluble substance (2.46%); the gum components adhered to the degummed ramie fibers with original strain were consisting of cellulose (88.38%), hemicellulose (8.64%), pectin (1.58%), lignin (0.21%), and water soluble substance (1.19%); the gum components adhered to the degummed ramie fibers with original strain were consisting of cellulose (87.52%), hemicellulose (8.78%), pectin (2.15%), lignin (0.28%), and water soluble substance (1.27%). It reflected that the bio-degumming ability of B. subtilis was decreased with the gene knocked-out of citT, and perhaps further illustrated that response regulator CitT was a key regulatory protein during the ramie fibers degumming process (Yang, Cheng, et al. Citation2022).
Figure 1. SEM analysis of the non-degummed ramie fibers (a) and degummed ramie fibers with original strain (b) or mutant strain (c).
HPLC analysis of monosaccharides composition in bio-degumming system
During the degumming process of ramie fibers by microbial technique, B. subtilis could degrade and utilize gummy non-cellulosic materials as the sole carbon source through carbohydrate metabolism pathways (Yang et al. Citation2022). The essence of microbial degumming was that the insoluble non-cellulosic polysaccharides compounds adhering to the ramie fibers were degraded into soluble monosaccharides which could be utilized by bacterial cells after the transmembrane transporting. After deletion of citT, more insoluble non-cellulosic polysaccharides compounds (gum) were remained on the ramie fibers (), which indicated that the degradation of polysaccharides was different between original strain and mutant strain. HPLC analysis was performed to investigate the difference on polysaccharides degradation between these two strains. HPLC analysis confirmed that multiple monosaccharides were produced during degumming of ramie fibers by B. subtilis. Chromatogram obtained by HPLC revealed the existence of 10 monosaccharides including ribose, mannose, glucuronic acid, rhamnose, glucose, galacturonic acid, xylose, galactose, fucose, and arabinose in the ramie fibers degumming systems performed with original strain and mutant strain respectively. N-acetyl-glucosamine and N-acetyl-galactose amino were not detected during microbial degumming process (). Results reflected that there was almost no difference on the monosaccharides composition in bio-degumming system with the deletion of citT. However, the result of chromatographic peak area calculating indicated that there was a big difference of the concentrations of monosaccharides in microbial degumming system between the original strain and the mutant strain ( and ). In the ramie fibers degumming system by microbial technique with original strain, the total concentration of monosaccharides was 102.72 μg/mL, while it was 82.46 μg/mL in the microbial degumming system treated with mutant strain. This result was consistent with the findings of the SEM observation. Moreover, the monosaccharide was defined as high abundant monosaccharide when its concentration was more than 10 μg/mL in the degumming system in this study. The more abundant monosaccharides in microbial degumming system treated with original strain were galactose (51.61 μg/mL), galacturonic acid (23.23 μg/mL), and arabinose (17.79 μg/mL); the more abundant monosaccharides in microbial degumming system treated with mutant strain were rhamnose (29.68 μg/mL), galacturonic acid (15.57 μg/mL), galactose (14.87 μg/mL), and arabinose (13.30 μg/mL). According to other research, the pectin obtained from raw ramie bast had high content of galacturonic acid (75.80%) and low content of neutral sugars such as rhamnose (12.60%), galactose (9.80%), and arabinose (2.10%). Galacturonic acid was the major component of the homogalacturonan region in raw ramie bast pectin, while neutral sugars were mostly from rhamnogalacturonan-I region in pectin (J. Wang et al. Citation2023). The HPLC analysis results probable supposed that more homogalacturonan region in ramie pectin was degraded by original strain and more rhamnogalacturonan-I region in raw ramie bast pectin was degraded by mutant strain. Because raw ramie bast pectin mostly consisted of homogalacturonan region and containing small amount of rhamnogalacturonan-I region, it may eventually lead to the down-regulation of the pectin degradation rate by mutant strain with the deficient in CitT from CitS/CitT two-component. Comparative proteomic analysis is usually executed to obtain insights into the metabolic regulatory mechanism in microorganism under diverse survival environment (S. S. Liu et al. Citation2017; Zhu et al. Citation2019), the internal and intrinsic reasons of CitT protein in regulating pectin degradation were further investigated by proteomic analysis on metabolic level in this study.
