Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in Bacillus licheniformis.

Extracellular polymeric substances (EPSs) are extracellular macromolecules in bacteria, which function in cell growth and show potential for mechanism study and biosynthesis application. However, the biosynthesis mechanism of EPS is still not clear. We herein chose Bacillus licheniformis CGMCC 2876 as a target strain to investigate the EPS biosynthesis. epsK, a member of eps cluster, the predicted polysaccharide synthesis cluster, was overexpressed and showed that the overexpression of epsK led to a 26.54% decrease in the production of EPS and resulted in slenderer cell shape. Transcriptome analysis combined with protein-protein interactions analysis and protein modeling revealed that epsK was likely responsible for the synthesis of teichuronic acid, a substitute cell wall component of teichoic acid when the strain was suffering phosphate limitation. Further cell cultivation showed that either phosphate limitation or the overexpression of teichuronic acid synthesis genes, tuaB and tuaE could similarly lead to EPS reduction. The enhanced production of teichuronic acid induced by epsK overexpression triggered the endogenous phosphate starvation, resulting in the decreased EPS synthesis and biomass, and the enhanced bacterial chemotaxis. This study presents an insight into the mechanism of EPS synthesis and offers the potential in controllable synthesis of target products.

Introduction

Bacterial extracellular polymeric substances (EPSs) have important applications for human beings. Due to their degradable, efficient, non-toxic and renewable properties, they are used in a wide range of applications in the pharmaceutical industry, the cosmetics industry, the food industry, agriculture and the bioremediation of pollution [[1], [2], [3]]. EPSs are composed of polysaccharides, proteins (e.g., γ-polyglutamic acid, γ-PGA), lipids, extracellular DNA, and humic substances, among which polysaccharides and proteins are the main components and the content can reach 75–90% [4,5]. Because of the complex composition and the many metabolic pathways involved, it is important to unravel the mechanisms of EPS synthesis for the exploitation and application of EPS.

We previously reported an eps cluster in the genome of Bacillus licheniformis CGMCC 2876, which was predicted to be responsible for the synthesis of exopolysaccharides [6]. In the cluster, several genes with demonstrated function have been identified, including encoding membrane sensors (epsA, epsC) [[6], [7], [8], [9]], tyrosine protein kinases (epsB, epsD) [[7], [8], [9]], glycosyl modifying enzymes (epsI, epsM, epsN, epsO) [[10], [11], [12]], and a bifunctional glycosyltransferase (epsE) [[13], [14], [15]]. We characterized a gene designated epsK, which was annotated as an α-helical membrane transporter gene in Bacillus subtilis [16]. Other studies found that EpsK contains the conserved domain Wzx, and the protein was postulated to be a flippase or glycosyltransferase. EpsK is most likely responsible for the transportation of polysaccharide repeating units from the cytoplasm to the periplasmic side [17,18]. Disruption of epsK led to an accumulation of poly-N-acetylglucosamine inside of B. subtilis cells [16]. During lipopolysaccharide synthesis, EpsK is responsible for the translation of the O-antigen, a repetitive glycan polymer which is subsequently added to rough lipopolysaccharide [19]. However, the function of epsK in B. licheniformis remains unclear.

In this study, we constructed an epsK overexpression strain of B. licheniformis and tried to shed light on the function of epsK, as well as the mechanism of EPS synthesis.

Materials and methods

Bacterial strains and growth conditions

B. licheniformis CGMCC 2876 was isolated by our laboratory [6]. Escherichia coli DH5α was grown in Luria–Bertani (LB) medium, which contained 1% tryptone, 0.5% yeast extract, and 1% NaCl, at 37 °C and 200 rpm. B. licheniformis was grown in LB or fermentation media at 37 °C and 200 rpm. Fermentation media refer to seed medium and EPS medium. LLP and LP medium provided phosphate starvation environment while HP150, HP200, HP250 and HP300 medium contained excess phosphate. Details of the medium composition were listed in Table S1.

For EPS production, B. licheniformis was primarily inoculated in seed medium and transferred to EPS medium as a 4% inoculum after 20 h.

