Whole-genome sequencing and phylogenomic analyses of a novel zearalenone-degrading Bacillus subtilis B72

Abstract

Bacillus strain B72 was previously isolated as a novel zearalenone (ZEN) degradation strain from the oil field soil in Xinjiang, China. The genome of B72 was sequenced with a 400 bp paired-end using the Illumina HiSeq X Ten platform. De novo genome assembly was performed using SOAPdenovo2 assemblers. Phylogenetic analysis using 16S rRNA gene sequencing demonstrated that B72 is closely related to the novel Bacillus subtilis (B. subtilis) strain DSM 10. A phylogenetic tree based on 31 housekeeping genes, constructed with 19 strains closest at the species level, showed that B72 was closely related to B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622. Detailed phylogenomic analysis using average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) demonstrated that B72 might be classified as a novel B. subtilis strain. Our study demonstrated that B72 could degrade 100% of ZEN in minimal medium after 8 h of incubation, which makes it the fastest degrading strain to date. Moreover, we confirmed that ZEN degradation by B72 might involve degrading enzymes produced during the initial period of bacterial growth. Subsequently, functional genome annotation revealed that the laccase-encoding genes yfiH (gene 1743) and cotA (gene 2671) might be related to ZEN degradation in B72. The genome sequence of B. subtilis B72 reported here will provide a reference for genomic research on ZEN degradation in the field of food and feed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03517-y.

Introduction

Zearalenone (ZEN) is a widespread source of contamination, with a structure similar to that of natural estrogen. ZEN can bind to estrogen receptors and cause reproductive system diseases, such as prostate, ovarian, cervical, and breast cancer (Gruber-Dorninger et al. 2021; Tatay et al. 2018; Zhang et al. 2019). Therefore, it is important to find safe and efficient methods for decontaminating ZEN from food. Through analysis of the structural characteristics of ZEN, detoxification of ZEN was found to be realized by hydrolyzing the lactone ring, breaking the C6 keto carbonyl, or cracking the diphenol ring (Zheng et al. 2018). Numerous physical and chemical methods have been used for in vitro detoxification of ZEN, including sorting, washing, thermal treatment, grain milling, ozone treatment, and sodium bicarbonate soaking (Wu et al. 2021). Most methods used in these efforts face major obstacles, including the loss of important nutrients and the associated high manufacturing costs. Therefore, biological detoxification has emerged as a promising method that has mild reaction conditions, strong specificity, high efficiency, and environmental friendliness (Abraham et al. 2022; Wu et al. 2021; Zhang et al. 2022).

Biological detoxification methods mainly depend on microbial enzymes that degrade mycotoxins. In recent years, several strains have been used for ZEN degradation, such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, Rhizopus spp., Aspergillus oryzae, Acinetobacter sp., and Saccharomyces cerevisiae (Borzekowski et al. 2019; Fu et al. 2016; Goko et al. 2021; Ju et al. 2019). They produce many types of enzymes or proteins that degrade ZEN, including lactone hydrolase, peroxidase, and laccase. Their degradation pathways mainly include the reduction of ketone carbonyl, modification of phenolic hydroxyl groups, hydrolysis of the lactone ring, and cleavage of the diphenol benzene ring (Wang et al. 2019a). Despite the important advances in microbial detoxification, the efficiency of this strain remains elusive, and relatively little research has focused on elucidating the degradation mechanism (Ji et al. 2021; Zheng et al. 2022).

Herein, with the aim of constantly exploring safe strains that can degrade ZEN efficiently and exploring its degradation mechanism, we isolated a strain (named B72) with ZEN-degrading ability from an oil field soil in Xinjiang, China. In recent years, there are many studies on microbial degradation of ZEN, but they report that the shortest degradation time is 24 h to completely degrade ZEN (Table 1). The results demonstrated that B72 could degrade 100% of ZEN in minimal medium after 8 h, which, to the best of our knowledge, is the fastest degrading strain identified to date. In addition, we proved that the degradation of ZEN is mediated by an enzyme secreted by B72. Although ZEN degradation by Bacillus species is an enzyme-catalyzed process (Wang et al. 2017; Xu et al. 2016), ZEN-degrading mechanisms from the molecular point of view have not been fully clarified. To broaden the current knowledge of ZEN degradation, this study investigated the (i) genome sequence of B72 using whole-genome sequencing (WGS) technology; (ii) ZEN degradation ability; (iii) candidate genes responsible for ZEN degradation.

