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Regulatory mechanism of C4-dicarboxylates in cyclo (Phe-Pro) production

Microbial Cell Factories volume 23, Article number: 255 (2024) Cite this article

Abstract

Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88’s culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061.

Introduction

Cyclic dipeptides, also known as 2,5-dioxopiperazines or 2,5-diketopiperazines (DKPs), are the smallest naturally occurring cyclic peptides, formed by the cyclization of peptide bonds between two amino acids [1]. These compounds have been found to possess various biological activities, including anti-fungal [2, 3], anti-bacterial [4], antioxidant properties [5], and cell signaling properties, which have attracted the interest of many researchers [6]. One particular cyclo (Phe-Pro) (cFP) has been identified to be produced by a variety of bacteria, such as Vibrio vulnificus [7], Lactobacillus reuteri [8] and Pseudomonas aeruginosa [9], cFP has been found to perform multiple functions, including inhibiting Staphylococcus aureus biofilms [10] and modulation of innate immune responses toward the pathogen [11], facilitating the protection of V. vulnificus against hydrogen peroxide [12], inhibiting interferon (IFN)-β production by interfering with retinoic-acid-inducible gene-I (RIG-I) activation [13], inducing DNA damage through elevation of ROS in mammalian cells [14].

C4-dicarboxylates play essential roles in both prokaryotic and eukaryotic central metabolism [15, 16]. The C4-dicarboxylate transport (Dct) system is the first regulatory system identified to monitor the external concentration of C4-dicarboxylic molecules [17, 18]. The Dct system consists of three proteins encoded by the Dct regulon: the DctA gene encodes a putative C4-dicarboxylate carrier, while the sensor-regulator pair DctB-DctD is expressed from the DctB-DctD operon [19, 20]. The DctB-DctD operon can be activated by the presence of C4-dicarboxylates in the periplasm, resulting in phosphorylated DctD protein. The regulator DctD is composed of an N-terminal phosphoryl receiver domain, a C-terminal DNA-binding domain, and a central ATPase domain [21]. The phosphorylation of DctD controls the activation of the σ54-dependent RNA polymerase holoenzyme (Eσ54), which regulates the transcription of DctA [22] .

The study of peptide synthetase regulation is important for understanding the biosynthesis and developing more efficient tools for synthesizing novel compounds. Cyclodipeptides are synthesized by two distinct enzyme families: non-ribosomal peptide synthetases (NRPS) and cyclodipeptide synthases (CDPS) [15, 16]. These enzymes are frequently linked with specific biosynthetic gene clusters that include additional enzymes responsible for modifying the cyclodipeptide scaffold [23]. It has been reported that cFP is produced as a byproduct of tyrocidine A by NRPS, due to the chemical instability of the intermediate bound [24, 25]. The discovery of genes involved in cFP synthesis has attracted considerable attention; however, the regulatory mechanism of cFP production remains elusive. Among the regulatory factors found in bacteria, Ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis, oxidative stress response, and virulence in bacteria [26, 27]. The ferric uptake regulator (Fur) blocks the transcription of target genes by binding to specific DNA sequences and utilizing Fe2+ or Mn2+ as a corepressor [28,29,30]. This protein is capable of globally regulating bacterial cells and is involved in multiple levels of regulation [31, 32]. For instance, it is reported that the ferric uptake regulator (Fur) controls the expression of T6SS4 in Yersinia pseudotuberculosis by responding to manganese ions (Mn²⁺) and exerts this negative regulatory effect by specifically binding to the promoter region of T6SS4 [33]. Fur also regulates the expression of several proteins in the tricarboxylic acid cycle (TCA) and the Fe2+-dependent super-oxide dis-mutase (SodB) [34].

Burkholderia seminalis strain R456 was isolated from the rhizosphere of rice and further characterized using multiple molecular approaches, exhibits robust antagonistic activity against Rhizoctonia solani [35, 36]. Its effectiveness in controlling rice sheath blight in greenhouse conditions was demonstrated, highlighting its promising potential for antibacterial applications [37]. Additionally, we previously found a newly NRPS cluster in R456 related to siderophore iron metabolism regulation which was acquired by horizontal gene transfer from alage [38]. In the present study, we unveil a novel regulatory aspect wherein C4-dicarboxylates exert a negative control over the production of cFP in strain R456. C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates [39]. Our findings elucidate the involvement of C4-dicarboxylates in modulating the activity of NRPS related to cFP production by interacting with the ferric uptake regulator (Fur), which functions as a repressor of NRPS. This insight into the regulatory mechanism governing cFP biosynthesis not only advances our understanding of microbial metabolism but also holds implications for environmental ecology and the development of synthetic tools.