Strain construction

Competent cell of E. coli was obtained from Ultra-Competent Cell Preps Kit (Sangon Biotech, Shanghai, China), while the competent cell of B. licheniformis CGMCC 2876 was prepared as reported [20]. Briefly, B. licheniformis was cultured in LB liquid medium containing 0.5 M sorbitol until mid-log phase, and the cells were collected at 4 ℃ and washed with pre-cooled electroporation medium (0.5 M sorbitol, 0.5 M mannitol and 10% glycerol) to prepare competent cells. The epsK, tuaB, tuaE genes were amplified using Phanta Master Mix (Vazyme, Nanjing, China), and the primers are listed in Table S2. Sequentially, the PCR products were cloned into the shuttle vector pHY300PLK-PamyL-TTamyL, a plasmid containing the promoter PamyL and terminator TTamyL constructed in our previous study [6], as KpnI/XhoI fragments and transformed into E. coli DH5α with selection on LB agar containing tetracycline (5 μg/ml). After verification, the correct plasmids were extracted from E. coli and transferred into B. licheniformis CGMCC 2876 by electroporation (25 kV/cm, 4 ms).

Purification of biopolymer and analysis of its composition

After the fermentation process, the biopolymer was extracted and purified using the method described in a previous study [21]. The content of total carbohydrates in the biopolymer was detected using the phenol–sulfuric acid method. Modified Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) was used to measure the protein content. The γ-PGA content was detected based on CTAB assay with incubation time adjusted to 10 min [22].

Analysis of biomass and bacterial morphology

To facilitate the determination of biomass, we measured the OD600 of the fermentation broth and subsequently collected the cells and lyophilised them for weighing, thus establishing a calibration curve relating biomass and optical density. The differences in morphology between the epsK overexpression strain OEK1 and wild-type strain 2876 were evaluated using SEM (S-4800, Hitachi, Tokyo, Japan) and the cells were collected after 24 h fermentation in EPS medium. The cells were washed with PBS (0.1 M, pH 7.2) three times before being fixed with 2.5% glutaraldehyde for 2 h. Additional washes were needed before hydrating the cells with 30, 50, 70, and 100% ethanol. The diameters of the bacteria were estimated using NanoMeasure software.

RNA-Seq

For RNA-seq, B. licheniformis CGMCC 2876 and OEK1 were primarily inoculated in seed medium and cultured for 20 h to the late logarithmic growth (OD∼2), then transferred to EPS medium and further incubated for 24 h. The cells were collected by centrifugation at 8000 rpm for 10 min at 4 °C for total RNA purification and cDNA library construction. Sequencing was performed using the Illumina HiSeqTM 2500 platform with paired-end 150 base pair reads by Gene Denovo Biotechnology Co.

Analysis of transcriptome data

Raw data were filtered before the quality-trimmed reads were mapped to the genome of B. licheniformis CGMCC 2876 using Bowtie2 [23].The gene expression level was analyzed according to FPKM (fragments per kilobase of transcript per million). The edgeR package (http://www.r-project.org/) was used to identify DEGs with fold changes (FC)≥2 and FDR<0.05.

STRING

We chose the STRING database [24] to predict PPIs between EpsK and other proteins in B. licheniformis. In our analysis, we used neighborhood and co-occurrence checkboxes with a high confidence score of 0.7, while other checkboxes showed no results.

Modeling of EpsK

The modeling of EpsK was performed using I-TASSER (https://zhanglab.dcmb.med.umich.edu/I-TASSER/) and PyMOL (https://pymol.org/2/). The substrate and pathway informations were obtained from BioCyc (https://biocyc.org/). IonCom (https://zhanglab.dcmb.med.umich.edu/IonCom/) was used to predict the ion ligand binding site of EpsK.

Gene expression level analysis

As with RNA-seq samples, overexpression strains were also collected after 24 h of incubation for RNA extraction and gene expression analysis. Total RNA was extracted using a Bacterial RNA Kit (Omega Bio-Tek, Guangzhou, China) and quantified by a NanoDrop 2000 (Thermo, CA, USA). First-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China), and qRT-PCR was performed with a qTOWER3 instrument (Analytik Jena AG, Jena, Germany) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reference gene was 16S rRNA, and the relevant primers are listed in Table S2.

Availability of data and materials

The sequences data reported in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive (SRA), with the accession number PRJNA675185.

