Promoted Spore Formation of Bacillus amyloliquefaciens fmbJ by its Secondary Metabolite Bacillomycin D Coordinated with Mn²⁺

Abstract

In Bacillus, the spore formation process is associated with the synthesis and release of secondary metabolites. A large number of studies have been conducted to systematically elucidate the pathways and mechanisms of spore formation. However, there are no studies have explored the relationship between secondary metabolites and spores. In this study, we investigated the relationship between its secondary metabolite bacillomycin D (BD) and spores using the simpler dipicolonic acid fluorimetry assay for spore counting in Bacillus amyloliquefaciens fmbJ. Our results showed that BD could promote the spore formation of B. amyloliquefaciens fmbJ and had a synergistic effect with certain concentrations of Mn²⁺. When 15.6 mg/L of BD and 1 mM of Mn²⁺ were added, the number of fmbJ spores increased from 1.42 × 10⁸ CFU/mL to 2.02 × 10⁸ CFU/mL after 36 h of incubation. The expressions of spore formation (kinA, kinB, kinC, kinD, kinE and spo0A) and Mn-related genes (mntA, mntH, mneS, mneP) were studied by RT-PCR. The results indicated that BD and Mn²⁺ promoted the spore formation of fmbJ by stimulating the transcription of kinB, kinD and increasing the influence of spo0F-spo0A phosphorylation transmission. This study provided a new idea to improve the spore production of B. amyloliquefaciens and laid the foundation for its industrial production.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01026-9.

Introduction

Spores are the product of Bacillus to cope with the complex environment. They can resist heat, radiation, drying, freezing, and chemical disinfectants [1]. Bacillus spores have important applications in improving enzyme stability, recombinant vaccines, adjuvants, drug delivery, biopesticides, etc. [2–7]. But Bacillus spores are difficult to form. Their formation is controlled by a series of regulatory and structural genes, the expression of which is tightly regulated [8].

Bacillus spore formation is usually caused by nutrient deficiency and high cell density [9]. It is regulated by a multi-component phosphorylase containing five histidine kinases (KinA-E) and two phosphorylated proteins (Spo0F and Spo0B) [10]. Among these kinases, KinA and KinB respond to impaired respiration, which may be sensed by the redox state of the cytochrome caa3 and bc complexes [11]. KinC responds to membrane damage and potassium ion leakage caused by a self-produced lipopeptide surfactin [12]. KinD is responsible for sensing the presence of glycerol and manganese through its extracellular cache domain [13]. KinE is involved in the process of spo0F to spo0A phosphorylation transmission and is associated with the expression of genes related to the cellular matrix [14]. Currently, various studies have been carried out to optimize the medium composition, pH, aeration and temperature to improve spore production of Bacillus [15–17]. However, few studies have been conducted on the effect of secondary metabolites of Bacillus on spore production.

Bacillus amyloliquefaciens, as a biocontrol bacterium, can produces a series of secondary metabolites, including lipopeptides with antimicrobial activity [18–20]. The synthesis of lipopeptides is closely related to sporulation. Spo0A is a control switch for the initiation of spore formation, not only promoting spore formation but also being linked to antimicrobial substances synthesis [21]. Surfactin can act as pheromones to regulate processes such as spore formation related to cell density dependence [22]. Bacillomycin D (BD), as a lipopeptide, consists of a hydrophilic portion of seven α-amino acids and a hydrophobic portion of a fatty acid chain of 14–17 carbon atoms [20]. It has many activities such as antioxidant, antitumor, antifungal, and surface activity, while being safe and biodegradable [22–25]. Recent studies have focused on the antifungal activity and synthetic mechanism of BD [26–28]. Previously, it was shown that the addition of inulin and L-glutamine increased BD production by stimulating comA, degU and spo0A expression [11, 29].

In the study, the effect of BD on spore formation in B. amyloliquefaciens fmbJ was investigated. First, the sub-toxic concentration of BD on fmbJ was determined. Then, the effect of BD with other metal ions on spore formation was explored. Finally, the mechanisms by which they affect spore formation were initially explored by comparing the expression of kinA-E and spo0A. The results of these studies will bring new explorations to Bacillus for improving spore production.