Materials and methods

Bacterial and fungal strains and growth conditions

The Burkholderia seminalis strain R456 was used in this study both as antifungal agent and the parental strain for genetic manipulations. Escherichia coli strains S17 or DH5α were used for suicide or expression plasmid replication, respectively. R456 were cultured in LB media or on agar plates at 30℃, while E. coli strains were cultured at 37℃. Antibiotics were added at the following concentrations when necessary: ampicillin (Amp),100 µg/mL; kanamycin (Km), 50 µg/mL; rifampicin (Rif), 50 µg/mL; and chloramphenicol (Chl), 3.4 µg/mL. Fungi were cultured in potato-dextrose agar (PDA) medium and incubated at 28℃. Minimum medium supplemented with various C4-dicarboxylates (oxaloacetate, succinate, malate or aspartate) was used for the sole carbon source assay to test the growth of R456 and its mutants [40]. The detailed sources of each strain and plasmid are listed in Table 1.

Table 1 Bacteria and plasmids used in this study
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Generation of random mutants

A random transposon library of B. seminalis strain R456 was generated using the EZTn5™< KAN-2 > Tnp Transposome™ Kit (Epicentre, Madison, WI, USA) according to the manufacturer’s instructions. Electroporation was carried out in a Bio-Rad Gene PulserXvell™ Electroporation System according to the manufacturer’s instructions. Cells were transformed onto LB plates with Km for isolation of putative transformants. The inserted region was identified using the Genome Walking Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. Sequencing, BLAST analysis, and alignment with the corresponding sequence from the R456 genome were also performed.

Antagonism assay

Single colonies of the Tn5 mutant strain and the wild-type R456 strain were picked and inoculated into 5 mL of LB liquid medium. The cultures were incubated in a shaker at 30 °C with shaking at 200 rpm/min for 5 days to allow for sufficient bacterial growth and metabolism. The cultures were centrifuged for 5 min at 8000×g and the supernatants were collected. For each test, 5 mL of PDA was mixed with 5 mL of the processed supernatant, and the mixture was poured onto Petri dishes to create agar plates. A 5 mm diameter plug of the activated Fg was inoculated at the center of each agar plate. A control group was set up by adding sterile water, following the above mentioned procedure. The plates were incubated at 28 °C for 5 days until the mycelium in the control group covered the entire plate. Each treatment was replicated three times. Antifungal assay of synthetic cyclo(Phe-Pro) was also conducted. Synthetic cyclo(Phe-Pro) powder (synthesized by Hangzhou Huahan Biotechnology Co., Ltd., China) was dissolved in sterile ddH2O to achieve concentrations of 1, 10, 100, 1000, and 10,000 ppm, which were stored for subsequent use. Nine mL of the PDA was mixed with 1 mL of the appropriately diluted cyclo(Phe-Pro) solution, and a 5 mm diameter plug of the activated Fg was inoculated at the center of each agar plate. Each treatment was replicated three times.

Construction of strain mutants and complementary strains

All the mutant strains were generated using an insertional mutagenesis method [41]. The complementary strains, on the other hand, were constructed by transferring a recombinational expression plasmid containing the full length of the gene and its upstream sequence of 500 bp into the corresponding mutants. Both the mutants and complementary strains were stored at -80℃ for further use.

Extraction and identification of secondary metabolites

To identify antifungal compounds, R456 was cultivated in LB broth at 30 °C for 4 days. Subsequently, the cell-free supernatant was obtained through centrifugation. The supernatant was extracted three times with ethyl acetate (v/v = 1:1) followed by evaporation. HPLC-MS was employed to separate and identify the desired compounds.

cFP production

Bacteria were inoculated from a single colony and cultured overnight until reaching an optical density (OD600) of 1.0, followed by a 1:100 re-inoculation into a new LB broth, and 100 µL of C4-dicarboxylates (1.0 mg/mL) was added as needed. One mL of bacterial culture was taken and filtered through a 0.22 μm membrane daily for five days. HPLC analyses were performed using a SHIMADZU LC-20AD HPLC system equipped with a CAPCELL PAK C18 column (250 mm× 4.6 mm, particle size of 5 μm) with the following program: solvent A consisting of 0.1% (v/v) formic acid and ddH2O, solvent B consisting of 100% acetonitrile; 2–25% B (0–20 min), 25–95% B (20–60 min), at a flow rate of 1 min− 1 and UV detection at 210 nm. cFPs were quantified based on their peak area at 210 nm using calibration curves obtained from authentic standards.