Table 1.

Recent studies on the use of microorganisms for ZEN degradation

Microorganism	ZEN concentration	Assay conditions	ZEN degradation rate	References
11 types of Bacillus strains	20ug/mL	At 30 °C,72 h,150 rpm	58–96.9% of ZEN	Gonzalez Pereyra et al. (2020)
Bacillus subtilis, Candida utilis, and Aspergillus oryzae	1ug/mL	At 37 °C,48 h,180 rpm	92.27–95.15% of ZEN	Liu et al. (2019)
Bacillus strains	5 mg/L	At 37 ◦C for 24 h	56% of ZEN	Chen et al. (2019)
Bacillus cereus	10 mg/L	At 37 °C for 24 h,	100% of ZEN	Wang et al. (2018)
Bacillus pumilus ES-21	17.9 mg/ml	At 30 °C, 24 h, 180 rpm	95.7% of ZEN	Wang et al. (2017)
8 of Lactobacillus pentosus strains	5.51–74.70 µg/mL	At 37 ºC for 24 h	81.69–83.17% of ZEN	Sangsila et al. (2016)
4 of Lactobacillus spp	12.3 g/mL	At 37 °C for 48 h	40–68.2% of ZEN	Vega et al. (2017)
Lactococcus lactis and Bifidobacterium sp.	130 μg/mL	At 37 °C for 24 h	90% of ZEN	Krol et al. (2018)
10 of Saccharomyces cerevisiae and 5 of S. pastorianus)	2800 μg/kg	At 20 °C for 96 h	70% of ZEN	Wall-Martinez et al. (2019)
Saccharomyces cerevisiae	2 µg/mL	at 37 ◦C for 24 h	57.0% of ZEN	Rogowska et al. (2019)
Saccharomyces cerevisiae	2 μg/mL	at 30 °C,72 h,150 rpm	90% of ZEN	Keller et al. (2015)
Aspergillus niger FS10	1 μg/mL	At 28 °C, 24 h,180 rpm	95% of ZEN	Ji et al. (2021)
Proteus mirabilis, Bacillus subtilis	10 μg/mL	At 37 °C, 48 h, 220 rpm	99.48%, 98.78%	Yang et al. (2022)

Materials and methods

Strain isolation and cultivation

The B72 strain was isolated from the soil of an oil field in Xinjiang, China, which had a history of environmental radioactivity levels. Specifically, 6 g of the soil of an oil field was transferred to an Erlenmeyer flask with 20 mL of PBS buffer, 200 rpm, 30 min. Then let the sample settle after standing for 1 h. 100 μL of supernatant was added to the minimal medium (MM) medium (pH 7.4) containing 10 μg/mL ZEN, 200 r/min, 37 ℃, 5 d. Then the culture solution was diluted to 10^–3, 10^–4 and 10^–5. Finally, 100 μL of diluents with different concentrations were, respectively, coated on the MM solid medium with 10 μg/mL ZEN, which cultured at 30 ℃ for 48 h. The single colony was cultured on LB solid medium, and then the target strain was isolated and purified. All single colony were, respectively, transferred to MM liquid medium with 10 μg/mL ZEN and cultured at 37 °C and 200 rpm for 72 h. Seven strains that can degrade ZEN were screened and isolated from the soil. Among them, strain B72 grew faster in a medium with ZEN as the sole carbon source. Strain B72 was incubated in lysogeny broth (LB broth) (Sangon Biotech, Shanghai, China) at 37 °C, 200 rpm for 12 h and preserved in 20% glycerol in a – 80 °C freezer (SIEMENS, Japan), located at the School of Food Science and Pharmaceutical Engineering, Nanjing Normal University. In addition, the strain was deposited in the Microbiological Culture Collection Center with the accession number CGMCC No. 22956.