Results and discussion

The epsK functions in biopolymer synthesis of B. licheniformis

In our previous study, we found that B. licheniformis CGMCC 2876 could produce polysaccharides and γ-PGA under different culture conditions [21]. EpsK was reported to play an important role in polysaccharide biosynthesis, however, its exact function has not been characterized [16]. As shown in Table 1, the crude biopolymer production of the epsK overexpression strain B. licheniformis OEK1 was decreased by 26.54% compared to that of the wild-type B. licheniformis CGMCC 2876, indicating that epsK overexpression inhibits the production of biopolymers in B. licheniformis. Further analysis of the biopolymer contents showed an evident reduction in the proportions of γ-PGA, total sugar, and protein in OEK1, as the proportion of γ-PGA dropped from 20.74% to 2.78%. This implied that OEK1 synthesizes more undetectable components. We tentatively determined that EPS also contains undetectable components such as the applicable surfactants, but more detailed characterization will be performed in our future study.

Table 1

Yields and compositions of biopolymers produced by Bacillus licheniformis.

		B. licheniformis CGMCC 2876	B. licheniformis OEK1
Production (g/L)		8.79 ± 0.26	6.56 ± 0.24
EPS Contents (%)	Protein	0.89 ± 0.05	0.49 ± 0.04
	Total sugar	3.48 ± 0.14	2.24 ± 0.09
	γ-PGA	20.74 ± 0.30	2.78 ± 0.08

The epsK functions in biomass and cell morphology

As shown in Fig. 1A, the epsK overexpression strain B. licheniformis OEK1 showed a 43.57% reduction in biomass compared to wild-type B. licheniformis CGMCC 2876 in the EPS medium at logarithmic phase and achieved a maximum at 20 h. In the mid-term period of fermentation, the biomass of the wild-type strain rapidly decreased. Meanwhile, the growth of OEK1 was relatively stable.

Fig. 1

Growth and morphology of B. licheniformis. (A) The growth curves of B. licheniformis strains in the EPS medium (mean ± standard error). (B) The statistics of bacterial length. (C) The statistics of bacterial width. (D) The SEM micrograph of B. licheniformis CGMCC 2876. (E) The SEM micrograph of B. licheniformis OEK1.

Scanning electron microscopy (SEM) was used to analyze the morphology of the cells collected at 24 h culture. The SEM micrographs showed that the morphology of the wild-type CGMCC 2876 tended to be short and thick, while the OEK1 cells were slender (Fig. 1D and E). It was calculated that the cell lengths of both strains were centralized at 2.5 μm, i.e., the cell length of the wild-type strain was ranging from 2.0 to 2.5 μm (33.33% of the total cell amount), while the length of OEK1 cells was mainly distributed between 2.5 and 3.0 μm (32.14% of the total cell amount) (Fig. 1B). In addition, there existed obvious difference in the cell width of the two strains. The cell width of 54.12% of the wild-type cells was distributed in the range of 0.5–0.7 μm, while the cell width of 77.01% of OEK1 cells was among 0.4–0.5 μm (Fig. 1C). It indicated that the epsK overexpression had effects on the cell propagation and cell morphology of B. licheniformis.

The epsK induces the global changes in the transcriptome

Principal component analysis (PCA) and Pearson correlation heat map analysis were performed to determine the correlation between the integrated data from the transcriptome data set (Figs. S1A and S1B). Quantitative real-time PCR (qRT-PCR) was performed to verify the credibility of transcriptome data (Figs. S1C and S1D). The results showed that the samples had high repeatability, and the data were suitable for subsequent analysis. We used the edgeR package to identify differentially expressed genes (DEGs) and obtained 1875 DEGs.

The analysis of GO (gene ontology) functional annotations revealed that DEGs were enriched for 29 functional terms. Among these terms, 14 terms were enriched in biological process, 8 terms in molecular function, and 7 terms in cellular component. Fig. 2A showed that 9 terms (single-organism process, response to stimulus, localization, locomotion, catalytic activity, transporter activity, membrane part, organelle part, and organelle) were significantly upregulated (≥60% genes). However, 7 terms (biological regulation, regulation of biological process, developmental process, transcription factor activity, protein binding, nucleic acid binding transcription factor activity, structural molecule activity, and macromolecular complex) exhibited obvious downregulation.

Fig. 2

Functional enrichment of DEGs in B. licheniformis CGMCC 2876 vs OEK1. (A) The functional categories of DEGs based on GO. (B) The significance analysis of top 20 Q-value pathways based on KEGG.

