Open AccessArticle

NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors

Jiaxin Qin

¹,

Ziqi Wang

¹,

Cheng Qian

¹,

Guohui Ji

¹,

Yizhuo Zhang

¹,

Zhanglei Cao

^1,†,

Bing Yan

¹ and

Jun Cai

^1,2,3,*

Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China

Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, China

Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300071, China

Author to whom correspondence should be addressed.

^†

Current address: Ningbo Health Gene Technologies Co., Ltd., Ningbo 315048, China.

Microorganisms 2025, 13(1), 212; https://doi.org/10.3390/microorganisms13010212

Submission received: 25 December 2024 / Revised: 12 January 2025 / Accepted: 15 January 2025 / Published: 20 January 2025

(This article belongs to the Section Molecular Microbiology and Immunology)

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Figure 1
NupR inhibits the expression of plcR during the stationary phase. (A) The 5′UTR of plcR. The conserved binding sites of Spo0A, PlcR, and NupR are shaded in blue, green, and gray. The ATG of PlcR is marked in pink. (B) β-galactosidase activities of PnupR-lacZ in BMB171 cultivated in LB and SSM media. (C) plcR mRNA levels in BMB171 and ΔnupR cultivated in LB medium. (D) β-galactosidase activities of PplcR-lacZ in BMB171 and ΔnupR cultivated in LB medium. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, non-significant. Data represent the mean ± SD of three samples. "> Figure 2
NupR can directly or indirectly regulate the expression of the plcR regulon. (A) β-galactosidase activities of Ppap-lacZ, Phemo-lacZ, and Pplc-lacZ in BMB171 and ΔnupR cultivated in the SSM medium. (B) β-galactosidase activities of Pmog-lacZ in BMB171 and ΔnupR cultivated in the SSM medium. (C) NupR binds directly to the promoter regions of mogR labeled by FAM. (D) BMB171, and ΔnupR strains were dripped on 0.5% and 0.3% soft agar plates (LB medium) and incubated at 28 °C. ** p < 0.01; *** p < 0.001. Data represent the mean ± SD of three samples. "> Figure 3
The effect of the culture supernatant of BMB171 and ΔnupR strains on the cell viability of Sf9 cells. * p < 0.05; ns, no significant. Data represent the mean ± SD of three samples. "> Figure 4
Expression of plcR is induced by glucose. (A) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 0.1% glucose. (B) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in LB medium with or without 0.1% glucose. *** p < 0.001; ns, no significant. Data represent the mean ± SD of three samples. "> Figure 5
Expression of plcR is Induced by Nucleosides. (A) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 1mM different nucleosides. An, adenosine; Cn, cytidine; Gn, guanosine; Un, uridine. (B) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 0.1% ribose. (C) plcR mRNA levels in BMB171 and ΔnupR cultivated in LB medium with or without 0.1% ribose. Ri, ribose. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant. Data represent the mean ± SD of three samples. "> Figure 6
Schematic representation of the regulation of PlcR. plcR is autoregulated and under the negative control of Spo0A~P and NupR. CodY positively controls the expression of plcR by regulating the expression of opp. The YvfTU two-component system is also involved in plcR expression via a yet unknown mechanism. Process lines are shown in black, and regulatory relationships are indicated by blue and red lines, with clipped heads for facilitation, horizontal lines for inhibition, solid lines for direct, and dashed lines for indirect. In addition, the red lines indicate processes influenced by ribose or glucose. ">

Versions Notes

Abstract

NupR is a nucleoside permease regulator belonging to the GntR family, mainly regulating nucleoside transport in Bacillus thuringiensis. A conserved binding site for NupR was found in the promoter region of plcR. This study aimed to investigate the regulation of the virulence regulator PlcR by NupR and its impact on Bt virulence. We demonstrated that NupR can directly repress the expression of plcR. The expression of plcR can be induced by glucose and nucleosides. Glucose impacts the expression of plcR mainly through Spo0A, while the induction effect of nucleosides may be due to the production of ribose through nucleoside catabolism. In addition, NupR regulates the expression of the PlcR regulon, including hemolysin, phospholipase C, papR, and oligopeptide permease, which could result in the culture supernatant of BMB171 being less virulent to sf9 cells compared to the nupR knockout strain. The results combine the nutritional status of cells with virulence to form a regulatory loop, providing new ideas and research foundations for the study of bacterial virulence.

Keywords:

PlcR; NupR; virulence; glucose; Bacillus thuringiensis

1. Introduction

The transcription factor PlcR regulates the expression of approximately 45 genes in the Bacillus cereus group of spore-forming Gram-positive bacteria [1,2]. In addition to the pathogenic Bacillus cereus, which causes foodborne and opportunistic infections, the Bacillus cereus group also includes six other species, such as the anthrax pathogen Bacillus anthracis (Ba) and the insect pathogen Bacillus thuringiensis (Bt). PlcR is truncated in Ba, rendering it inactive [1,3,4]. Most of the genes controlled by PlcR in B. cereus and B. thuringiensis encode proteins related to food supply and virulence (phospholipases, proteases, hemolysins, toxins, etc.), cell protection, and environmental sensing. Deletion of plcR significantly reduces the virulence of B. thuringiensis (against insects) and B. cereus (in mouse infection models) [5].