Degradation of ZEN by B72

According to the previous reports (Wang et al. 2017), 4% (v/v) of the cell suspension was inoculated to 50 mL of minimal medium [1.52 g/L KH₂PO₄, 0.05 g/L CaCl₂, 2.44 g/L, 0.2 g/L MgSO₄·7H₂O, Na₂HPO4, 0.5 g/L (NH₄)₂SO₄, pH 7.4] supplemented with 10 μL/mL of ZEN, which was grown at 37 °C and 200 rpm. The initial pH of the minimal medium (MM) medium with 10 μg/mL ZEN we used was 7.4. After 24 h of degradation, the measured pH was 7.29. In addition, we also investigated the effect of pH on the growth and degradation of ZEN by strain B72. It can be seen from Table 2 that when the pH was within the range of 5–9, the strain grown well. In the pH range of 7–9, strain B72 could completely degrade ZEN in the medium within 8 h. We have optimized the temperature and found that 37 ℃ was the most appropriate growth temperature for ZEN degradation. The optimum degradation temperature range was 30–45 ℃ (Table 3). Within this temperature range, B72 strain can completely degrade ZEN in MM medium within 8 h. Too high temperature (55 ℃) or too low temperature (25 ℃) was not conducive to the growth of strain B72 or the degradation of ZEN (Table 3). Aliquots were collected 0, 4, 8, 12, and 24 h after incubation. ZEN concentrations were quantified using high-performance liquid chromatography (HPLC) (Xiang et al. 2016). An equal volume of methanol was transferred to the collected samples for 5 min at 5 °C and then centrifuged at 12,500 rpm for 10 min. A 0.22 μm syringe filter was used to filter the supernatant. An Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 m) was used for HPLC. The mobile phase was acetonitrile/water (60:40; v/v) with a flow rate of 1 mL/min. The detection wavelength was set at 254 nm. The injection volume was 20 μL and the column was maintained at 30 °C. The standard is ZEN of 0.25, 0.5, 1, 2, 4, 8, 16 µg/mL, and then the concentration of ZEN is measured by HPLC. With the concentration of ZEN as the abscissa and the peak area of ZEN as the ordinate, the standard curve is drawn as shown in Fig. 1. The linear equation of the standard curve is: Y = 47.9329x + 3.76149, R² = 0.999.

Table 2.

Effects of different pH values on the growth and ZEN degradation efficiency of B72

pH	OD 600 (nm)	ZEN degradation (%)
3.0	0.0667 ± 0.008	8.2 ± 1.6
4.0	0.0679 ± 0.009	56.1 ± 2.9
5.0	0.0860 ± 0.012	90.9 ± 1.8
6.0	0.0872 ± 0.015	99.1 ± 0.3
7.0	0.0858 ± 0.016	100
8.0	0.0843 ± 0.007	100
9.0	0.0893 ± 0.008	100
10.0	0.0634 ± 0.010	50.1 ± 1.6

Table 3.

Effects of different temperature on the growth and ZEN degradation efficiency of B72

Temperature	OD 600 (nm)	ZEN degradation (%)
25	0.0689 ± 0.012	47.1 ± 1.8
30	0.0799 ± 0.009	100
37	0.0903 ± 0.021	100
45	0.0853 ± 0.008	100
55	0.0423 ± 0.013	20.9 ± 2.0

Fig. 1.

Standard curve of ZEN

To investigate the effect of heat treatment on ZEN degradation by cell extracts, the cell extracts were treated at 100 °C for 10 min. Simultaneously, the effects of 1% sodium dodecyl benzene sulfonate (SDS), protease K (1 mg/ml), and protease K + SDS on the degradation of ZEN by the cell extracts was analyzed. As described by Gao et al. (Gao et al. 2022), the cells were treated at 37 °C for 12 h. Phosphate buffered saline (PBS) was used as a blank control.

Whole-genome sequencing

As previously described (Anani et al. 2019), the genomic DNA of strain B72 was extracted by E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). The draft genome was obtained using an Illumina HiSeq X Ten platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China) with a 150 bp paired-end strategy. Finally, the sample provided no less than 100 copies of the genome × covered the raw data of the depth and assembled multiple genome scaffolds.