Materials and Methods

Microorganism, Culture Condition, and Reagents

B. amyloliquefaciens fmbJ (CGMCC 0943) used in this study was obtained from the laboratory of enzyme engineering, Nanjing Agricultural University. The strain was inoculated in Luria-Bertani (10.0 g/L peptone, 5.0 g/L yeast extract, and 10.0 g/L NaCl, pH 7.2) medium at 37 °C with 180 rpm for 36 h to detect spores. The Landy medium (glucose 20.0 g/L, L-glutamic acid 5.0 g/L, yeast extract 1.0 g/L, KH₂PO₄ 0.5 g/L, MgSO₄·7H₂O 0.5 g/L, KCl 0.5 g/L, CuSO₄·5H₂O 0.15 mg/L, FeSO₄·7H₂O 1.2 mg/L, MnSO₄ 5.0 mg/L, pH 7.0) was used as a fermentation medium for the preparation of BD [11]. The fermentation conditions were 33 °C, 180 rpm, and 72 h. For RNA extraction, the strains were inoculated in LB at 37 °C, 180 rpm for 24 h. All the chemicals and culture medium compositions were bought from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China.

Determination of Strain Growth and Spore Formation

Bacillomycin D (BD) was purified using high performance liquid chromatography (Agilent 1260, Agilent Technologies Co. Ltd. USA) in the previous work [30]. The effect of BD on the growth of B. amyloliquefaciens fmbJ were assayed in sterile 96-well plates [31]. The strains (1 × 10⁷ CFU/mL) were added to LB medium containing different concentrations of BD (1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 0 mg/L) and incubated at 37 °C for 24 h. The OD₆₀₀ was measured using a SpectraMax 190 Microplate Reades (Molecular Devices Corporation, USA).

The strains were inoculated into LB medium containing BD and incubated for 36 h at 37 °C to form spores. The strain was then stained using the Schaeffer-Fulton cold staining method [32]. 1 μL of bacterial solution and 100 μL of sterile water were taken, placed on a clean slide, heat fixed, stained with saturated malachite green for more than 15 min, washed with water and then re-stained with 0.5% of saffron solution, rinsed, dried, and then observed microscopically.

Determination of the Number of Spores

Follow-up spore’s counts were performed using the method of detecting dipicolinic acid (DPA) in spores. The spores were treated with high temperature and pressure so that the DPA in the spores could be fully released [33]. The supernatant was obtained by centrifugation. EuCl₃ (2 mM) and Cy-DTA (2 mM) were added to the supernatant in a ratio of 4.5:4.5:1. An equal amount of Tri-HCl was used as a control instead of the supernatant. Fluorescence spectra were used uses Hitachi F-7000 (Hitachi, Japan). The parameter settings refer to those set by Ren et al. [34].

Bacterial count standard curve and fluorescent DPA standard curve were plotted using two-fold dilution. The absorbance of the bacterial solution at OD₆₀₀ was measured using a UV spectrophotometer (MAPADA-v-1100D, Shanghai MAPADA Instruments Co., Ltd. China). The standard curve of bacterial count was: y = 0.0002x + 0.0078, R² = 0.999 (Fig. S1). Also, the curve of DPA was plotted using the same method. There was a good linear correlation between the spore concentration from 4.32 × 10⁷ CFU/mL to 3.4 × 10⁸ CFU/mL and the corresponding DPA fluorescence intensity from 0 to 1000 AU. y = 0.318x + 10.37, R² = 0.9986 (Fig. S2). Initial counting of spores was performed using heat shock to kill asexual cells, followed by counting of surviving CFUs.

Determination of Metal Ions and BD on the Spore Formation

Mn²⁺, Fe²⁺, Mg²⁺, and Ca²⁺ were added to the LB broth containing 1% fmbJ to a final concentration of 1 mM, respectively, and incubated at 37 °C for 36 h. The number of spores was then determined using the above method. Based on these results, Mn²⁺ was selected for concentration optimization. The final concentrations of Mn²⁺ in the LB medium were set at 0.5, 1 and 2 mM. Also, with regard to the determination of BD and Mn²⁺ on spore formation, 62.5 mg/L BD and different concentrations of Mn²⁺ (0.5, 1, and 2 mM) were added separately to the medium, as well as 1 mM Mn²⁺ and different concentrations of BD (15.6, 62.5, and 250 mg/L) were added to the medium. The bacteria were incubated using the same conditions and then the spores were determined. The main procedure of the experiment was shown in Fig. S3.