It has been demonstrated that PlcR can bind to DNA at specific sequences known as “PlcR boxes”, located upstream of the controlled genes and at varying distances in front of the −35 box of the promoter [1,6,7]. Transcription of plcR begins shortly before the stationary phase at T0 and reaches a plateau two hours later (T2) [8]. Transcription of plcR is autoinduced [8], and it is inhibited by the sporulation factor Spo0A [9]. In addition, the expression of plcR is also regulated by the YvfTU two-component signal system located near its genetic locus in Bacillus cereus. During the high expression of the PlcR regulon phase, the expression of plcR in the yvfTU mutant is only 50% of that in the wild-type strain. Moreover, the yvfTU mutant exhibits slightly lower virulence in the Galleria mellonella insect model than the wild strain [10]. It has been reported that the expression of plcR and PlcR-dependent genes in B. cereus requires the global regulator CodY [11,12]. The impact of CodY on the expression of virulence factors is not achieved through the direct binding of CodY to the promoter regions of plcR or PlcR-dependent genes. Instead, it participates in the expression of virulence factors in B. thuringiensis through its role in the import of the quorum-sensing signal peptide PapR.

PlcR requires activation by PapR. This peptide, which is expressed as a precursor under the control of PlcR, is exported outside the cell. After being processed by proteases such as NprB in the periplasmic space, PapR can become the mature pentapeptide PapR₇, which accumulates gradually in the periplasmic space as a signaling molecule, responding to the density and state of the bacterial community and is imported into the bacterial cell through the bacterial surface oligopeptide permease OppABCDF after accumulating to a certain concentration. Once transported into the cell, PapR₇ binds to PlcR, causing a conformational change in PlcR, forming a PlcR-PapR complex dimer that recognizes and binds to the target and activates the expression of downstream genes [6,13,14,15]. Thus, the three partners—PlcR, OppABCDF, and PapR—act as a quorum-sensing system.

Previous research has shown that in the Bacillus thuringiensis BMB171 strain, the GntR/HutC family transcriptional regulator NupR (nucleoside permease regulator) can directly bind to the 5’ noncoding region of plcR. The NupR transcriptional regulator is highly conserved in the Bacillus cereus group, and NupR-like proteins are also widely present in Bacillus subtilis, Clostridium difficile, Pseudomonas aeruginosa, and Streptococcus pneumoniae. Whether this protein is involved in regulating plcR, the mechanism of regulation, and its significance are still unknown [16].

Therefore, in this study, we investigated the regulatory effects of NupR on plcR and its regulon at different time points and assessed the impact of nupR deletion on the virulence of Bt. NupR (nucleoside permease regulator) can directly regulate the expression of four nucleoside permeases. Moreover, glucose can induce nupR expression through CcpA. Thus, we conducted further investigations into the response of plcR to glucose and nucleosides.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

The bacterial strains used in this research were stored in our laboratory at −80 °C and are listed in Supplementary Table S1. The strains were removed from the −80 °C freezer, activated in 5 mL Luria–Bertani (LB) liquid medium, and subsequently streaked onto LB agar plates containing the appropriate antibiotics. They were then cultivated in 5 mL LB medium containing the appropriate antibiotics for experimental investigations. B. thuringensis BMB171 and its derived strains were cultured in LB or Schaeffer’s sporulation medium (SSM) [17] at 28 °C with shaking at 200 rpm. Escherichia coli strains were cultured in LB media at 37 °C with shaking at 200 rpm.

2.2. RNA Extraction and RT–qPCR

The BMB171 strain and its derivative strains were cultured in SSM to the early stationary phase. Following the supplementation of the culture medium with the inducers, a 30 min induction period was implemented to allow for the activation of the targeted genetic pathways. The bacterial solution was centrifuged to remove the supernatant, and the cell pellet was resuspended in 1 mL of RNAiso Plus. Zirconium beads were added for cell disruption. After grinding, 200 µL of RNA extraction solution was added to the homogenate and it was mixed gently and allowed to stand for phase separation. It was centrifuged at 12,000 rpm for 20 min at 4 °C, and the upper aqueous phase was transferred to a new RNase-free 1.5 mL centrifuge tube. An equal volume of pre-chilled isopropanol was added and incubated at −20 °C to enhance RNA precipitation. It was centrifuged again at 12000 rpm for 20 min at 4 °C, the supernatant was discarded, and the pellet was washed twice with anhydrous ethanol. Finally, the centrifuge tube was placed in a 37 °C metal bath to evaporate any remaining alcohol. The RNA pellet was dissolved in RNase-free water and its concentration was determined.

For reverse transcription, 1 µg of RNA was converted to cDNA using a reverse transcription kit (Takara, Biotechnology Corporation, Dalian, China). The cDNA was then used as a template for quantitative PCR (qPCR) with TB Green Premix Ex Taq™ II (Tli RNaseH Plus) (Takara). The 16S rRNA gene was used as an internal control [16,18,19].