Genome assembly and annotation

The short sequence assembly software SOAPdenovo version 2.04 (http://soap.genomics.org.cn/) was used to splice multiple k-mer parameters of the optimized sequences in the genome scanning map of B72 to obtain the optimal contings assembly results, which then compared the reads to contings. The genome completion map of B72 was assembled using Unicycler software (version 0.5.0). During the assembly process, the sequence was corrected using Pilonjin software (version 1.14). If there was an overlap of more than a certain length at both ends of the final assembly sequence, the sequence was looped, and the overlap sequence at one end was truncated. Finally, the complete chromosome sequences were obtained. The assembly software SOAPdenovo v2.04 was designed to construct the bacterial genome scanning map. The bacterial genome scanning map adopts the strategy of small-segment library building, deep sequencing, and preliminary genome assembly, which can obtain the genome information of the samples to be tested with high-cost performance and can meet the basic needs of bacterial genome research. All the obtained genome sequences were at the scaffold level (i.e., there was a gap between few fragmented sequences and the scaffold). The assembly software Unicycler v0.4.8 was used to obtain the bacterial completion diagram. The bacterial genome completion map comprehensively utilizes first-, second-, and even third-generation sequencing technology to formulate the optimal strategy according to the specific situation of the strain, and finally, obtains a complete genome sequence (1 scaffold, ≤ 3 gaps) with no gaps between scaffolds. The coding sequences (CDS) in the genome were predicted using Glimmer version 3.02, GeneMarkS version 2.3.0, and Prodigal version 2.6.3. Glimmer version 3.02 was used to predict the assembly results of the scanning map, and GeneMarkS version 2.3.0 was used to predict the plasmid genome. The tRNA in the genome was predicted by the tRNAscan-SE software (version 2.0) (http://trna.ucsc.edu/software/) to subsequently obtain the nucleotide sequence, anti-codon, and secondary structure information of tRNA in each sample genome. The rRNA was predicted using the software Barrnap (version 0.9), and we obtained the location, type and sequence information of all rRNA in each sample genome. The genome of B72 had been submitted to the National Center for Biotechnology Information (NCBI) GenBank database under accession number JAMDFN000000000.1.

16S ribosomal RNA gene analysis

The 16S rRNA gene was used to identify strain B72, and was genomic DNA of the strain B72 was isolated (Li et al. 2022). The 16S rRNA gene sequence PCR universal primer 27F (5′-A GAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-AA GGAGGTGATCCAGCCCGCA-3′) was used to amplify the experimental strain fragments. The PCR-amplified products were purified, recovered, and sent to Shanghai Sangon Biotech company for sequencing. The taxonomy of strain B72 was identified using phylogenetic tree analysis. The 16S rRNA sequences was constructed the 16S rRNA phylogenetic tree from B72 and 12 other Bacillus species. We downloaded the 16S rRNA gene sequences of the 12 Bacillus species from the NCBI GenBank database. Subsequently, a phylogenetic tree was obtained related on neighbor joining (NJ) and the maximum likelihood (ML) model with 500 bootstrap replicates by MEGA X (Kumar et al. 2018).

Phylogenetic tree of house-keeping genes

By comparing with the local database, 19 strains closest to each other at the species level were selected based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB,rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC,rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf), and the phylogenetic tree was obtained by the NJ method with MEGA X. Species groupings were conducted according to a previous study (Lalucat et al. 2020).

Average nucleotide identity (ANI) and genome-to-genome distance (GGDC)

The DNA-DNA hybridization (dDDH) value in GGDC (https://ggdc.dsmz.de/) was calculated for the genomes including B72 and the different Bacillus species to confirm the identity of the B72 genome (Meier-Kolthoff et al. 2022). Moreover, the ANI calculator (Yoon et al. 2017) was used to calculate the genome of all strains, and the threshold of 95% ANIb was used as the cutoff point for species. ANI values greater than the threshold of 95% indicated that the two bacteria belonged to the same species (Lalucat et al. 2020).

According to Gomila et al. GGDC functions of GGDC were used to determine genetic distances and generate high-scoring segment pairs (HSPs). Then, we obtained the GGDC value using GGDC formula 2 from the HSPs (set species cutoff point at 70%) (Meier-Kolthoff et al. 2013a, b). Moreover, the subspecies boundaries and corroborate species were further analyzed using GGDC. Three formulas (Formulas 1, 2, and 3) were found in the GGDC for the calculation of DDH, and the sum of all identities in high-scoring pairs (HSP) was calculated. It was divided by the length of the HSP and was independent of the genome size. We defined the species boundary using a cutoff value of 70% in Formula 2 and the subspecies boundary of 79% (Meier-Kolthoff et al. 2013a, b).