Primers, RNA extraction and qRT-PCR assay

The primers used for quantitative real-time (qRT)-PCR were listed in Table 2. Total RNA was extracted using RNAiso Plus (TAKARA, China) following to the manufacturer’s protocol. Reverse transcription of total RNA was carried out using the PrimeScript™RT reagent Kit with gDNA Eraser (TAKARA, China). qRT-PCR was performed on an ABI PRISM 7500 Sequence Detection System using ChamQ SYBR qPCR Master Mix Q311-02 (Vazyme, China). The recA gene served as the internal control. Each 20 µL reaction volume contained 500 ng cDNA, 1 × SYBR Green Mix, and 5 µM of the forward and reverse primers. Each sample and experiment were conducted in triplicate.

Table 2 Primers for cFP production regulation related genes
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Yeast one-hybrid assay

The predicted Fur-box transcription factor binding regions on the target gene promoter, as determined by the Softberry website were cloned into the pAbAi vector. The resulting recombinant plasmid, pAbAi-furbox, was linearized using the Bbsl restriction endonuclease and transformed into the Y1HGold yeast strain in the sensitive state. This yeast strain was selected on SD/-Ura/-AbA medium. After evaluating the AbA expression in the reporter strain Y1HGold/pAbAi-furbox, utilizing the SD/-Ura plate with gradient AbA concentration (0, 100, 200, 300, 400, 500, 1000 ng/mL) to determine the minimal inhibitory concentration, the Fur gene was constructed onto the expression vector pGADT7 and subsequently transformed into Y1HGold/pAbAi-furbox. Following this, Y1HGold/pGADT7-Fur + pAbAi-furbox was selected on SD/-Leu plates supplemented with screening AbA concentration (0, 200, 250, 300 ng/mL), along with 100 µM MnCl2 and 0.4 mM Malic acid, one of the C4-dicarboxylates.

Statistical analysis

All experiments were executed following a complete randomized design. The General Linear Model (GLM) procedure was employed to examine the significant differences among primary treatments, and individual comparisons between mean values were conducted using the Least Significant Differences (LSD) test (P < 0.05). Data underwent analysis of variance (ANOVA) using SPSS software version 16.0 (SPSS Inc., Chicago, IL).

Results

R456 mutant with high production of cFP

We previously observed that strain R456 demonstrated pronounced antagonistic activity against Fg [35]. To determine the specific active substances responsible for the antagonistic effects of strain R456, as well as to investigate the related genes that control the synthesis and secretion of these active substances, we conducted Tn5 transposon mutagenesis in the wild-type strain of R456. After two rounds of antifungal screening of 997 Tn5 insertion mutants of R456, the inhibition effects of 13 mutants were detected either increased or decreased. As shown in Fig. 1a, eight mutants exhibited a stronger inhibitory effect compared to the wild-type strain, while 5 mutants weakened the inhibition effect. The relative inhibition rate was used to demonstrate the change in inhibitory effect of the mutants relative to the wild-type strain (Table 3). Specifically, the eight mutants that exhibited an inhibitory effect were No. 88, 113, 117, 255, 268, 495, 885, and 189. These mutants showed an increase of inhibition to 147.58%, 124.51%, 132.27%, 131.50%, 135.68%, 123.53%, 128.11%, and 130.56%, respectively. On the other hand, the five mutants that demonstrated a reduced inhibitory effect was No. 85, 123, 216, 224, and 225. These mutants exhibited a decrease of inhibition to 59.62%, 67.66%, 63.50%, 68.18%, and 73.51%, respectively.