Figure 2. Compositions and contents of monosaccharides in bio-degumming system analyzed by HPLC. The fermentation broth was obtained from original strain or mutant strain in ramie fibers bio-degumming system after cultivating for 5 h (the late logarithmic phase).
Table 1. Concentrations of monosaccharides in microbial degumming system.
Proteomics analysis during degumming of ramie fibers
The proteome of original strain and mutant strain (lacking of gene citT) cultured under ramie degumming system was quantitatively examined to globally define the regulation function of CitT protein. Based on the enzymatic digestion of bacterial intracellular proteins, the resulting peptides from original strain and mutant strain samples were isotopically labeled and mixed in equal amounts before RPLC separation and LC-MS/MS detection. A total of 2739 proteins were identified from three biological replicates, and only 14 and 15 proteins were up- and down-regulated respectively in the citT mutant compared with its parental strain. Therefore, these altered proteins were likely to be regulated either directly or indirectly by the CitS/CitT two-component system under ramie degumming system. For special, proteins with higher or lower expression levels in the citT-deletion mutant strain were the potential candidates which was repressed or activated by CitT protein. All DEPs was provided in Table S1, and DEPs were classified into different functional clusters to gain a global insight of CitT-controlled metabolic processes (). Among the CitT-repressed proteins, proteins were required for two-component system, phosphotransferase system (PTS), flagellar assembly, peptidoglycan biosynthesis, energy production and conversion, arginine degradation, as well as bacterial secretion system. On the other hand, CitT seemed to activate a series of proteins which had related relationship with two-component system, histidine metabolism, pectin degradation, ABC transporters, and histidine transporters. In addition, several CitT-regulated proteins were involved in category function unknown, reflecting that the current understanding of the CitS/CitT two-component regulatory system was not complete enough. Other research showed that the amino acid sequence of the response regulatory protein CitT in the CitS/CitT two-component system was highly conserved in genus Bacillus, it was an important transcription factor related to the regulation of carbon metabolism (Lensbouer and Doyle Citation2010). Studies suggested that CitT protein mainly regulated citrate metabolism, a strain deficient in citT gene could not grow on the minimal plates containing citrate as the sole carbon source (Repizo et al. Citation2006; Yamamoto, Murata, and Sekiguchi Citation2000). This study found that the CitT could regulate the histidine biosynthesis pathway during ramie degumming process, which has enriched the comprehending on the regulatory function of pectin degradation.
Table 2. Functional classification of DEPs according to KEGG.
Network analysis of protein–protein interactions among CitT-regulated DEPs
Network analysis with DEPs by using the STRING database was performed to understand the functional associations or interactions among these proteins regulated by CitT () (Z. Wang et al. Citation2018). There were five unconnected proteins among the proteins regulated by CitT. Three of them, FliT, YopD, and YjpA were all up-regulated in mutant strain. FliT was a protein participated in flagellar assembly, which was required for flagellar biosynthesis (Sanchez et al. Citation2022). YopD was a protein involved in bacterial secretion system, and YjpA was belonged to unknown function category. YqxJ and YuiF were down-regulated in mutant strain, the function of YqxJ was unknown according to current research, while YuiF possibly constituted a new family of histidine transporters, and linked to histidine biosynthesis or utilization pathways (Leyn et al. Citation2013; Vitreschak et al. Citation2004). However, the interaction between YuiF and enzymes participated in histidine biosynthesis were not reflected in the STRING networks, perhaps more experiments were needed to explore the intrinsic connection among these proteins. In addition, there were several distinct networks with the most significant one related to the histidine biosynthesis, two-component system, pectin degradation, ABC transporters, arginine degradation, peptidoglycan biosynthesis, and energy production and conversion ( and Table S1). What’s more, a small cluster including those required for PTS was suggested their repression by CitT, this small cluster was connected to the protein YflJ. However, the function of YflJ was not clear and needed more exploration.
Figure 3. Network analysis of protein–protein interactions of DEPs in the gene citT knocking-out mutant strain. The color of nodes reflected the up- or down-regulation information of DEPs in mutant strain. Red sphere represented this protein was up-regulated in mutant strain, which was CitT-repressed protein. Green sphere represented this protein was down-regulated in mutant strain, which was CitT-activated protein. Empty spheres represented proteins of unknown 3D structures, while filled spheres presented these proteins with known or predicted 3D structures. The red bulb closed to CitT was used for indicating that CitT was absent in the mutant strain.