Results and discussion

Whole-genome sequencing of B72

We constructed a fragment with an insertion of ~ 400 bp from qualified DNA samples and sequenced B72 with 150 bp paired-end reads based on Illumina sequencing. Among them, reads of 150 bp by single-end sequencing and a total of 7358914 raw reads and 1111196014 raw bases were obtained using Illumina sequencing. The raw read qualities of Q20 and Q30 were 96.94% and 92.09%, respectively. We used Cutadapt software to filter poor-quality reads (Kechin et al. 2017). Subsequently, we obtained 7205742 bp high-quality clean reads and 1097491376 bp clean raw reads, with an average read length of 152.31 bp. The clean raw read qualities of Q20 and Q30 were 97.46% and 92.71%, respectively.

Genome assembly of B72

The genome completion map of B72 was used in the assembly software Unicycler (version 0.5.0) for three-generation sequence assembly. During the assembly process, the sequence was corrected using the Pilonjin (version 1.14) software. The middle high-quality sequencing region was selected and the genome size was evaluated using the k-mer value, which was set to 17. In addition, the best assembled genome was selected according to the N50 value and total number of overlaps in the assembled genome. Finally, in the assembled genome, the total number of scaffolds per genome was 33, the length of scaffold N50 was 661700 bp, and the length of scaffold N90 was 193812 bp. The total length (i.e., genome size) of all scaffolds was 4038661 bp, and the GC content of all scaffolds in each genome was 43.74% (Table 4).

Table 4.

Strain B72 genome features

Property	Scaffold
Total number	33
Total length(bp)	4,038,661
N50(bp)	661,700
N90(bp)	193,812
GC content (%)	43.74

Gene annotation of B72

Previous studies mainly focused on microbial degradation of ZEN; however, the main enzymes involved in ZEN degradation and its related mechanisms are still unknown. Therefore, we used the Clusters of Orthologous Groups of Proteins (COG) annotation scheme to annotate the gene and coding sequence of B72. We observed 4 categories and 20 types in the COG annotation, and 3284 genes were encoded by COG (Table S1). The COG annotation of the B72 strain genome protein-coding gene function is shown in Fig. 2. We found that the first three system categories of B72 were amino acid transport and metabolism (308), transcription (268), and carbohydrate transport and metabolism (255) (Fig. 2).

Fig. 2.

B72 strain genome protein-coding gene function COG annotation by comparing with the six major databases

The species and quantity of carbohydrate-Active (CAZy) enzymes in strain B72 were analyzed using diamond and HMMscan software and quantitatively compared. Secretory proteins with obvious differences were analyzed (Table 5). There were 54 glycoside hydrolases (accounting for 36% of the total predicted ORF), 49 glycosyltransferases (32.67%), 31 carbohydrate esterases (20.7%), and five auxiliary active enzymes. In addition, three sugar-binding modules and eight polysaccharide lyases were found in the CAZy enzyme. The discovery of genomic information related to strain B72 will lay the foundation for a follow-up study on the mechanism of ZEN.

Table 5.

Carbohydrate active enzyme annotation of strain B72

Class definition	Genes No.	Percentage (%)
Auxiliary activities	5	3.33
Carbohydrate-binding modules	3	2.0
Carbohydrate esterases	31	20.67
Glycoside hydrolases	54	36
Glycosyl transferases	49	32.67
Polysaccharide lyases	8	5.33

Phylogenetic analysis of B72

To confirm the results of the 16S rRNA phylogenetic tree, we performed BLAST homology analysis using NCBI. The 16S rDNA sequence with high homology (12 strains) was selected, and MEGA X software was used for phylogenetic tree analysis (Fig. 3a). The results showed that strain B72 was closest to B. subtilis DSM 10 (GenBank ID: NR027552.1) and B. tequilensis 10b (GenBank ID: NR104919.1). To further determine the classification of strain B72, we compared it with the local database and selected 19 strains closest at the species level based on 31 housekeeping genes to construct a phylogenetic tree. We found that B72 was closely related to B. subtilis 168 (GCF_ 000009045.1), B. licheniformis PT-9 (GCF_002153395.1), and B. tequilensis KCTC 13622 (GCF_000507145.1) (Fig. 3b, Table S2). Therefore, we inferred that strain B72 may belong to B. subtilis, B. licheniformis, and B. tequilensis.