2.3. Determination of β-Galactosidase Activity

Overnight cultures were transferred to 100 mL of SSM or LB medium and cultured at 200 rpm and 28 °C until the cells reached the end of the exponential growth period (T0). A total of 1 mL of sample was taken to determine the OD₆₀₀ of the bacterial solution. Then, 2 mL samples were taken and centrifuged to determine the β-galactosidase activity at the desired time. The method has been described elsewhere [16,20]. The cell pellet was resuspended in 1 mL of Z buffer; then zirconium beads were added and the cells disrupted using a tissue breaker. The resulting supernatant was collected as the reaction solution. Next, 800 µL of Z buffer was added to a 2 mL tube, which was placed in a 37 °C metal bath. Then, 200 µL of the reaction solution and 200 µL of ONPG was added to initiate the reaction, recording the start time as T0. Finally, 500 µL Na₂CO₃ (1M) was added to terminate the reaction, recording the stop time as T1. After the reaction was stopped, the optical density at 420 nm was measured. The Miller unit was calculated using the following formula: Miller unit = 1000 × OD₄₂₀/(T1 − T0) × OD₆₀₀ × V. V = 200 µL; T1 − T0 = reaction time.

2.4. Electrophoretic Mobility Shift Assay (EMSA)

The purification of NupR and the electrophoretic mobility shift assay were performed as previously described [16]. The purified NupR-His protein was incubated with FAM-labeled PmogR fragments in a buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol (DTT), and 4% glycerol) at 28 °C for 25 min. To verify whether the binding of the protein to DNA is specific, 3 µg of salmon sperm DNA and 3 µg of unlabeled PmogR DNA fragments were added separately. Next, the mixture was loaded onto a 6% native polyacrylamide gel, which had been pre-electrophoresed in 0.5× TBE buffer for 30 min. The gel was run at 140 V for 90 min at 4 °C. After electrophoresis, the bands of the biotin-labeled DNA were visualized using the ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) molecular imager.

2.5. Determination of Cell Viability

Spodoptera frugiperda ovarian Sf9 cells were used to determine the cell viability and cultivated as described previously [21]. The BMB171 strain and ΔnupR strain were cultivated in Luria–Bertani (LB) medium to the T2 phase, after which the culture supernatant was collected by centrifugation. The supernatant was sterilized and diluted 10 times with PBS. Subsequently, 20 microliters of the diluted supernatant were added to 200 microliters of Sf9 insect cell culture. The cell viability was determined at 12-, 24-, and 48-h post-culture using the Cell Counting Kit-8 (CCK-8), following the manufacturer’s instructions.

2.6. Motility Assay

Overnight cultures were transferred to 100 mL of LB medium and cultured at 200 rpm and 28 °C until the cells reached the T2 phase. The samples at this point were used for motility assy. Swimming assays were performed on LB soft agar plates as described [22]. Freshly made soft agar (0.5% soft agar plates: 0.75 g agar/100 mL LB, 0.3% soft agar plates: 0.5 g agar/100 mL) was kept at 55 °C until the beginning of the assay. Twenty milliliters of soft agar were poured into a Petri dish and allowed to sterilize and dry for 10 min. Two microliters of the diluted culture were then spotted in the center of a Petri dish for inoculation, followed by incubation at 28 °C.

2.7. Statistical Analyses

The data were subjected to one-way analysis of variance using the Student’s t-test. Significance thresholds were specified as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001, and ns, no significant. Data represent the mean ± SD of three samples.

3. Results

3.1. NupR Inhibits the Expression of plcR During the Stationary Phase

Previous results showed that NupR directly binds to the intergenic region of plcR and baci [16]. Based on the NupR boxes, we predicted the DNA sequence (AGTGGTATGACAACTCAAAA) that NupR directly binds to, which is located upstream of the RBS of plcR (Figure 1A), closer to the start codon of plcR than that of baci. Therefore, we speculate that NupR directly regulates the expression of plcR. To verify this hypothesis, we first determined the expression phase of nupR. The results showed that in either rich medium LB or minimal medium SSM, the promoter activity of nupR reached the highest level at T1, suggesting that it mainly plays a regulatory role during the stationary phase (Figure 1B).

To explore the difference in plcR expression between the BMB171 strain and the ΔnupR strain, we measured the mRNA levels of plcR in both strains. The results showed that the mRNA level of plcR in the ΔnupR strain was significantly higher than that in the BMB171 strain during the stationary phase (Figure 1C). In addition, we also connected the promoter and 5’ noncoding region of plcR to the lacZ reporter gene and transferred it into the BMB171 strain and the ΔnupR strain. The β-galactosidase activity assay results showed that the activity of the plcR promoter in the BMB171 strain was significantly lower than that in the ΔnupR strain, which is consistent with the qRT-PCR results (Figure 1D). In summary, the expression of plcR is negatively regulated by NupR during the stationary phase.

3.2. NupR Can Directly or Indirectly Regulate the Expression of the plcR Regulon

Given that NupR directly downregulates the expression of plcR, it may indirectly control the transcription of PlcR-dependent genes. It has been reported that PlcR positively regulates the transcription of various virulence genes, such as phospholipase C [23], hemolysin [4], and papR [2]. Therefore, expression vectors with the promoters of these genes fused to lacZ were constructed and introduced into the BMB171 strain and the ΔnupR strain to measure β-galactosidase activity. The results showed that the plc, hemolysin, and papR promoter activities significantly increased in the ΔnupR strain (Figure 2A). NupR may reduce the transcription of these three genes by inhibiting plcR.

mogR is the only gene experimentally verified to be directly downregulated by PlcR, which encodes the motility gene repressor protein [24,25]. The activity of the mogR promoter was measured in the BMB171 strain and the ΔnupR strain. The results indicated that the promoter activity was significantly increased in the ΔnupR strain, suggesting that NupR represses mogR expression (Figure 2B).