Reverse Transcription Quantitative Real-Time PCR (RT-qPCR)

The total RNA of B. amyloliquefaciens fmbJ was extracted by the Trizol method according to the instructions [35]. The concentrations of total RNA were detected by Nano DropOne (Thermo Scientific, USA). The synthesis of cDNA was carried out according to the kit instructions (Vazyme Biotech Co., Ltd., Nanjing, China). DNA was amplified by a RT-qPCR System (7500, Applied Biosystems, Foster City, USA) and the expressions of target genes (kinA, kinB, kinC, kinD, kinE, spo0A, mntA, mntH, mneP, mneS) were analyzed by the following PCR Program. The program included pre-denaturation at 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Table S1 showed the primers used to amplify the target genes and the reference gene16s RNA. Evaluation of the relative fold changes in target genes expression was carried out by 2^−△△Ct calculation. RT-qPCR system software (7500, Applied Biosystems, Foster City, USA) provided the threshold cycle (C_t) values.

Statistical Analysis

All statistical analyzes were performed using SPSS (SPSS version 16.0, IBM, USA) for one-way analysis of variance (ANOVA). Duncan’s test was used to examine significance below 0.05 and 0.01. All results were expressed as mean ± standard deviation (SD) with 3 replicates.

Results and Discussion

Effect of BD on the Growth of B. amyloliquefaciens fmbJ

The visual colorimetric method [36] and spectrophotometry [37] were used to compare the effect of BD on the growth of B. amyloliquefaciens fmbJ (Fig. S4). When BD was added at low concentrations (0, 4, 8, and 16 mg/L), there was no significant effect on the growth of fmbJ. With increasing BD concentration (32.5 and 62.5 mg/L), there was a significant negative effect on the growth of fmbJ. When the concentration of BD reached 1000 mg/L, its OD₆₀₀ value was only 0.41 ± 0.08. The above results indicated that the BD concentration in the medium at 32.5–62.5 mg/L had a sub-toxic effect on fmbJ.

Effect of BD on the Spore Formation of B. amyloliquefaciens fmbJ

Fig. S5 showed that the addition of BD (0–62.5 mg/L) to the medium was effective in increasing spore production, but too high a BD concentration (250 mg/L) had a negative effect on fmbJ growth, leading to a decrease in the number of spores. Fig. S5A showed that 62.5 mg/L of BD in the medium was the optimal concentration, with a fluorescence intensity of 535.41 ± 9.98 AU. Furthermore, results of spore staining indicated a corresponding trend in the number of spores and BD concentration in the medium (Fig. S5B). Combined with the previous results, we concluded that the BD concentration of 62.5 mg/L in the medium had a slight inhibitory effect on the growth of fmbJ, but significantly promoted its spore formation. Considering the economy and reasonableness of the subsequent experiments, the concentration of BD in the medium was 62.5 mg/L.

Effect of Metal Ions on the Spore Formation of B. amyloliquefaciens fmbJ

Table S2 showed that Ca²⁺, Mg²⁺, Fe²⁺, and Mn²⁺ significantly increased the spore production of fmbJ. Their fluorescence intensities was 503.32 ± 9.5 AU, 525.20 ± 13.53 AU, 542.98 ± 6.46 AU, and 615.97 ± 6.43 AU, respectively. Among them, Mn²⁺ had the most significant promotion effect on spore production with (1.93 ± 0.02) × 10⁸ CFU/mL, it was 1.37 times more the control ((1.42 ± 0.05) × 10⁸ CFU/mL).