Fig. 3.

a Neighbor-joining (NJ) phylogenetic tree based on 16S rRNA gene sequence shows the phylogenetic position of strain B72 among closely related taxa. Bootstrap values (expressed as percentages of 1000 replications) above 75% are shown at branch points. Bar, 0.01 nucleotide substitutions per position; b Neighbor-joining (NJ) phylogenetic tree based on comparing with 19 strains closest to each other at the species level with 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB,rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC,rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). Bootstrap values (expressed as percentages of 1000 replications) above 75% are shown at branch points. Bar, 0.05 nucleotide substitutions per position

Analysis of ANI and GGDC

To further identity the B72 genome, we performed DNA-DNA hybridization (dDDH) and calculated the ANI based on BLAST (ANIb) values. We selected the reference genomes based on the closest neighbor to B72 in the phylogenetic tree: B. subtilis 168 (GCF_ 000009045.1), B. licheniformis PT-9 (GCF_002153395.1), and B. tequilensis KCTC 13622 (GCF_000507145.1). ANIb analysis showed that the similarity between B72 and B. subtilis, B. licheniformis and B. tequilensis was 98.39%, 91.67%, and 92.34%, respectively. The results of the phylogenetic analysis confirmed that B72 may be B. subtilis (Fig. 4a). GGDC analysis showed that the dDDH values of B72 with B. subtilis, B. licheniformis and B. tequilensis were 86.1%, 47.3%, and 44.7%, respectively. This also supports our findings that the GGDC values of B. licheniformis and B. tequilensis for B72 were lower than the 70% species cutoff value proposed (Meier-Kolthoff et al. 2013a, b), while the value of B. subtilis was higher than the 70% species cutoff value (Fig. 4b). Therefore, it was further confirmed that B72 was B. subtilis.

Fig. 4.

a ANIb analysis of strain B72 and 3 Bacillus (B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622) reference genomes. The species cutoff value is 95%. b GGDC analysis of strain B72 and 3 Bacillus (B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622) reference genomes. The species cutoff value is 70%

Degradation of ZEN by Bacillus subtilis B72

As shown in Fig. 5a, B. subtilis B72 grew rapidly within 0–4 h and degraded 94.46% of ZEN in minimal medium after 4 h. Accordingly, the ZEN residue in minimal medium was decreased from 10 to 0 μg/mL after treatment with B. subtilis B72 at 37 °C for 8 h (Fig. 5a). Strain B72 grew gradually from 4 to 8 h, and we inferred that the slow growth of strain B72 after 8 h might be due to the lack of ZEN as the sole carbon source in the culture medium. Our results showed that strain B72 is the fastest ZEN-degrading strain identified to date. Wu et al. showed that Stappia sp. WLB 29 could degrade 98.8% of ZEN after 60 h in minimal medium (Wu et al. 2022).

Fig. 5.

a The growth curve of strain B72 was calculated by OD600nm at 0–24 h and measurements of ZEN degradation by B72 in minimal medium with 10 μg/mL ZEN at 0–24 h; b B72 was cultured in minimal medium with 10 μg/mL ZEN and incubated under different conditions for 24 h: PBS (control), protease K, cell extracts + 100 ℃, cell extracts + protease K, cell extracts + protease K + SDS and cell extracts. Data represent means ± SD of three independent replicates (b). Statistical significance was determined by Student’s t test (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 (b)

To further determine the enzymatic degradation of ZEN, the cell extract of strain B72 was treated with heat, protease K, and protease K + SDS. As shown in Fig. 5b, the degradation rate of ZEN by the cell extract of B72 after heat treatment was only 1.6%, which might be due to the denaturation and aggregation of proteins caused by heat treatment, ultimately resulting in the loss of enzyme activity. After the cell extract was treated with protease K, its ability to degrade ZEN was only 10.36%, whereas treatment with protease K and protease K + SDS almost completely eliminated the ability of the cell extract to degrade ZEN (Fig. 5b). We inferred that SDS could destroy the spatial structure of proteins, whereas protease K could destroy the peptide bond between amino acids in proteins (Zhang et al. 2020). Therefore, we believe that there is a protein or enzyme in the cells of strain B72 that can degrade ZEN. Recently, several strains have been shown to degrade ZEN through enzyme secretion. For instance, Rhizopus spp. could catalyze the glycosylation on phenolic hydroxyl group in ZEN to produce low-toxicity zearalenone-4-beta-d-glucopyranoside (Kamimura 1986). Aspergillus niger hydrolyzes the sulfation of zearalenone at the C-4 hydroxyl group to reduce ZEN toxicity (Sun et al. 2014). We inferred that ZEN degradation by B72 might involve degrading enzymes produced during the initial period of bacterial growth and that the enzyme was relatively stable. However, further analysis and investigation of the specific degradation mechanisms are needed.