If NupR modulates the expression of mogR through PlcR, the expression of mogR in the ΔnupR strain should theoretically be downregulated. However, the experimental results failed to confirm this hypothesis. Thus, an EMSA assay was conducted to investigate the regulation of mogR gene expression by NupR. The result indicated that the NupR protein can directly bind to the promoter sequence of the mogR gene (Figure 2C). Thus, NupR can directly inhibit the expression of mogR. Additionally, the impact of the nupR deletion on the motility of the strain was measured. The results showed that the motility of the ΔnupR was significantly reduced (Figure 2D). MogR represses the synthesis of flagella, so the reduction in motility may be due to the upregulation of mogR expression in ΔnupR, which is consistent with the enzymatic activity results.

3.3. Effect of nupR Deletion on the Virulence of Bacillus thuringiensis

Since NupR influences the expression of virulence factors, the deletion of nupR may affect the production of virulence factors of the strain, which can change the virulence of the strain. The Bacillus thuringiensis in this study did not contain insecticidal proteins. The toxicity of the strain culture supernatant in the stable phase was determined against S. frugiperda Sf9 cells, the ovary cells of the grass-coveting nightshade moth, to test the effect of nupR deletion on the virulence of the BMB171 strain. The results showed that the toxicity of the ΔnupR supernatant was higher than that of the BMB171 strain at 24 h and 48 h (Figure 3). Therefore, NupR may attenuate its virulence by inhibiting the expression of the cytotoxicity regulator.

3.4. Expression of plcR Is Induced by Glucose

The expression of nupR is positively regulated by CcpA and is induced significantly by glucose [16]. NupR directly inhibits the expression of plcR, suggesting that the expression of plcR may be repressed by glucose. The effect of glucose on plcR expression was determined. The strains were cultured in LB or SSM medium until the early stationary phase and induced with 0.1% glucose for 30 min. The mRNA levels of plcR in the BMB171 strain and the ΔnupR strain were measured. The results showed that in the SSM medium, the mRNA level of plcR was upregulated 1.7-fold in the BMB171 strain and 5.3-fold in the ΔnupR strain. In the LB medium, it was upregulated 9.5-fold in the BMB171 strain and 12.5-fold in the ΔnupR strain (Figure 4). After the deletion of nupR, the expression of plcR was no longer inhibited by NupR. It became more sensitive to glucose, with a higher fold increase in expression than the BMB171 strain.

Glucose still induces the expression of plcR in the ΔnupR strain. Therefore, the promoting effect of glucose on the expression of plcR is evidently due to other reasons. It has been reported that the expression of plcR is inhibited by Spo0A. A conserved binding site for Spo0A was predicted in the promoter region of plcR (Figure 1A). Moreover, the mRNA level of plcR in the Δspo0A strain is 15 times higher than in the BMB171 strain (Figure 5B). The additional glucose may promote the expression of plcR by reducing the inhibition of Spo0A. The mRNA level of plcR in the Δspo0A strain under glucose induction was measured. The results showed that in the absence of spo0A, the expression of plcR was no longer promoted by glucose (Figure 4). However, the expression level of spo0A in the BMB171 strain did not change in the presence of glucose, leading us to speculate that glucose may reduce the phosphorylation of Spo0A, weakening its inhibition of plcR and resulting in an increase in the expression of plcR.

3.5. Expression of plcR Is Induced by Nucleosides

nupR encodes a regulator of nucleoside permeases, affecting the utilization of guanosine, adenosine, uridine, and cytidine [16]. Therefore, we hypothesized that nucleosides may act as regulators in virulence modulation. Like the induction method with glucose, after adding 1 mM of different nucleosides to the culture medium, the mRNA levels of plcR in both the BMB171 strain and the ΔnupR strain were measured. The results showed that the expression of plcR in both strains significantly increased after adding different nucleosides, with guanosine showing the most pronounced effect (Figure 5A). The expression of plcR was upregulated 6-fold in the BMB171 strain and 10-fold in the ΔnupR strain by guanosine. Since all these nucleosides contain ribose, we speculate that the effect of nucleosides on plcR may primarily be due to ribose.

Similarly, after adding 0.1% ribose to the culture medium, the expression level of plcR was measured. The results showed that the expression of plcR increased in both strains after adding ribose. In the SSM medium, the mRNA level of plcR was upregulated 1.3-fold in the BMB171 strain and 1.9-fold in the ΔnupR strain (Figure 5B). In the LB medium, the mRNA level of plcR was upregulated three-fold in the BMB171 strain and five-fold in the ΔnupR strain (Figure 5C). In addition, the effect of ribose on plcR expression was measured in the Δspo0A strain, and the results showed that after the deletion of spo0A, ribose no longer affected the expression of plcR (Figure 5B). Overall, Spo0A plays a leading role in glucose and ribose induction.

4. Discussion

This study discovered that the nucleoside permease regulator NupR can directly inhibit the transcription of plcR during the stationary phase, thereby impacting the expression of PlcR-dependent virulence factors. After nupR deletion, the strain supernatant’s toxicity to Sf9 cells was significantly enhanced. This further proves the regulatory role of NupR on the virulence of Bt.