Metal ions are one of the essential factors for the formation of spores. Metal ions play a major role in activating the enzyme system necessary for sporulation [15]. Mn and Fe elements are not only involved in the synthesis of secondary metabolites such as antibiotics and lipopeptides, but are also among the essential elements for sporulation [38–40]. Ca can be chelated with DPA to become Ca-DPA to help spores gradually dehydrate to form mature spores and to help improve heat resistance [8]. Mg²⁺ at 2 mM significantly increased the spores of B. amyloliquefaciens BS-20 from (9.97 ± 0.49) × 10⁸ CFU/mL to (1.54 ± 0.77) × 10⁸ CFU/mL[15]. There was a good agreement between our results and these studies. In addition, we found that Mn²⁺ was more favourable for spore formation. Therefore, Mn²⁺ were chosen for the follow-up study.

Effect of BD and Mn²⁺ on the Spore Formation of B. amyloliquefaciens fmbJ

As seen in Table S3, the spore formation of fmbJ showed a trend of increasing and then decreasing with the increase of Mn²⁺ concentration in the culture medium. The optimal Mn²⁺ concentration was 1 mM. However, when the Mn²⁺ concentration increased to 2 mM, the rate of spore formation decreased and its fluroescence intensity was only 474.55 AU and the spores were reduced to 1.48 × 10⁸ CFU/mL.

The effect of BD and Mn²⁺ on spores production of fmbJ was researched by the one-way method. Firstly, BD concentration (62.5 mg/L) was fixed in the medium and varying Mn²⁺ concentration (0.5, 1, and 2 mM) were used as single factors. From Table S3, it could be seen that in the medium containing 62.5 mg/L BD, the number of fmbJ spores showed a trend of increasing and then decreasing with increasing Mn²⁺ concentration. The optimal Mn²⁺ concentration in the medium was 0.5 mM, and the spore production was 1.38 times higher than the control. In contrast, high Mn²⁺ concentration (1 and 2 mM) led to a decrease in the number of fmbJ spores. When the Mn²⁺ concentration was 1 mM, the effect of different concentrations of BD synergistic Mn²⁺ on spore formation was compared. The results indicated that 15.6 mg/L BD and 1 mM Mn²⁺ promoted the spore formation of fmbJ ((2.02 ± 0.03) × 10⁸ CFU/mL), and its fluorescence intensity was as high as 643.36 ± 9.99 AU. However, the interaction between increasing BD concentration (62.5 and 250 mg/L) and 1 mM Mn²⁺ changed from a synergistic to an inhibitory effect. This phenomenon might be due to the increased toxic effect on fmbJ caused by the increased BD concentration in the presence of Mn²⁺, resulting in lower spore production.

Improving spore production usually required optimization of several factors, including the composition of the solid substrate, the amount of additional nutrients in the liquid substrate [15]. The maximum yield of spores in this study was lower than other reports, and their spore production ranged from 5.93 × 10⁸ CFU/mL to 3.82 × 10⁹ CFU/mL [41, 42]. In this study, only one factor in the medium was optimized and was not fully optimized, resulting in low spore yield. However, these results suggested that BD could promote spore production in B. amyloliquefaciens fmbJ, and this effect was further enhanced when Mn²⁺ was present.

Effects of BD and Mn²⁺ on the Expression of Genes Related to Spore Formation

The effects of different concentrations of BD on the relative expression of histidine kinase genes (kinA, kinB, kinC, kinD and kinE) and spore initiation genes (spo0A) in fmbJ were presented in Fig. 1a. From the results, it can be seen that the relative expression of the spo0A gene showed a trend of increasing and then decreasing with the increase of BD concentration. Meanwhile, the trend of the relative expression of kinB and kinD was similar to that of spo0A. The expression of kinB, kinD and spo0A in fmbJ increased 3.0, 2.5 and 2.1-fold, respectively, after the addition of 62.5 mg/L BD compared with the control group.

Fig. 1.

Effects of BD and Mn²⁺ on the expression of histidine kinase genes (kinA, kinB, kinC, kinD and kinE), spore initiation gene (spo0A) and Mn transport related genes (mntA, mntH, mneS, and mneP) in B. amyloliquefaciens fmbJ. * and ** were significantly different from controls at 0.05 and 0.01

Figure 1b demonstrated the effect of different concentrations of Mn²⁺ on the relative expression of genes related to spore formation. Compared to the control, the addition of 1 mM Mn²⁺ significantly increased the relative expression of kinA-E as well as spo0A. The relative expression of kinB and kinD was 3.7 and 2.7 times higher than that of the control, respectively. As the concentration of Mn²⁺ increased, the relative expression of spo0A showed a trend of first increasing and then decreasing. Moreover, we found that the effect of increasing Mn²⁺ concentration on the relative expression of kinB, kinC and kinD was consistent with that of spo0A.