Metabolic pathways of DEPs
A total of 23 DEPs were participated in multiple metabolic pathways, including transmembrane transport system, two-component system, amino acid metabolism, flagellar assembly, peptidoglycan biosynthesis, histidine transporters, and energy production and conversion (Table S1). Among these proteins, five DEPs were involved in various metabolic pathways individually, that were PelA, FliT, PbpF, YuiF, and FdhD. FliT and YuiF were participated in flagellar assembly and histidine transporters pathways respectively, they were also unconnected protein after STRING analysis and have been discussed above (). PbpF was a penicillin-binding protein involved in peptidoglycan biosynthesis and played redundant roles in sporulation (McPherson, Driks, and Popham Citation2001), but the relationship between PbpF and the degumming of ramie fibers was not clear yet. FdhD was consisted in energy production and conversion pathway and required for formate dehydrogenase activity, it was a protein response to acid stress in B. subtilis (Wilks et al. Citation2009), FdhD was connected to CitS/CitT two-component system and was repressed by CitT, the down-regulation of CitS/CitT two-component system perhaps was the main reason for its up-regulation in mutant strain (). In extracellular environment, B. subtilis degraded pectin by secreted pectate lyases, PelA catalyzed the second step of pectin degradation pathway to form soluble small molecule compound galacturonate (Zou et al. Citation2013). PelA was connected to CitS/CitT two-component system and activated by CitT, the down-regulation of PelA perhaps was the main reason to decrease the degradation rate during degumming of ramie fibers by mutant strain (). However, PelA was a secreted enzyme to perform function in extracellular environment, the extracellular proteomic analysis was also needed to reflect the total protein expression level of PelA in further study ().
Figure 4. Metabolic pathways which were participated by DEPs. Names in red or green indicated proteins were up-regulated or down-regulated in mutant strain, respectively. Names in black represented these proteins almost had no significant difference in expression abundant between original strain and mutant strain.
Transmembrane transport systems
Transmembrane transport system was very important for cells to uptake soluble carbohydrate substances from extracellular culturing environment, PTS and ABC transporters were the main pathways for monosaccharide transporting. Three DEPs YfmC, BglP, and NagP participated in transmembrane transport system were differently expressed after deleting of gene citT during the degumming process of ramie fibers. YfmC was part of the ABC transporter complex YfmCDEF involved in citrate-dependent Fe3+ import (), it could bind citrate-dependent Fe3+ and deliver it to the surface of YfmDE (Miethke, Kraushaar, and Marahiel Citation2013; Ollinger et al. Citation2006). As CitS/CitT two-component system was participated in regulation of citrate metabolism, perhaps it was the reason why YfmC was activated by CitT. BglP and NagP were involved in the phosphoenolpyruvate-dependent sugar PTS, a major carbohydrate active-transport system, catalyzed the phosphorylation of incoming sugar substrates concomitantly with their translocation across the cell membrane (Gaugue et al. Citation2013; Kruger and Hecker Citation1995). The BglP system was involved in beta-glucoside transport and also had a minor activity in glucose transport (Schilling et al. Citation2006). The NagP system was involved in N-acetyl-glucosamine transport (Y. F. Liu et al. Citation2013). Both BglP and NagP were up-regulated in mutant strain, which were repressed by CitT (). However, the concentration of glucose had no significant difference in the ramie fibers degumming system incubated with the original strain and mutant strain, and the N-acetyl-glucosamine was not detected in neither bio-degumming system (). The essential connections between these three DEPs involved in transmembrane transport system and degumming of ramie fibers needed further exploration by metabonomics and membrane proteomics.