Six genes encoding oligopeptide permease were found to be differentially expressed in the transcriptomic data of ΔnupR and BMB171. Oligopeptide permeases are ATP-binding cassette transporters consisting of five proteins: two membrane-integrating proteins that form the actual pore (OppB and OppC), two ATPases that bind to the membrane proteins to provide the energy required for transport (OppD and OppF), and an extracellularly oriented, membrane-anchored substrate-binding protein (SBP) (OppA). OppBCDF has been reported to be required for PapR import [26,27]. In addition, OppA is not the only SBP involved in recognizing PapR, and several other OppA-like proteins can import this peptide [28]. Therefore, oligopeptide permease is indispensable in PlcR activation. The mRNA levels of these six permeases were determined in the BMB171 strain and the ΔnupR strain. The results showed that two permease (oppAs) genes were significantly upregulated during the stable phase (Figure S1). Therefore, after nupR deletion, the mRNA level of plcR was elevated, and its activation efficiency was also likely increased by the high expression of the oligopeptide permeases and papR.

The expression of plcR is induced by glucose, and its induction fold increased after nupR was deleted. After the deletion of spo0A, plcR expression was no longer affected. Therefore, the inducing effect of plcR by glucose is caused by Spo0A. NupR does, indeed, have an inhibitory effect on the expression of plcR under the action of glucose, but this effect is no longer evident after the deletion of spo0A. In the absence of spo0A, the expression of plcR is upregulated approximately 15 times. In this case, the inhibitory effect of NupR becomes insignificant. As a regulator of nucleoside permeases, NupR can directly affect the utilization of nucleosides by bacteria. The expression of plcR is significantly increased after adding different nucleosides, and the breakdown product of nucleosides, ribose, may cause this effect.

The global regulatory factor CodY, prevalent in low-GC-content gram-positive bacteria, regulates early stationary phase genes and initiates sporulation [29]. CodY positively regulates the expression of oligopeptide permeases, oppABCDF, and several other Opp-like proteins, which influence the transport of PapR, leading to the activation of PlcR [28]. Consequently, CodY exerts a stimulatory effect on the transcription of PlcR-dependent genes. In contrast to CodY, NupR exerts a suppressive influence on the expression of oppA, inhibiting the activation of plcR. Hence, the regulatory impacts of CodY and NupR on plcR expression are antagonistic. When the culture medium is replete with glucose, resulting in reduced levels of branched-chain amino acids (BCAAs), the function of CodY may be diminished [29]. Consequently, in the presence of glucose, the expression of the plcR gene may be reduced via CodY.

The pleiotropic regulator PlcR promotes the transcription of virulence factor genes during the stationary phase, such as degradation enzymes, antibiotics, toxins, etc., ensuring the production of specific compounds necessary for spore formation [9]. Figure 6 shows a schematic representation of the regulation of PlcR based on diagrams drawn by Slamti et al. [30]. The expression of plcR is controlled by Spo0A and the nucleoside permease regulator NupR. As bacteria enter the late exponential phase and the nutrients in the culture medium are consumed, the strain transitions from exponential growth to a transitional state, where the concentration of Spo0A-P increases sharply and represses plcR expression [31,32,33]. NupR further suppresses the expression of plcR during the stationary phase. If additional glucose is added, it could reduce the concentration of Spo0A-P by inhibiting the Calvin cycle, resulting in a high expression of plcR [34]. Glucose will also suppress the expression of plcR by inducing the expression of NupR, compensating for the reduced effect of Spo0A. Besides, the promotion of plcR by CodY is diminished under glucose conditions. NupR and CodY are auxiliary modulators within this regulatory framework, whereas Spo0A assumes the primary regulatory function. This is underscored by the observation that following the deletion of spo0A, the expression of plcR becomes independent of glucose influence.

This research identifies the regulator NupR as a component of the regulatory network governing plcR, which directly modulates plcR expression and may also exert indirect effects on PlcR activation through the regulation of oppA expression. It has significant implications for the virulence of the BMB171 strain. We elucidated, for the first time, the role of NupR in regulating virulence. Furthermore, we demonstrated that plcR expression can be induced by glucose and nucleosides, highlighting the nuanced regulatory role of NupR and the decisive role of Spo0A in this process. This regulatory circuit may establish a connection between strain virulence, nutritional conditions, and fate decisions, thereby coordinating bacterial behavior. Besides, this study revealed that the additional inclusion of glucose and nucleosides in the LB medium or the nupR deletion significantly enhances the expression of plcR, thereby increasing the virulence of the bacteria. This finding provides a theoretical foundation for future studies on bacterial toxin production or related research.

5. Conclusions

We determined that plcR is regulated by NupR (nucleoside permease regulator), a member of the GntR family, which is a novel component of the regulatory network governing plcR. Besides, additional glucose and nucleosides can induce plcR expression mainly through Spo0A. This regulatory circuit may establish a connection between strain virulence, nutritional conditions, and fate decisions, thereby coordinating bacterial behavior.