Fig. 1
figure 1

cFP was identified as the effective substance for inhibition of B. seminalis strain R456 against F. graminearum. (a) The inhibitory effect of culture supernatant of thirteen representative Tn5 mutants of B. seminalis strain R456 against F. graminearum. (b) Culture filtrate of Tn5 mutants of B. seminalis R456 were analyzed by HPLC. And mass spectrogram of cFP showed the first order mass spectrometry on the left side and secondary mass spectrometry on the right side. (c) Detection of the cFP production of 13 antagonistic Tn5 mutants. Different letters on the Bar chart represents significant differences between samples. The error bars represent standard errors of the means (n = 3). (d) Antagonistic effect of synthetic cFP on F. graminearum

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Table 3 Inhibition rate of thirteen Tn5 mutants against Fusarium graminearum
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Since we found mutant 88 have the highest antifungal effect, we further identified the effective substance of mutant 88 responsible for the antagonistic activity of R456. Here, we conducted a HPLC analysis on the culture supernatants of the bacterial culture. The results revealed a significant increase in the peak at 19 min in mutant 88 compared to the wild-type strain (Fig. 1b). Moreover, this peak was isolated through preparative chromatography. LC-MS analysis was performed on the differential peak, revealing a relative molecular mass of 245.12 in the primary mass spectrum (Fig. 1b). The secondary mass spectrum exhibited 7 fragment peaks at m/z 65.04, 70.06, 92.05, 154.07, 172.11, 217.13, and 245.12. Based on this data, the chemical formula of the differential peak was determined to be chemical composition C14H16N2O2. Following a search of the Reaxys database and the expected relative molecular mass (245.12) by LC-MS, the compound was identified as cyclo(Phe-Pro), which can exist as two enantiomers, cyclo(L-Phe-L-Pro) and cyclo(D-Phe-D-Pro). Confirmation was further achieved based on the antagonistic activity of the two chemically synthesized enantiomers, which showed that Fg can be inhibited by the former but not by the latter. Therefore, it can be inferred that the active compound was cyclo(L-Phe-L-Pro). Subsequent HPLC analysis revealed that the retention times of the synthetic cFP and the differential peak were identical. This confirms the accuracy of the assigned structure for the differential peak. The levels of cFP in the supernatant filtrate of the 13 mutants screened for altered antagonism were measured using HPLC. The cFP level in the supernatant filtrate of mutant 88 (71.9 ppm) was significantly higher compared to the wild type (23.1 ppm) with a significance of P < 0.001. However, the cFP levels in the supernatant filtrates of mutants 113, 117, 189, 255, 268, 495, 885, 85, 123, 216, 224 and 225 were 21.9, 17.6, 18.5, 18.2, 18.2, 19.1, 18.0, 17.0, 16.0, 16.2, 16.6 and 15.1 ppm, respectively, and no significant difference was found compared to the wild type R456 (Fig. 1c). Additionally, we chemically synthesized cFP and tested its antagonistic effect. As shown in Fig. 1d, the chemically synthesized cFP exhibit a significant inhibition effect on the growth of Fg, and the inhibition effect increased with the concentration of cFP. Therefore, we can conclude that cFP is one of the substances in the R456 culture filtrate that effectively inhibit Fg, and R456 mutant 88 has a higher production of cFP.

Dct system regulates cFP production in R456

Functional annotation of mutant 88 revealed it to be a histidine kinase DctB, which act as a sensor for C4-dicarboxylate transport. DctB belongs to the Dct system, which is responsible for transporting C4-dicarboxylates. Upon analyzing the R456 genome, we identified three genes related to the Dct system: DctB, DctD and DctA (Fig. 2a). To investigate the potential connection between the Dct system and R456’s antifungal activity, we constructed the site-inserted mutants of the Dct genes (Table 1) and examined their antifungal activity. Disruption of each of the dctA, dctB and dctD genes resulted in a significant increase in antifungal activity compared to the wild type (Fig. 2b). Interestingly, when we complemented these mutants with the wild- type Dct genes, their phenotype was restored to resemble that of the wild type. Additionally, the analysis of cFP production revealed that all three Dct mutants produced significantly higher levels of cFP compared to both the wild-type and complementary strains (Fig. 2c). These findings strongly suggest that the each gene in Dct system plays a role related to cFP production of R456.