We collected the top 20 pathways with low Q value for significance analysis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) (Fig. 2B) and obtained the four most significantly enriched pathways belonging to Metabolism, including starch and sucrose metabolism, the tricarboxylic acid cycle (TCA cycle), and carbon metabolism, which indicated an enhanced energy metabolism induced by epsK overexpression. There were also two enriched pathways belong to cellular processing, containing flagellar assembly and bacterial chemotaxis. Combined with the changes in the morphology of OEK1 cells, it suggested epsK overexpression not only led to a slenderer cell shape, but also resulted in an activated bacterial movement. In addition, aminoacyl-tRNA biosynthesis pathways were enriched as well, which may facilitate the protein synthesis of cells. We noted that some genes were also enriched and annotated to the pathway of photosynthesis and plant-pathogen interaction, which does not imply that the bacterium is photosynthetic or a plant pathogen.

Functional speculation of epsK

To better understand the disturbance caused by epsK overexpression, the STRING database and Cytoscape were introduced to construct a network of protein-protein interactions (PPIs) between EpsK and other proteins (Fig. 3A and Table S3). It is noteworthy that 21 out of 42 genes were related to flagellar assembly, bacterial chemotaxis, and glycan metabolism, and most of these genes were upregulated. However, 21 genes associated with cell growth, transcription, transporters, and other pathways were downregulated. When combining PPIs and gene expression analyses, we noticed that the gene elements in the eps cluster, which interacted with epsK, only epsC, epsG, epsL, and epsJ showed detectable expression. This was associated with the decrease of polysaccharide content in the EPS.

Fig. 3

Functional prediction of EpsK. (A) The differential expression levels of the genes included in the predicted PPIs of epsK. The network was constructed using Cytoscape. Each node represents a gene, and each line refers to an interaction. The circle colors revealed the expression level of the genes. (B) The protein model of EpsK (transport channel is marked in blue, while the protein is light gray). (C) The modeled sugar repeating unit. (D) Binding sites of EpsK and Zn2+. Predicted binding residue: E40 (marked in red). (E) Binding sites of EpsK and Ca2+. Predicted binding residues: G38, E40, S262, H342, L346 (marked in red). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

It was interesting to observe that the trends in expression of tuaE and tuaF genes were consistent with epsK. As reported, the Wzy-type polymerase TuaE polymerizes repeating units in the periplasm [25]. TuaE is functionally and structurally homologous to the PssT protein in Rhizobium leguminosarum bv. trifolii. Disruption of PssT in R. leguminosarum leads to an increase in the level of EPS production and the proportion of high-molecular-weight EPS [26]. TuaF contains a Wzz-domain, which acts as a co-polymerase that regulates the molecular weight of polysaccharides [27]. In B. subtilis, tuaE and tuaF belong to the operon required for the polymerization of teichuronic acid [28]. Teichuronic acid, like teichoic acid, is an important component of the cell wall. Under phosphate-limiting conditions, teichuronic acid is synthesized and replaces teichoic acid reduce the demand for phosphate [[28], [29], [30]].

Further analysis of gene expression in the teichuronic acid synthesis pathway revealed that epsK overexpression also led to the upregulation of tuaD and downregulation of tuaB. It was demonstrated that tuaD encoded UDP-glucose 6-dehydrogenase in B. subtilis 168, which converts UDP-glucose to UDP-glucuronate, and subsequently UDP-glucuronate was involved as a precursor substance in teichuronic acid synthesis [28]. The TuaB, on the other hand, was mainly responsible for the transmembrane transport of teichuronic acid repeating units [31]. Because TuaB shared the same Wzx structural domain as EpsK [17,18,28], it was speculated that TuaB may compete with EpsK during the synthesis of teichuronic acid. Therefore, it exhibited an upregulation of epsK expression along with a downregulation of tuaB expression in the OEK1 strain. To summarize, we postulated that epsK is responsible for teichuronic acid synthesis and may be involved in the bacterial response to phosphate starvation.

In addition, the expression level of ylqG was upregulated due to the overexpression of epsK. However, ylqG has been rarely reported, except one reported that ylqG is part of the transcriptional organization of succinyl coenzyme A synthase (α subunit) [32]. ylqG may therefore be associated with the synthesis of succinyl coenzyme A synthase and the enhanced expression of ylqG may be responsible for the enhancement of TCA cycle in B. licheniformis OEK1.