Two-component systems
A total of six DEPs respectively belonged to CitS/CitT, LiaS/LiaR, and DctS/DctR two-component systems were induced by the deletion of gene citT during degumming of ramie fibers (). All of proteins involved in CitS/CitT two-component system were down-regulated in mutant strain, which revealed that the deficiency of CitT would bring the down-regulation of CitM and CitS. LiaS and LiaH were members of the two-component system LiaS/LiaR, which probably involved in response to a subset of cell wall-active antibiotics that interfere with the lipid II cycle in the cytoplasmic membrane (bacitracin, nisin, ramoplanin, and vancomycin) (Kobayashi et al. Citation2001). In addition, LiaS was also involved in response to cationic antimicrobial peptides and secretion stress, which was activated LiaR by phosphorylation (Kesel et al. Citation2013), and performed function with LiaH (Bernal-Cabas et al. Citation2020). It speculated that the mutant strain would face more survival stress when degumming of ramie fibers, and the up-regulation of LiaS and LiaH was devoted to survive in the bio-degumming system while B. subtilis was deficient in CitT. DctA was a protein responsible for the transport of succinate and fumarate (but not malate) across the membrane, though it was not directly involved in the DctS/DctR two-component system (Asai et al. Citation2000). Studies also reported that members of a set of paralogous two-component regulatory systems in B. subtilis, CitS/CitT and DctS/DctR, were involved in a related function-uptake (and metabolism) of the tricarboxylic acid cycle (TCA cycle) intermediates-but with distinct substrate specificities (Tanaka, Kobayashi, and Ogasawara Citation2003). It perhaps revealed that DctA was performed synergistic effect with CitS/CitT during the regulation of pectin degradation and utilization in degumming process of ramie fibers, and the loss of function of CitS/CitT in mutant strain highlighted the role of DctA in regulation of carbohydrate metabolism.
Amino acid metabolism
A new finding was discovered in this study, that was the deletion of gene citT would significantly influence the amino acid metabolism during degumming of ramie fibers by B. subtilis. Most enzymes participated in histidine biosynthesis were down-regulated in mutant strain, including HisI, HisB, HisZ, HisA, HisG, HisD, HisF, and HisH (). The eight DEPs catalyzed the histidine biosynthesis pathway from phosphoribosyl pyrophosphate (PRPP) to L-histidine. Only HisC and HisJ had no significant difference in protein abundant, which catalyzed two steps from imidazole-acetol-P to L-histidinol-P and from L-histidinol-P to L-histidinol, respectively. The connection between histidine biosynthesis and carbohydrate metabolism was constructed through pentose phosphate pathway via compound PRPP, which probably indicated that the pectin degradation was closely related to histidine biosynthesis with the regulation of CitT. Another enzyme NosA, which catalyzed the degradation of arginine to form nitric oxide (Adak, Aulak, and Stuehr Citation2002), was up-regulated in the mutant strain (). Interestingly, the DEPs involved in histidine biosynthesis pathways were connected to CitS/CitT two-component system via NosA (). These results possibly revealed that CitT regulated the degumming process of ramie fibers combined with down-regulation of histidine biosynthesis and up-regulation of arginine degradation.
qRT-PCR determination of transcripts
To study whether the changes in the proteome were correlated with differences at the mRNA level or not, the expression of eight selected genes, essential for different metabolic processes, was analyzed by qRT-PCR. The corresponding eight proteins were involved in ABC transporters (YfmC), two-component system (LiaS, LiaH, DctA, CitT), PTS (NagP, BglP), and pectin degradation (PelA) respectively. qRT-PCR revealed that most detected genes (except gene pelA) had similar trend with proteomic quantification results, showed a positive correlation between changes at translational and transcriptional expression level, and demonstrated that the trends of the changes in transcriptional expression of the selected genes were in accordance with their corresponding proteins (). The result of qRT-PCR analysis of gene pelA was consistent with previous research, i.e. the transcriptional level of PelA had little significant difference between original strain and mutant strain (Yang, Cheng, et al. Citation2022). It revealed that the deletion of gene citT had little impact on the transcription level of PelA from different strains when degumming of ramie fibers. However, the expression level of PelA was significantly decreasing in mutant strain, which was consistently matched with the enzymatic activity test in previous study, i.e. the pectinase activity was significantly decreased in ramie fibers degumming system with mutant strain as compared to original strain (Yang, Cheng, et al. Citation2022). In addition, because pectinases are extracellular enzymes and supposed to the most important and crucial enzymes in the degradation and utilization of pectin (Basu et al. Citation2009), perhaps the extracellular proteomic analysis was essentially to explore the expression levels of PelA both in intracellular and extracellular environment, and more studies were needed to reveal the intrinsic relationship between CitT and PelA.