Fig. 2
figure 2

Dct pathway is closely related to the production of cFP of B. seminalis strain R456. (a) Arrangement of DctB, DctD, DctA genes in Dct systems. (b) Antagonistic effect of filtrate of DctB, DctD, DctA mutants and complements of B. seminalis R456 on Fg. (c) The production of cFP of DctB, DctD, DctA mutants and complements of B. seminalis R456. Different letters on the Bar chart represents significant differences between samples. The error bars represent standard errors of the means (n = 3)

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Dct system was responsible for transport of C4-dicarboxylates

To determine the function of the Dct system in R456 and its connection to C4-dicarboxylates utilization, bacterial growth was assessed using C4-dicarboxylates as the sole carbon source. As shown in Fig. 3, R456 displayed significantly better compared to the Dct mutants when cultured in minimum medium amended with oxaloacetate, succinate, malate or aspartate as the sole carbon source. The wild-type strain reached a higher growth plateau than the Dct mutants, indicating that Dct system plays a crucial role in transporting C4-dicarboxylates from the external environment. The similar growth patterns of the three mutants suggest that mutations in any of the Dct genes can disrupt the functionality of the Dct system. Interestingly, mutations in the Dct system did not completely eliminate the ability of R456 to utilize C4-dicarboxylates as a carbon source, suggesting the presence of other potential transport systems in R456.

Fig. 3
figure 3

Growth curve of wild type and ΔDctB, ΔDctD, ΔDctA mutants of B. seminalis R456 in medium with four C4-dicarboxylates as the only carbon source

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C4-dicarboxylates suppress the production of cFP

Previous studies have shown that cFP is linked to the Dct transport system, responsible for importing C4-dicarboxylates into the cell. To investigate how exogenous C4-dicarboxylates affect cFP production in R456, we measured cFP levels in bacterial cultures supplemented with oxaloacetate, succinate, malate, or aspartate. As illustrated in Fig. 4a, cFP production by R456 significantly decreased in the presence of C4-dicarboxylates. Compared to the control, cFP production on day five was reduced by 79.73% with oxaloacetate, 80.76% with succinate, 83.61% with malate, and 77.96% with aspartate. Since we have proven that Dct system is responsible for the uptake of C4-dicarboxylates in R456, we determined the cFP production of the Dct mutants treated with or without C4-dicarboxylates. There were no significant differences between the control and C4-dicarboxylates treatments in all Dct mutants, except for strain ΔDctD treated with aspartate (Fig. 4b, c,d). Thus, the regulation of C4-dicarboxylates on cFP production was essentially cut off through genetic mutations in the Dct system.

Fig. 4
figure 4

C4-dicarboxylates suppress the production of cFP in B. seminalis strain R456. (a) The effects of four C4-dicarboxylates on cFP production of wild type of B. seminalis R456. (b, c, d) The effects of four C4-dicarboxylates on cFP production of ΔDctB, ΔDctD and ΔDctA mutants of B. seminalis R456 respectively. The error bars represent standard errors of the means (n = 3) (*, P < 0.01; ***, P < 0.001; NS, not significant)

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C4-dicarboxylates-mediated suppression of cFP need the collaboration of Fur

As cFP was produced as a byproduct of tyrocidine A by NRPS, we found NRPS cluster in R456 which was confirmed to have been obtained through horizontal gene transfer from algae in our previous work [38]. To investigate how C4-dicarboxylates regulate cFP production, we employed targeted gene knockout within the NRPS cluster to explore the link between cFP and NRPS activity. As illustrated in Fig. 5a, cFP production in R456 significantly decreased upon mutation of gene 2061 within the NRPS cluster compared to the wild type. In contrast, mutations in other NRPS genes did not lead to noticeable changes in cFP levels. These findings suggest that NRPS gene 2061 plays a critical role in cFP biosynthesis in R456. Bioinformatic analysis using the Softberry website identified a putative Fur binding box (Fur box) upstream of the cFP biosynthetic gene 2061 (Fig. 5b). This finding prompted us to investigate whether the transcription factor Fur regulates cFP production through interaction with the Fur box. To verify the presence of a functional Fur box and potential Fur binding, we employed a yeast one-hybrid (Y1H) assay, which identified transcription factors that bind to specific DNA sequences.