We observed that epsK also interacted with 17 genes related to flagellar assembly and bacterial chemotaxis through ylqG and caused significant upregulation of 16 out of them [[33], [34], [35]].These 16 genes were fliI-M, fliP-R, fliY-Z, yvyG, flnA-B, flgK, ylxG and flgG. Bacterial chemotaxis, the targeted motions of cells following a gradient of chemo attractants, is often regarded as a bacterial starvation response induced upon nutrient depletion, or as a bacterial response to escape from a harmful environment [36]. It was speculated that there was an interconversion between the formation of biofilm and the maintenance of motility during bacterial growth, and that inhibition of bacterial motility would facilitate biofilm formation [37].

The modeling of EpsK was constructed to further elucidate its function (Fig. 3B). It was shown that there is a transmembrane transport channel in the EpsK protein (marked in blue), and the bottleneck of the channel is 3.006 Å. According to the pathway for the synthesis of teichuronic acid in B. subtilis 168 provided on BioCyc, the sugar repeat unit transported by flippase was β-d-glucuronosyl-(1 → 4)-β-d-glucuronosyl-(1 → 3)-N-acetyl-α-d-galactosaminyl-(1 → 6)-N-acetyl-α-d-galactosaminyl-diphospho-ditrans, octacis-undecaprenol (Fig. 3C). Since B. licheniformis is highly homologous to B. subtilis, they may share the same teichuronic acid repeat unit. The maximum width of the molecular model of the repeating unit, as measured by PyMOL, is about 13.3 Å, indicating that EpsK needs to change the configuration to achieve the transport of sugar repeating units.

It has been reported that when transporting the sugar repeating units, the Wzx domain contained in EpsK first binds to the cations from the periplasm to realize the configuration transformation and opens the transport channel toward the inside of the cell, and then binds to the sugar repeating units. Subsequently, the bound cations fall off, and the configuration of Wzx changes again toward the periplasm, thus realizing the reversal of sugar repeat units [27]. Using IonCom to predict the ion ligand binding site of EpsK, it was found that it may bind to Zn2+ at E40 (Fig. 3D) and Ca2+ at G38, E40, S262, H342, and L346 (Fig. 3E). This is consistent with the transport function of Wzx. Therefore, we speculated that EpsK was responsible for the export of teichuronic acid repeating units in B. licheniformis CGMCC 2876.

Putative mechanism of EPS synthesis

The above analysis indicated that the overexpression of epsK is correlated to teichuronic acid synthesis and phosphate starvation. As we know, starvation response is related to quorum sensing (QS), cell chemotaxis, Therefore, we selected the genes related to phosphate starvation response, QS, bacterial chemotaxis, and extracellular polymer synthesis for heatmap analysis. The possible metabolic and regulation mechanism of epsK in B. licheniformis CGMCC 2876 was constructed based on the heatmap analysis (Fig. 4).

Fig. 4

The metabolic mechanism of EPS biosynthesis in B. licheniformis. The metabolic pathway was reconstructed based on BioCyc and KEGG analyses. Transcripts of B. licheniformis CGMCC 2876 and OEK1 are shown aside the pathway as a heat map based on fpkm in the transcriptome data. Note: (β-D-GlcA-(1 → 4)-β-D-GlcA-(1 → 3)-α-D-GalNAc-(1 → 6)-α-D-GalNAc-PP-Und represents β-d-glucuronosyl-(1 → 4)-β-d-glucuronosyl-(1 → 3)-N-acetyl-α-d-galactosaminyl-(1 → 6)-N-acetyl-α-d-galactosaminyl-diphospho-ditrans, octacis-undecaprenol).

Since teichuronic acid is an alternative to teichoic acid, teichoic acid synthesis pathway was analyzed and the expression of tagA, tagB, tagD-F, tagG and tagO were found to be decreased. It was reported that TagO, TagA, TagB, and TagD catalyzed the initial steps of teichoic acid synthesis to form lipid-anchored disaccharide glycerol 3-phosphate. TagE modified teichoic acid with α-glucose using UDP-glucose as a donor. TagF was responsible for adding 45–60 glycerol-3-phosphate units to the TagB product to form a polymer. TagG transported teichoic acid or teichoic acid precursor across the membrane [38,39]. Therefore, in B. licheniformis OEK1, teichuronic acid synthesis was enhanced accompanied with a weakened synthesis of teichoic acid.