As illustrated in Fig. 1c, the relative expression of kinB, kinD and spo0A showed the same trend of being significantly higher than the control when the same concentration of BD and different concentrations of Mn²⁺ were added. Moreover, the expression of these three genes was highest when 62.5 mg/L BD and 1 mM Mn²⁺ were added compared to the other samples. Then, the simultaneous addition of BD and different concentrations of Mn²⁺, however, caused different degrees of decrease in the relative expression of kinA, kinC and kinE, except for the expression of kinE in the sample group with the addition of 62.5 mg/L BD and 1 mM Mn²⁺.

As can be seen in Fig. 1d, the addition of 1 mM Mn²⁺ and different concentrations of BD prompted a significant increase in the relative expression of spo0A at low concentrations of BD addition, whereas at 250 mg/L BD addition, it acted as a repressor of its expression. In addition, the relative expression of kinB, kinC, kinD, and kinE were increased to varying degrees in these samples.

These results above indicated that the addition of either BD or Mn²⁺ alone increased the relative expression of spo0A, which in turn could promote the formation of spores. When both were added simultaneously, this synergistic promotion of spore formation was dependent on the concentration of each. When Mn²⁺ or BD were added in excess, this synergistic effect rapidly disintegrated.

Histidine kinase stimulates the phosphorylation reaction of spo0F-spo0B-spo0A by sensing external stimuli, thus promoting the formation of spores [43]. It was obvious from the results that the addition of BD and Mn²⁺ promoted spore formation by promoting the expression of the histidine kinases KinB and KinD, which led to a significant increase in the transcription of spo0A (Fig. 1a–d). Xu et al. [44] found that BD could affect the phosphorylation level of Spo0A to form biofilms by sensing external stimuli mainly through KinB. In addition, glycerol and Mn²⁺ enhanced spore production of B. subtilis by stimulating kinD to promote the expression of spo0A [13].

Effects of BD and Mn²⁺ on the Expression of Mn Transporter Genes

The Mn²⁺ uptake system of Bacillus mainly consists of two systems: (1) MntH is an insert encoding a divalent metal ion transporter of the NRAMP family. Therefore, it is regarded as the main Mn²⁺ importing system under high Mn²⁺ conditions. (2) Another transport system for Mn is the ATP-dependent MntABC transporter protein system. MneP acts as the primary efflux pump for Mn²⁺, while MneS plays a secondary role [45].

Figure 1e indicated the effects of BD and Mn²⁺ on the relative expression of Mn transporter genes (mntA, mntH, mneS, and mneP) in fmbJ. From the results of Fig. 1e, it was observed that the relative expression of Mn transporter-related genes (mntA, mntH) and Mn efflux genes (mneP) increased with increasing BD concentration. However, another gene associated with Mn efflux (mneS) showed the opposite change.

As shown in Fig. 1f, the addition of different concentrations of Mn²⁺ obtained significant inhibitory effects on the expression of mntA and mntH. With the addition of 0.5 and 1 mM Mn²⁺, the expression of mneS was significantly increased, promoting the excretion of Mn from the bacteria. Nevertheless, when the concentration of Mn²⁺ reached 2 mM, the relative expression of mneS did not change significantly compared to the control. In addition, the expression of mneP significantly decreased with increasing Mn²⁺ concentration.

As depicted in Fig. 1g, when the same concentration of BD and different concentrations of Mn²⁺ were added, the relative expression of both mntA and mntH was significantly reduced compared to the control. With the addition of both Mn²⁺ and BD, the relative expression of mneS was significantly higher than that of the control, especially, when 1 mM Mn²⁺ and 62.5 mg/L BD were added, the relative expression was up to 7.5 times higher than that of the control. In contrast, the relative expression of mneP was significantly lower in all of them at this time.