Figure 5. qRT-PCR determination of selected gene transcripts. The differential expression patterns of eight chose genes (yfmC, liaS, liaH, dctA, citT, nagP, bglP, and pelA) were substantiated by qRT-PCR analysis. mRNA levels from different samples were denoted as relative values to 16S rRNA, the ratio values for the sample original strain were arbitrarily set to 1. Six independent determinations of mRNA abundance were executed to calculate error bars in each sample.
Conclusions
This study is the first comprehensive analysis of the response regulator CitT from CitS/CitT two-component system of B. subtilis strain 168 during degumming process of ramie fibers by quantitative proteomic method, results showed that multiple DEPs were differentially regulated with the deletion of gene citT. The proteomic data clarified that the deficient in CitT mainly leaded to the influence on amino acid metabolism, transmembrane transport system, and two-component system: (i) down-regulation of enzymes participated in histidine biosynthesis, and up-regulation of NosA involved in arginine degradation; (ii) down-regulation of YfmC belonged to ABC transporter complex YfmCDEF, and up-regulation of BglP and NagP in PTS system; (iii) for two-component systems, the CitS/CitT was down-regulated, while LiaS/LiaR and DctS/DctR were up-regulated. Interestingly, the pectin degradation enzyme PelA was significantly down-regulated in mutant strain, it was in accordance with the down-regulation of the pectin degradation rate with the deficient in CitT protein. Furthermore, there are several types of pectinases that can play a role in the ramie degumming, such as polygalacturonase and pectinesterase. The specific regulatory function for different pectinases need a more in-depth research of the possible role of CiT protein by chromatin immunoprecipitation sequencing (ChIP-Seq) and electrophoretic mobility shift assay (EMSA) in the future. In summary, the data acquired here provided a first insight into the global proteomic changes occurring in B. subtilis cells with the deletion of gene citT under ramie fibers degumming environments. Our study enhanced the current understanding of metabolic regulatory function of CitT protein to pectin degradation process via comparative quantitative proteomic analysis of B. subtilis cells in ramie fibers degumming process, and may provide an important and valuable scientific basis for constructing engineering strains in target to be applied in bio-degumming of ramie fibers.
Abbreviations
| DEPs | = | differentially expressed proteins |
| HPLC | = | high performance liquid chromatography |
| SEM | = | scanning electron microscope |
| PBS | = | phosphate buffer solution |
| SDS | = | sodium dodecyl sulfate |
| TMT | = | tandem mass tags |
| DTT | = | dithiothreitol |
| TEAB | = | triethylammonium bicarbonate |
| ACN | = | acetonitrile |
| RPLC | = | reversed phase liquid chromatography |
| RP | = | reversed phase |
| MS | = | mass spectrometry |
| FA | = | formic acid |
| AGC | = | automatic gain control |
| HCD | = | higher-energy collisional dissociation |
| NCE | = | normalized collision energy |
| FDR | = | false discovery rate |
| STRING | = | STRING, functional protein association networks (http://string-db.org) |
| KEGG | = | KEGG, Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp/) |
| qRT-PCR | = | quantitative real-time polymerase chain reaction |
| TCA cycle | = | tricarboxylic acid cycle |
| PRPP | = | phosphoribosyl pyrophosphate |
| PTS | = | phosphotransferase system |
Highlights
Proteomic delineation of the CitT regulator discovers its novel function in bio-degumming of ramie fibers.
The deficient of CitT protein could down-regulate histidine biosynthesis.
The disrupting the citT gene could up-regulate arginine degradation.
CitT protein influences the transmembrane transport system when bio-degumming of ramie fibers.
CitT protein probably has a synergistic effect on regulation of bio-degumming with two-component systems LiaS/LiaR and DctS/DctR.
Authors’ contribution
All authors conceived and designed the study. QY, YH, and SD carried out the proteomic studies, qRT-PCR analysis, and drafted the manuscript. LC, XF, and GX contributed to the proteomic data analysis and helped to draft the manuscript. KZ, ZP, and FH participated in SEM observation and HPLC analysis. All authors read and approved the final manuscript.
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Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15440478.2024.2334414.
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