Fig. 5
figure 5

(a) The cFP production of wild type and NRPS cluster mutants. The error bars represent standard errors of the means (n = 3) (***, P < 0.001; NS, not significant). (b) Schematic diagram of the binding sites of Fur transcription factor

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We constructed an Y1H reporter strain harboring the plasmid pAbAi-furbox, containing the putative Fur box sequence. We initially tested this reporter strain for self-activation by exposing it to various concentrations of the antibiotic AbA (0, 100, 200, 300, 400, 500, and 1000 ng/mL) on selective media (Fig. 6a). The results revealed that the Y1HGold/pAbAi-furbox strain grew normally on plates lacking AbA, indicating no inherent resistance or self-activation. However, no colonies were observed on plates containing increasing AbA concentrations. This absence of growth suggests that the reporter strain alone is not resistant to AbA and requires additional activation for growth in the presence of the antibiotic. Fur, a ferric uptake regulator, requires Fe2+ to form active dimers, however, Fe2+ readily undergoes oxidation to Fe3+ in the natural environment. To address this, we employed 100 µM MnCl2 to the plate instead of Fe2+ with a gradient concentration of AbA (Fig. 6b). As expected, the absence of Mn2+ completely abolished yeast growth in the presence of AbA, indicating no Fur binding to the Fur box under this condition (treatment 1). Interestingly, the addition of 100 µM Mn2+ (treatment 2) partially restored yeast growth at the lowest gradient (100) with 300 ng/mL AbA. This suggests that high Mn2+ concentrations resulted in the binding of Fur to the Fur box. Furthermore, in the third treatment where Mn2+ and Malate were added, yeast was able to grow in five gradients (100, 10− 1, 10− 2, 10− 3, 10− 4) on plates with AbA concentrations of 0, 200 and 250 ng/mL. However, this decreased to four gradients (100, 10− 1, 10− 2, 10− 3) on plates with 300 ng/mL, indicated that the binding effects between Fur and Fur box were improved by the addition of external both of 100 µM Mn2+ ions and C4-dicarboxylates (Fig. 5b). Collectively, these findings demonstrate that Fur can bind to the Fur box located upstream of NRPS gene 2061. Moreover, the presence of C4-dicarboxylates appears to enhance Fur binding efficiency, potentially influencing cFP production regulation.

Fig. 6
figure 6

(a) Detection of self-activation of reporter strain Y1HGold/pAbAi-furbox in SD/-Ura medium plate with differnet concentrations of AbA (0, 100, 200, 300, 400, 500, 1000 ng/mL). (b) Detection of combination between Fur and Fur-box before R456_2061 using strain Y1HGold/pGADT7-Fur + pAbAi-furbox in SD/Leu medium plate with 0, 200, 250, 300 ng/mL of AbA in different treatments, added with 100 µM MnCl2 in the second treatment and additional 0.4 mM Malate in the third treatment

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Since C4-dicarboxylates enhanced the binding efficiency of Fur and the Fur box, we investigated whether Fur regulated NRPS gene 2061 expression and how C4-dicarboxylates might influence this regulation. Specifically, we found that Fur mutation led to a significant up-regulation of the expression level of NRPS gene 2061 (Fig. 7a). An increase in the production of cFP production was also detected, which indicated that Fur could inhibit the NRPS gene 2061 transcription (Fig. 7b). Furthermore, with the addition of C4-dicarboxylates, there was no significant difference in the expression level of the Fur protein, but the expression level of the NRPS gene 2061 was significantly down-regulated (Fig. 7c, d). These results were consistent with the decrease in cFP production upon the addition of C4-dicarboxylates. Therefore, we have demonstrated that C4 negatively regulates cFP production by enhancing the binding efficiency of Fur to the Fur binding box upstream of the NRPS gene 2061 involved in cFP synthesis.

Fig. 7
figure 7

Regulatory role of the C4-dicarboxylates with Fur and NRPS gene 2061 in synthesis of cFP. (a) Relative expression of NRPS gene 2061 were compared between the wild type (WT) and theΔR456_Fur mutant as well as the complemented mutant (ΔR456_Fur-com) (b) cFP production were detected in wild type (WT) and theΔR456_Fur mutant as well as the complemented mutant (ΔR456_Fur-com). (c) Relative expression of gene encoding Fur were compared between wild type (WT) and wild type added with four different C4-dicarboxylates. (d) Relative expression of gene encoding NRPS 2061 were compared between wild type (WT) and wild type added with four different C4-dicarboxylates. All the error bars represent standard errors of the means (n = 3) (***, P < 0.001; NS, not significant)