The increase of teichuronic acid and the decrease of teichoic acid, which are the two important components of the cell wall of OEK1, may be the reason for the change of cell morphology. It was reported that the deletion of teichoic acid synthetase genes in B. subtilis inhibited teichoic acid production and changed the morphology of the mutants [40]. MreBH was demonstrated to be involved in the control of teichoic acid synthesis and the regulation of lytE. The lytE mutation of B. subtillis formed a longer cell chain and the cell diameter was smaller than that of the wild-type strain [41,42]. The change of cell wall compositions caused by the epsK overexpression resulted in the downregulation of mreBH and lytE in OEK1 strain, which made the cells slenderer than the wild-type strain.

As mentioned earlier, the overxpression of epsK enhanced teichuronic acid and inhibited teichoic acid synthesis, which was consistent with the physiological changes under phosphate starvation. We then analyzed the expression level of the key genes in bacterial phosphate starvation response, phoP, phoR, and resABCDE, which encode transcriptional activator PhoP, sensor kinase PhoR, and the two-component systems, respectively [43,44]. It was shown that these genes were upregulated in the strain of OEK1, which suggested the overexpression of epsK results in the phosphate starvation response. However, the alkaline phosphatase genes, phoB and phoD, which are responsible for harvesting phosphate from exogenous environment [43], were downregulated in OEK1. The results indicated that the response to phosphate starvation in OEK1 was caused by endogenous factors rather than exogenous factors, i.e., the strain generated phosphate starvation response in the absence of phosphate depletion.

QS system senses the signals from both the external environment and the cell itself, and then regulates the physiology changes of the cell, such as the EPS synthesis and the sporulation. QS is regulated by cell density: ComX is an extracellular cell density autoinducer, which is secreted to the periplasm by ComQ [45,46]. Periplasmic ComX binds to ComP and promotes the phosphorylation of ComP, ComA, DegQ and DegU sequentially, thereby enhance the synthesis of γ-PGA and surfactin and activate the phosphorylation of Spo0A [47,48]. Spo0A relieves the inhibition of SinR on eps gene cluster via increasing the expression of SinI, an anti-repressor of SinR, thus promoting the production of the extracellular matrix [49]. In B. licheniformis OEK1, comQ, comA, degU, and spo0A were downregulated compared to CGMCC 2876, resulted in the inhibitory effect of SinR on eps gene cluster. The gene expression analysis revealed that the CapBCA complex genes (capA, capB, and capC), which are responsible for the polymerization of γ-PGA [50], were downregulated, while the eps genes for polysaccharides synthesis were inhibited due to the suppression effect by SinR, leading a decrease of extracellular polymeric substances.

As we know, QS system is also regulated by CSF (competence and sporulation factor), which is induced by starvation and other external stimuli. Phosphorylation of ComA is inhibited by a certain concentration of intracellular CSF transported via oligopeptide permease Opp [45,46]. In the transcriptome of B. licheniformis OEK1, the oligopeptide permease gene oppABCDE was significantly upregulated, indicating that Opp may be transporting a large amount of CSF produced by phosphate starvation. It was reported that starvation signals bring the cell into a stable phase [46]. This may be the reason why B. licheniformis OEK1 entered the stable stage earlier and the biomass was lower than CGMCC 2876.

Additionally, the starvation response caused by nutrient consumption will enhance the chemotaxis of bacteria [36]. In B. subtilis, fla/che operon contains flagellum genes, chemotaxis genes, and regulatory genes sigD and swrB. SigD and SwrB are responsible for regulating the transcription of genes related to flagella synthesis outside the operon [51]. In strain of OEK1, the expression of fla/che operon and the genes regulated by sigD were significantly upregulated. The results indicated that B. licheniformis OEK1 exhibited an enhancement of bacterial chemotaxis under phosphate starvation response.

EPS production of B. licheniformis under phosphate starvation

Based on the previous speculations, we applied different concentrations of phosphate to B. licheniformis CGMCC 2876 for EPS production and further constructed tuaB and tuaE overexpression strains, B. licheniformis OETB and OETE, to investigate the relationship among techuronic acid synthesis, phosphate starvation, and EPS synthesis.