Supplementary Materials

The following supporting information can be downloaded online: https://www.mdpi.com/article/10.3390/microorganisms13010212/s1. Figure S1: opp mRNA levels in BMB171 and ΔnupR cultivated in SSM medium (T2); Table S1: Bacterial strains and plasmids used in this study. References [16,35] are cited in the supplementary materials.

Author Contributions

Conceptualization, J.Q., B.Y. and J.C.; Methodology, J.Q., Z.W., C.Q. and Y.Z.; Investigation, J.Q., C.Q., G.J., Z.C. and J.C.; Resources, J.C.; Data curation, J.Q., Z.W., G.J., Y.Z. and Z.C.; Writing—original draft, J.Q.; Writing—review & editing, J.C.; Visualization, J.C.; Supervision, B.Y. and J.C.; Project administration, J.C.; Funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by grants from the National Natural Science Foundation of China [32272610].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available at Figshare (https://doi.org/10.6084/m9.figshare.27211629).

Conflicts of Interest

Author Zhanglei Cao was employed by the company Ningbo Health Gene Technologies Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Agaisse, H.; Gominet, M.; Økstad, O.A.; Kolstø, A.; Lereclus, D. PlcR Is a Pleiotropic Regulator of Extracellular Virulence Factor Gene Expression in Bacillus thuringiensis. Mol. Microbiol. 1999, 32, 1043–1053. [Google Scholar] [CrossRef] [PubMed]
Gohar, M.; Faegri, K.; Perchat, S.; Ravnum, S.; Økstad, O.A.; Gominet, M.; Kolstø, A.B.; Lereclus, D. The PlcR Virulence Regulon of Bacillus cereus. PLoS ONE 2008, 3, e2793. [Google Scholar] [CrossRef] [PubMed]
Raymond, B.; Johnston, P.R.; Nielsen-LeRoux, C.; Lereclus, D.; Crickmore, N. Bacillus thuringiensis: An Impotent Pathogen? Trends Microbiol. 2010, 18, 189–194. [Google Scholar] [CrossRef] [PubMed]
Mignot, T.; Mock, M.; Robichon, D.; Landier, A.; Lereclus, D.; Fouet, A. The Incompatibility between the PlcR- and AtxA-Controlled Regulons May Have Selected a Nonsense Mutation in Bacillus anthracis. Mol. Microbiol. 2001, 42, 1189–1198. [Google Scholar] [CrossRef] [PubMed]
Salamitou, S.; Ramisse, F.; Brehelin, M.; Bourguet, D.; Gilois, N.; Gominet, M.; Hernandez, E.; Lereclus, D. The PlcR Regulon Is Involved in the Opportunistic Properties of Bacillus thuringiensis and Bacillus cereus in Mice and Insects. Microbiology 2000, 146, 2825–2832. [Google Scholar] [CrossRef]
Slamti, L.; Lereclus, D. A Cell-Cell Signaling Peptide Activates the PlcR Virulence Regulon in Bacteria of the Bacillus cereus Group. EMBO J. 2002, 21, 4550–4559. [Google Scholar] [CrossRef]
Grenha, R.; Slamti, L.; Nicaise, M.; Refes, Y.; Lereclus, D.; Nessler, S. Structural Basis for the Activation Mechanism of the PlcR Virulence Regulator by the Quorum-Sensing Signal Peptide PapR. Proc. Natl. Acad. Sci. USA 2013, 110, 1047–1052. [Google Scholar] [CrossRef]
Lereclus, D.; Agaisse, H.; Gominet, M.; Salamitou, S.; Sanchis, V. Identification of a Bacillus thuringiensis Gene That Positively Regulates Transcription of the Phosphatidylinositol-Specific Phospholipase C Gene at the Onset of the Stationary Phase. J. Bacteriol. 1996, 178, 2749–2756. [Google Scholar] [CrossRef]
Lereclus, D.; Agaisse, H.; Grandvalet, C.; Salamitou, S.; Gominet, M. Regulation of Toxin and Virulence Gene Transcription in Bacillus thuringiensis. Int. J. Med. Microbiol. 2000, 290, 295–299. [Google Scholar] [CrossRef]
Brillard, J.; Susanna, K.; Michaud, C.; Dargaignaratz, C.; Gohar, M.; Nielsen-Leroux, C.; Ramarao, N.; Kolstø, A.B.; Nguyen-The, C.; Lereclus, D.; et al. The YvfTU Two-Component System Is Involved in PlcR Expression in Bacillus cereus. BMC Microbiol. 2008, 8, 183. [Google Scholar] [CrossRef]
Lindbäck, T.; Mols, M.; Basset, C.; Granum, P.E.; Kuipers, O.P.; Kovács, Á.T. CodY, a Pleiotropic Regulator, Influences Multicellular Behaviour and Efficient Production of Virulence Factors in Bacillus cereus. Environ. Microbiol. 2012, 14, 2233–2246. [Google Scholar] [CrossRef] [PubMed]
Frenzel, E.; Doll, V.; Pauthner, M.; Lücking, G.; Scherer, S.; Ehling-Schulz, M. CodY Orchestrates the Expression of Virulence Determinants in Emetic Bacillus cereus by Impacting Key Regulatory Circuits. Mol. Microbiol. 2012, 85, 67–88. [Google Scholar] [CrossRef] [PubMed]
Gominet, M.; Slamti, L.; Gilois, N.; Rose, M.; Lereclus, D. Oligopeptide Permease Is Required for Expression of the Bacillus thuringiensis PlcR Regulon and for Virulence. Mol. Microbiol. 2001, 40, 963–975. [Google Scholar] [CrossRef] [PubMed]
Declerck, N.; Bouillaut, L.; Chaix, D.; Rugani, N.; Slamti, L.; Hoh, F.; Lereclus, D.; Arold, S.T. Structure of PlcR: Insights into Virulence Regulation and Evolution of Quorum Sensing in Gram-Positive Bacteria. Proc. Natl. Acad. Sci. USA 2007, 104, 18490–18495. [Google Scholar] [CrossRef]