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Discussion

This study unveils a novel regulatory mechanism for cFP production in R456. We demonstrated that the Dct system, responsible for C4-dicarboxylates uptake, negatively regulates cFP biosynthesis (Fig. 8). C4-dicarboxylates, acting as signaling molecules beyond their metabolic roles, appear to influence cFP production through a Fur- mediated pathway. Upon importing via the Dct system, C4-dicarboxylates likely contribute to the transcriptional regulation of the NRPS gene cluster responsible for cFP synthesis. Our findings suggest that C4-dicarboxylates enhance Fur binding to the Fur box located upstream of a key NRPS gene (gene 2061), leading to its increased transcriptional repression. This ultimately results in decreased cFP production. This work provides significant insights into the complex interplay between C4-dicarboxylate metabolism, Fur-mediated regulation, and cFP biosynthesis in R456.

Fig. 8
figure 8

The putative model for the negatively regulation of C4-dicarboxylates with Fur to the production of cFP in B. seminalis strain R456

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The rhizosphere microbiome harbors a wealth of bacteria with the potential to act as biological control agents against plant pathogens [42]. Notably, strain R456, isolated from rice rhizosphere soil displays a broad antifungal spectrum [36]. Our findings demonstrate its strong inhibitory activity against the previously reported rice pathogen Rhizoctonia solani [37]. The potent inhibitory effects on the fungal pathogens suggest that strain R456 holds immense potential for controlling diseases in rice crops.

Historically, cyclic dipeptides have been recognized for their ability to influence numerous important biological processes [43]. These compounds are commonly found in protein and polypeptide hydrolysates and are also produced by various organisms such as bacteria, lichens, and fungi [44]. cFP has been previously identified in Burkholderia species and reported to exhibit antifungal and nematocidal activities [45]. Our study presents the first identification of cFP in B. seminalis (strain R456), suggesting its potential prevalence within this genus. Functional characterization of the cFP produced by R456 confirmed its strong antifungal activity, aligning with observations from other studies [46, 47]. Notably, we observed minimal antibacterial activity, evident from the lack of prominent inhibition zones (data not shown). This finding is consistent with reports by [48], who employed a microplates assay to assess cFP’s antibacterial effects. Collectively, these findings suggest that the differential inhibitory effects of R456 against fungi and bacteria can be attributed to the selective antimicrobial spectrum of cFP it produces. Additionally, among the 13 Tn5 mutants screened in this study, all displayed either enhanced or reduced antagonistic effects. Notably, apart from mutant 88, which showed a significant increase in cFP content, the other mutants did not exhibit noticeable changes in cFP levels. As shown in Fig. 1a and c, other mutants that does not produce more cFP (e.g. 113, 117, 189, 255, 268, 495, 885) has greater antifungal activity than native strain R456. This suggests that the supernatant of R456 contains not only cFP but also potentially other active substances, additional factors are involved in the antagonistic activity of R456 strain, consistent with previous findings that identified a diverse array of bioactive secondary metabolites contributing to the antifungal capabilities of Bacillus species [49], which require further investigation.

Compared with chemical synthesis, the biosynthesis research of DKPs has not been fully studied. NRPS and cyclodipeptide synthases (CDPSs) are the two most important enzyme-related pathways in DKPs biosynthesis based on current researches [50]. Previous work explored fermentative cFP production using the heterologous host Escherichia coli [51]. This study identified factors influencing cFP production, including variations in genetic setups medium composition, and temperature. However, the cFP production might be reduced due to the decrease of bacterial growth.

In our study, we determined the impact of C4-dicarboxylates on cFP production. Importantly, we observed the uptake of C4-dicarboxylates played a crucial role in reducing cFP production. It was reported that LapD and phosphotransfer protein HptB indirectly modulated the levels of cyclic di-GMP (c-di-GMP), a crucial secondary messenger involved in bacterial virulence and biofilm formation [52]. This was somewhat similar to our signaling perception pathway, but our findings introduced some innovations, suggest that the Dct system is not only crucial for strain R456 signaling perception of transporting and utilizing C4-dicarboxylates, but it is also working together with Fur protein and closely linked to the production of cFP.