Like B. licheniformis OEK1, the overexpression of tuaB and tuaE induced the decrease in EPS production (Fig. 5A). When cultured in LLP medium, the crude biopolymer production of B. licheniformis CGMCC 2876 was 2.92 ± 0.43 g/L, a decrease of 66.09% compared to that of the strain cultured in EPS medium, which was 8.61 ± 0.51 g/L. The crude biopolymer production of B. licheniformis CGMCC 2876 increased with increasing phosphate concentration and reached a maximum at the highest phosphate concentration (HP300 medium) (Fig. 5B). The results indicated that phosphate starvation inhibited bacterial production of EPS.

Fig. 5

The effect of phosphate starvation on the metabolism of B. licheniformis. (A) Crude biopolymer yield of B. licheniformis CGMCC 2876, OETB and OETE. (B) Crude biopolymer yield of B. licheniformis cultured under different concentrations of phosphate. (C) Analysis of gene expression in the phosphate starvation response pathway of B. licheniformis CGMCC 2876 and OETB. (D) Analysis of gene expression in the teichuronic acidsynthesis pathway of B. licheniformis CGMCC 2876 and OETB. (E) Analysis of gene expression in the phosphate starvation response pathway of B. licheniformis CGMCC 2876 and OETE. (F) Analysis of gene expression in the teichuronic acidsynthesis pathway of B. licheniformis CGMCC 2876 and OETE. (G) Analysis of gene expression in the phosphate starvation response pathway of B. licheniformis CGMCC 2876. (H) Analysis of gene expression in the teichuronic acid synthesis pathway of B. licheniformis CGMCC 2876. (I) Analysis of gene expression in the osmotic stress response pathway of B. licheniformis CGMCC 2876. Relative expression was determined by 2-△△Ct, *p < 0.05 versus 2876.

Transcriptional analysis was performed using qRT-PCR to compare the related gene expression levels of B. licheniformis CGMCC 2876, OETB and OETE strains. As shown in Fig. 5C and D, the expression levels of phosphate starvation response genes were increased by 1.44~4.21-fold in B. licheniformis OETB, as well as teichuronic acid synthesis genes tuaD and tuaE. Differently, epsK was slightly downregulated (0.82-fold) due to the overexpression of tuaB, implying a possible functional competition between epsK and tuaB. In B. licheniformis OETE (Fig. 5E and F), the phosphate starvation response genes phoP, resD were moderately upregulated (1.45 and 1.21-fold respectively) while others were downregulated. The expression of epsK and tuaB were activated by the tuaE overexpression. When incubated at different phosphate concentrations, the expression of phosphate starvation response genes in B. licheniformis CGMCC 2876 showed a correlation with the changes in phosphate concentration, i.e., enhanced phosphate starvation response was exhibited at both low and high phosphate concentrations (Fig. 5G). Meanwhile, the expression of teichuronic acid synthesis genes showed a similar correlation (Fig. 5H), which indicated that teichuronic acid synthesis and phosphate starvation response might also play a role in the osmotic stress response generated by high phosphate concentrations, and this hypothesis was confirmed by expression analysis of osmotic stress response genes [52] (Fig. 5I). Overall, we hypothesized that EpsK, like a flippase is involved in teichuronic acid synthesis and phosphate starvation response, and the overexpression of epsK had complex effects on cellular metabolism.

Conclusion

In summary, we found that overexpression of epsK in B. licheniformis CGMCC 2876 inhibited the synthesis of EPS and altered cell morphology and reproduction. Combining transcriptomics and PPIs analysis, we speculated that epsK is a flippase transporting teichuronic acid repeating units and the overexpression of epsK in B. licheniformis CGMCC 2876 stimulated endogenous phosphate starvation response, which then played roles in bacterial growth and morphology, EPS synthesis, bacterial chemotaxis, and QS. Further construction of tuaB and tuaE overexpression strains, as well as cell cultivation corroborated our suspicions above. Our results provided the putative functions of the epsK gene in B. licheniformis, which will benefit the researchers in manipulating the expression of stress response genes to make bacteria missense their metabolic states instead of imposing stress conditions, thereby regulating the synthesis of target products. This work also paves the way for further understanding the mechanism of EPS synthesis.

Funding

This work was financially supported by the ( 31871779 and 32170061).

CRediT authorship contribution statement

Yiyuan Xu: Conceptualization, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. Lijie Yang: Investigation, Validation. Haiyan Wang: Validation. Xiaoyu Wei: Investigation. Yanyan Shi: Visualization. Dafeng Liang: Writing – review & editing. Mingfeng Cao: Conceptualization, Project administration, Writing – review & editing. Ning He: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no competing interests.