It can be seen from Fig. 1h that different concentrations of BD and the same concentration of Mn²⁺ both significantly inhibited the expression of the transporter genes of Mn, while significantly increasing the relative expression of mneS, with the expression of mneS reaching 10 times that of the control. Interestingly, when the concentration of BD was low (15.6 mg/L), the expression of mneP increased by 2.4 times.

The above results suggested that the addition of BD alone stimulated the cells to enhance their uptake of Mn and that the addition of Mn²⁺ drove the expression of its efflux genes. The expression of mneS was significantly increased with the assistance of BD, slowing down the toxic effect of Mn on cells, while it could be seen that it was mneS that played a major role in the efflux of Mn.

Metal ions and lipopeptides are inextricably linked to each other. The increase of metal ions effectively increases the production of lipopeptides. The addition of 0.01 mM Mn²⁺ increased the surfactin production of B. subtilis ATCC 21,332 from 0.33 to 2.6 g/L [46]. Zhao et al. [17]. found that Mg²⁺ significantly enhanced the synthesis of iturin A. The specificity of lipopeptide permeability is also related to metal ions [47]. Enniatin can form complexes with cations such as K⁺, Na⁺ and Ca²⁺, and changes in intracellular ion concentrations disrupt cellular functions [48]. In the study, we found that low concentrations of Mn²⁺ (0.5 mM) and BD (62.5 mg/L) synergistically promoted the formation of fmbJ spores. When the same concentration of Mn²⁺ (1 mM) was added, the synergistic effect of BD and Mn²⁺ to promote spore formation gradually diminished with the increase of the added concentration (Table 1). Xu et al. [44] demonstrated that BD promotes transcription of the iron ABC transporter FeuABC by binding to its transcription factor Btr. Our results showed that the addition of BD promoted the transcription of mntA and mntH and increased the intracellular concentration of Mn²⁺, while BD increased Mn efflux by promoting the transcription of mneS in the presence of sufficient Mn²⁺ (Fig. 1e–h). Furthermore, the possible pathways of BD and Mn²⁺ influencing spore formation in fmbJ were presented in Fig. 2. BD and Mn²⁺ might promote the spore formation of fmbJ by stimulating the phosphorylation of spo0F-spo0B-spo0A through affecting kinB and kinD transcription.

Table 1.

Interactions between different concentration of BD and Mn²⁺

Number	Increased DPA fluorescence intensity (AU)											Interaction
	BD (mg/L)			Mn2+ (mM)			BD (mg/L) + Mn2+ (mM)
	15.6	62.5	250	0.5	1	2	15.6 + 1	62.5 + 1	250 + 1	62.5 + 0.5	62.5 + 2
1	33.16				170.96		198.35					Additive effect
2		61.30			170.96			153.16				Inhibitory effect
3			90.40		170.96				63.29			Inhibitory effect
4		61.30		63.88						179.91		Synergistic effect
5		61.30				29.54					107.50	Additive effect

Additive effect, the interaction of the two factors increases but less than the sum of their effects. Synergistic effect, the interaction of the two factors is greater than the sum of their separate effects. Inhibitory effect, the interaction of the two factors was less than the separate effect

Fig. 2.

The hypothetical pathways by which BD and Mn²⁺ promote the spore formation in B. amyloliquefaciens fmbJ

Conclusion

In conclusion, we have conducted a preliminary study of the mechanism by which BD to influence the spore formation of B. amyloliquefaciens fmbJ. BD and Mn²⁺ have synergy and facilitation and affect the spore formation through stimulation via the kinB and kinD transcription. Further, we will combine this with other conditions such as media optimization to obtain higher spore productions of B. amyloliquefaciens fmbJ. To lay the foundation for the subsequent industrial production application of B. amyloliquefaciens fmbJ spores.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Jing Sun and Yingjian Lu conceived and designed research. Jin Zhang and Xiaojiao Luo conducted experiments. Jing Sun and Jin Zhang analyzed data and wrote the manuscript. Xinyi Pang and Xiangfei Li revised the manuscript. All authors read and approved the manuscript.

Availability of data and material

The data used to support the findings of this study are available from the corresponding author upon request.

Declarations

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with humans or animals performed by any of the authors.