Pomerantsev, A.P.; Pomerantseva, O.M.; Leppla, S.H. A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence. Infect. Immun. 2004, 72, 5814–5823. [Google Scholar] [CrossRef]
Qin, J.; Cao, Z.; Cai, X.; Fang, Y.; An, B.; Li, X.; Zhang, Y.; Tian, H.; Hu, W.; Yan, B.; et al. NupR Responding to Multiple Signals Is a Nucleoside Permease Regulator in Bacillus thuringiensis BMB171. Microbiol. Spectr. 2022, 10, e0154322. [Google Scholar] [CrossRef]
Schaeffer, P.; Millet, J.; Aubert, J.P. Catabolic Repression of Bacterial Sporulation. Proc. Natl. Acad. Sci. USA 1965, 54, 704–711. [Google Scholar] [CrossRef]
Galimand, M.; Courvalin, P.; Lambert, T. Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S RRNA Methylation. Antimicrob. Agents Chemother. 2003, 47, 2565–2571. [Google Scholar] [CrossRef]
Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2^−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
Hirata, H.; Negoro, S.; Okada, H. Molecular Basis of Isozyme Formation of β-Galactosidases in Bacillus stearothermophilus: Isolation of Two β-Galactosidase Genes, BgaA and BgaB. J. Bacteriol. 1984, 160, 9–14. [Google Scholar] [CrossRef]
Hou, X.; Han, L.; An, B.; Zhang, Y.; Cao, Z.; Zhan, Y.; Cai, X.; Yan, B.; Cai, J. Mitochondria and Lysosomes Participate in Vip3Aa-Induced Spodoptera Frugiperda Sf9 Cell Apoptosis. Toxins 2020, 12, 116. [Google Scholar] [CrossRef]
Michaux, C.; Holmqvist, E.; Vasicek, E.; Sharan, M.; Barquist, L.; Westermann, A.J.; Gunn, J.S.; Vogel, J. RNA Target Profiles Direct the Discovery of Virulence Functions for the Cold-Shock Proteins CspC and CspE. Proc. Natl. Acad. Sci. USA 2017, 114, 6824–6829. [Google Scholar] [CrossRef] [PubMed]
ØKstad, O.A.; Gominet, M.; Purnelle, B.; Rose, M.; Lereclus, D.; Kolstø, A.B. Sequence Analysis of Three Bacillus cereus Loci Carrying PIcR-Regulated Genes Encoding Degradative Enzymes and Enterotoxin. Microbiology 1999, 145, 3129–3138. [Google Scholar] [CrossRef] [PubMed]
Shen, A.; Higgins, D.E. The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria Monocytogenes. PLoS Pathog. 2006, 2, e30. [Google Scholar] [CrossRef] [PubMed]
Smith, V.; Josefsen, M.; Lindbäck, T.; Hegna, I.K.; Finke, S.; Tourasse, N.J.; Nielsen-LeRoux, C.; Økstad, O.A.; Fagerlund, A. MogR Is a Ubiquitous Transcriptional Repressor Affecting Motility, Biofilm Formation and Virulence in Bacillus thuringiensis. Front. Microbiol. 2020, 11, 610650. [Google Scholar] [CrossRef]
Higgins, C.F. ABC Transporters: From Microorganisms to Man. Annu. Rev. Cell Biol. 1992, 8, 67–113. [Google Scholar] [CrossRef]
Hiles, I.D.; Gallagher, M.P.; Jamieson, D.J.; Higgins, C.F. Molecular Characterization of the Oligopeptide Permease of Salmonella Typhimurium. J. Mol. Biol. 1987, 195, 125–142. [Google Scholar] [CrossRef]
Slamti, L.; Lemy, C.; Henry, C.; Guillot, A.; Huillet, E.; Lereclus, D. CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis. Front. Microbiol. 2016, 6, 1501. [Google Scholar] [CrossRef]
Geiger, T.; Wolz, C. Intersection of the Stringent Response and the CodY Regulon in Low GC Gram-Positive Bacteria. Int. J. Med. Microbiol. 2014, 304, 150–155. [Google Scholar] [CrossRef]
Slamti, L.; Perchat, S.; Huillet, E.; Lereclus, D. Quorum Sensing in Bacillus thuringiensis Is Required for Completion of a Full Infectious Cycle in the Insect. Toxins 2014, 6, 2239–2255. [Google Scholar] [CrossRef]
Chibazakura, T.; Kawamura, F.; Takahashi, H. Differential Regulation of Spo0A Transcription in Bacillus subtilis: Glucose Represses Promoter Switching at the Initiation of Sporulation. J. Bacteriol. 1991, 173, 2625–2632. [Google Scholar] [CrossRef] [PubMed]
Matsuno, K.; Blais, T.; Serio, A.W.; Conway, T.; Henkin, T.M.; Sonenshein, A.L. Metabolic Imbalance and Sporulation in an Isocitrate Dehydrogenase Mutant of Bacillus subtilis. J. Bacteriol. 1999, 181, 3382–3391. [Google Scholar] [CrossRef]
Yamashita, S.; Kawamura, F.; Yoshikawa, H.; Takahashi, H.; Kobayashi, Y.; Saito, H. Dissection of the Expression Signals of the SpoA Gene of Bacillus subtilis: Glucose Represses Sporulation-Specific Expression. Microbiology 1989, 135, 1335–1345. [Google Scholar] [CrossRef] [PubMed]
Ireton, K.; Jin, S.; Grossman, A.D.; Sonenshein, A.L. Krebs Cycle Function Is Required for Activation of the Spo0A Transcription Factor in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 1995, 92, 2845–2849. [Google Scholar] [CrossRef] [PubMed]
He, J.; Shao, X.; Zheng, H.; Li, M.; Wang, J.; Zhang, Q.; Li, L.; Liu, Z.; Sun, M.; Wang, S.; et al. Complete genome sequence of Bacillus thuringiensis mutant strain BMB171. J. Bacteriol. 2010, 192, 4074–4075. [Google Scholar] [CrossRef] [PubMed]