Identification of ZEN-degradation gene of Bacillus subtilis B72

To identify the genes involved in ZEN degradation, the ZEN-degrading enzymes reported before against the genome of B72 were compared using BLAST with six protein databases (NR, Swiss-Prot, Pfam, COG, GO, KEGG), and the setting threshold was E value ≤ 1e⁻⁵ (Bairoch and Apweiler 2000; Finn et al. 2008; Jensen et al. 2008; Kallberg and Persson 1999; Kanehisa and Goto 2000; Vesztrocy and Dessimoz 2017). Three main types of biological ZEN-degrading enzymes can be distinguished based on the difference of three groups on the molecule: laccase, lactone hydrolase, and peroxidase. The first reaction site is located on the long lactone ring, especially the C–C bond near the carbonyl group. The second position is the ester bond of the lactone ring, and the resulting molecule inactivates its estrogen-mimicking features once the lactone ring be opened. Moreover, another way to degrade ZEN is opened up the aromatic ring (Kriszt et al. 2012). As shown in Table S3, two genes were identified with laccase function, that is, the laccase-encoding genes yfiH (gene 1743) and cotA (gene 2671). Laccase utilized oxygen as an electron acceptor to oxidize polyphenol, methoxy substituted phenol, bisphenol, aromatic diamine, and related substances (Chaurasia et al. 2015; Couto & Herrera 2006). Laccase is a multicopper oxidase protein that degrades ZEN (Qin et al. 2021; Wang et al. 2019b). The results showed that laccase produced by B. subtilis B72 could hydrolyze the lactone ring of ZEN, resulting in its degradation. In summary, laccase in B. subtilis B72 might be related to ZEN degradation, and further research is needed to verify the conjecture.

Conclusions and possible challenges

In this study, a novel ZEN degradation by B72 isolated from the soil of an oil field in Xinjiang, China, was reported. The genome size of B72 is about 4.0 Mbp. Phylogenomic analyses demonstrated that strain B72 is a novel Bacillus species that is closely related to B. subtilis. Whole-genome sequencing of B72 was performed to identify the enzymes for ZEN degradation, and functional genome annotation revealed that B72 has the laccase-encoding genes yfiH (gene 1743) and cotA (gene 2671), which might be responsible for ZEN degradation by B72. Our investigation will provide an insightful insight for the genomic features and facilitate the valid reference data on ZEN degradation by the genus B. subtilis B72.

Through the analysis of the growth and degradation characteristics of strain B72 and found that B72 could efficiently degrade ZEN, but the degradation products of ZEN were not analyzed. The analysis of degradation products could better understand the degradation mechanism of strain B72 to ZEN. Moreover, we also found that two key genes of B72 degrading ZEN, but the specific mechanism needs to be verified by molecular means.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

NW and KL conceived the idea. KL & JYJ wrote the manuscript. KL & QX performed the experiments and analyses. All authors reviewed and gave suggestions to improve the manuscript.

Funding

This work was financially supported by the National Key Research and Development Program of China (No. 2020YFC1606800) and China Postdoctoral Science Foundation (No. 211100B62102).

Data availability

The whole-genome sequence of Bacillus subtilis B72 have been uploaded to the NCBI database. The BioProject number is PRJNA835306, the BioSample number is SAMN28099767, and the GenBank accession number is JAMDFN000000000.1. Our MIxS standard information have been uploaded in NCBI and the MIxS table can be found at https://submit.ncbi.nlm.nih.gov/subs/biosample/SUB11545850/overview. This announcement describes the first version of the genome assembly.

Declarations

Conflict of interest

All authors declare no conflict of interest.