We previously identified a new nonribosomal peptide synthetase (NRPS) cluster that was acquired through horizontal gene transfer. This cluster plays a crucial role in iron metabolism by affecting the production of all major siderophores. It likely exerts its influence by regulating the transcriptional expression of the ferric uptake regulator (Fur) [38]. However, the relationship between NRPS and Fur is still not clear. Here the hypothesis of C4-dicarboxylates closely realted to cFP production was further supported by our observation that exogenous C4-dicarboxylates were involved in the regulatory process of NRPS and suppressed the production of cFP by cooperating with the ferric uptake regulator (Fur). These results suggest that C4-dicarboxylates and Fur likely play critical roles in regulating bacterial cFP production. Given cFP’s antifungal properties, this study sheds light on the complex regulatory mechanisms governing bacterial secretion of this essential compound.

Conclusions

In this study, we demonstrate the regulation of cFP production by C4-dicarboxylates in B. seminalis strain R456. We demonstrate that C4-dicarboxylates, taken up by the Dct system, negatively regulate cFP biosynthesis. Disrupting the Dct system through targeted mutations led to a significant increase in cFP production, supporting its role in cFP regulation. Furthermore, exogenous C4-dicarboxylates significantly suppressed cFP production in the wild-type strain. Mechanistic investigations revealed that C4-dicarboxylates enhance the binding of the transcription factor Fur to the Fur box located upstream of a key NRPS gene (gene 2061) involved in cFP synthesis. This enhanced binding likely results in Fur-mediated repression of NRPS gene 2061 transcription, ultimately leading to decreased cFP production. Our findings provide new insights into the intricate interplay between C4-dicarboxylate metabolism, Fur-mediated regulation, and cFP biosynthesis in B. seminalis. This work paves the way for further studies to elucidate the detailed mechanisms by which C4-dicarboxylates modulate Fur activity and its interaction with the NRPS promoter region. Unraveling these intricate regulatory processes will not only deepen our understanding of cFP production but also shed light on the broader mechanisms governing cFP biosynthesis and regulation in bacteria.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP2024R123), King Saud University, Riyadh, Saudi Arabia. In addition, we want to acknowledge all our research team for being supportive during the research period.

Funding

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (LZ24C140004), National Natural Science Foundation of China (32072472, 32372614), Key Research and Development Project in Zhejiang Province (2019C02035), Hangzhou Science and Technology Development Plan Project (20231203A05), State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (grant number 2021DG700024-KF202415); “San Nong Jiu Fang” Science and Technology Plan439Project (2023SNJF040). This work was funded by the Researchers Supporting Project number (RSP2024R123), King Saud University, Riyadh, Saudi Arabia.

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Author notes

  1. Xinyan Xu and Liu Liu contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Rice Biology and Breeding, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang Key Laboratory of Biology and Ecological Regulation of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China

    Xinyan Xu, Liu Liu, Yang Zhang, Rahila Hafeez, Munazza Ijaz, Temoor Ahmed & Bin Li

  2. Institute of Eco-Environmental Protection, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China

    Lihui Xu

  3. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Hayssam M. Ali

  4. Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khoud 123, Muscat, Oman

    Muhammad Shafiq Shahid

  5. Xianghu Laboratory, Hangzhou, 311231, China

    Temoor Ahmed

  6. Department of Life Sciences, Western Caspian University, Baku, Azerbaijan

    Temoor Ahmed

  7. Faculty of Agriculture, University of Zagreb, Svetošimunska Cesta 25, Zagreb, 10000, Croatia

    Gabrijel Ondrasek

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Contributions

X.X. and L.L.: Writing – original draft, methodology, investigation, formal analysis, data curation and conceptualization. L.X., Y.Z. and R.H.: Writing – review & editing, validation and formal analysis. M.I.: Formal analysis, investigation and data curation. H.M.A.: Writing – review & editing, validation and investigation. M.I., M.S.S., T.A. and G.O.: Writing– review & editing, project administration, formal analysis. B.L.: Writing – review & editing, supervision, project administration, methodology, funding acquisition, formal analysis and conceptualization.

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Xu, X., Liu, L., Xu, L. et al. Regulatory mechanism of C4-dicarboxylates in cyclo (Phe-Pro) production. Microb Cell Fact 23, 255 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02527-6

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Microbial Cell Factories

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