Figure 1. NupR inhibits the expression of plcR during the stationary phase. (A) The 5′UTR of plcR. The conserved binding sites of Spo0A, PlcR, and NupR are shaded in blue, green, and gray. The ATG of PlcR is marked in pink. (B) β-galactosidase activities of PnupR-lacZ in BMB171 cultivated in LB and SSM media. (C) plcR mRNA levels in BMB171 and ΔnupR cultivated in LB medium. (D) β-galactosidase activities of PplcR-lacZ in BMB171 and ΔnupR cultivated in LB medium. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, non-significant. Data represent the mean ± SD of three samples.

Figure 2. NupR can directly or indirectly regulate the expression of the plcR regulon. (A) β-galactosidase activities of Ppap-lacZ, Phemo-lacZ, and Pplc-lacZ in BMB171 and ΔnupR cultivated in the SSM medium. (B) β-galactosidase activities of Pmog-lacZ in BMB171 and ΔnupR cultivated in the SSM medium. (C) NupR binds directly to the promoter regions of mogR labeled by FAM. (D) BMB171, and ΔnupR strains were dripped on 0.5% and 0.3% soft agar plates (LB medium) and incubated at 28 °C. ** p < 0.01; *** p < 0.001. Data represent the mean ± SD of three samples.

Figure 3. The effect of the culture supernatant of BMB171 and ΔnupR strains on the cell viability of Sf9 cells. * p < 0.05; ns, no significant. Data represent the mean ± SD of three samples.

Figure 4. Expression of plcR is induced by glucose. (A) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 0.1% glucose. (B) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in LB medium with or without 0.1% glucose. *** p < 0.001; ns, no significant. Data represent the mean ± SD of three samples.

Figure 5. Expression of plcR is Induced by Nucleosides. (A) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 1mM different nucleosides. An, adenosine; Cn, cytidine; Gn, guanosine; Un, uridine. (B) plcR mRNA levels in BMB171, ΔnupR, and Δspo0A cultivated in SSM medium with or without 0.1% ribose. (C) plcR mRNA levels in BMB171 and ΔnupR cultivated in LB medium with or without 0.1% ribose. Ri, ribose. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant. Data represent the mean ± SD of three samples.

Figure 6. Schematic representation of the regulation of PlcR. plcR is autoregulated and under the negative control of Spo0A~P and NupR. CodY positively controls the expression of plcR by regulating the expression of opp. The YvfTU two-component system is also involved in plcR expression via a yet unknown mechanism. Process lines are shown in black, and regulatory relationships are indicated by blue and red lines, with clipped heads for facilitation, horizontal lines for inhibition, solid lines for direct, and dashed lines for indirect. In addition, the red lines indicate processes influenced by ribose or glucose.

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Qin, J.; Wang, Z.; Qian, C.; Ji, G.; Zhang, Y.; Cao, Z.; Yan, B.; Cai, J. NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors. Microorganisms 2025, 13, 212. https://doi.org/10.3390/microorganisms13010212

AMA Style

Qin J, Wang Z, Qian C, Ji G, Zhang Y, Cao Z, Yan B, Cai J. NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors. Microorganisms. 2025; 13(1):212. https://doi.org/10.3390/microorganisms13010212

Chicago/Turabian Style

Qin, Jiaxin, Ziqi Wang, Cheng Qian, Guohui Ji, Yizhuo Zhang, Zhanglei Cao, Bing Yan, and Jun Cai. 2025. "NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors" Microorganisms 13, no. 1: 212. https://doi.org/10.3390/microorganisms13010212

APA Style

Qin, J., Wang, Z., Qian, C., Ji, G., Zhang, Y., Cao, Z., Yan, B., & Cai, J. (2025). NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors. Microorganisms, 13(1), 212. https://doi.org/10.3390/microorganisms13010212

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