Abstract
Aflatoxin B[1] (AFB[1]) contamination is a food safety issue
threatening human health globally. Biodegradation is an effective
method for overcoming this problem, and many microorganisms have been
identified as AFB[1]-degrading strains. However, the response
mechanisms of these microbes to AFB[1] remain unclear. More degrading
enzymes, especially of new types, need to be discovered. In this study,
a novel AFB[1]-degrading strain, DDC-4, was isolated using coumarin as
the sole carbon source. This strain was identified as Bacillus
halotolerans through physiological, biochemical, and molecular methods.
The strain’s degradation activity was predominantly attributable to
thermostable extracellular proteins (degradation rate remained
approximately 80% at 90 °C) and was augmented by Cu^2+ (95.45% AFB[1]
was degraded at 48 h). Alpha/beta hydrolase (arylesterase) was selected
as candidate AFB[1]-degrading enzymes for the first time as a gene
encoding this enzyme was highly expressed in the presence of AFB[1].
Moreover, AFB[1] inhibited many genes involved in the nucleotide
synthesis of strain DDC-4, which is possibly the partial molecular
mechanism of AFB[1]’s toxicity to microorganisms. To survive under this
stress, sporulation-related genes were induced in the strain.
Altogether, our study identified a novel AFB[1]-degrading strain and
explained its response mechanisms to AFB[1], thereby providing new
insights for AFB[1] biodegradation.
Keywords: aflatoxin B[1], Bacillus halotolerans, biodegradation,
thermostable extracellular proteins, response mechanisms
1. Introduction
Aflatoxins are a group of noxious difuran coumarin derivatives and are
mainly produced by the Aspergillus species (e.g., As. flavus and As.
parasiticus) [[36]1]. They primarily spread through contamination of
various foodstuffs (e.g., nuts, corn, and oil by-products) during crop
growth, harvest, and storage [[37]2,[38]3,[39]4]. Moreover, this toxin
can barely be degraded naturally because of its high stability. Thus,
approximately five billion people are at the risk of chronic exposure
to aflatoxin worldwide [[40]5]. Among the identified aflatoxins,
aflatoxin B[1] (AFB[1]) is regarded the most toxic, carcinogenic, and
mutagenic because of the C8-C9 double bond of the difuran ring and the
lactone ring within the coumarin ring [[41]6].
In the last decade, several physical, chemical, and biological
approaches have been reported for AFB[1] degradation [[42]5]. Compared
with other methods, biodegradation is the most promising alternative
because of its high specificity, eco-friendliness, and harmlessness to
nutritional and organoleptic properties of food [[43]6]. Until now,
many AFB[1]-degrading strains have been identified, such as Bacillus
subtilis UTBSP1 [[44]7], Pseudomonas putida [[45]8], Mycobacterium
smegmatis mc^2 155 [[46]9], Rhodococcus pyridinivorans [[47]10], As.
niger FS10 [[48]11], Zygosaccharomyces rouxii [[49]12], Armillariella
tabescens [[50]13], and Trametes versicolor [[51]14]. However, the
response mechanisms related to AFB[1] toxicity, degradation, and
adaptation in degrading strains remain unknown.
Degrading strains chiefly mediate AFB[1] degradation by producing
enzymes that convert this toxin into less toxic or nontoxic
metabolites. Most of the reported degrading enzymes are
oxidoreductases, including oxidase (e.g., aflatoxin oxidase enzyme, AFO
[[52]13] and laccases [[53]15]), peroxidase (e.g., manganese
peroxidase, MnP) [[54]16], and reductases (e.g., F[420]/H[2]-dependent
reductases) [[55]9]. The degradation mechanisms of oxidase and
peroxidase to AFB[1] are mainly oxidation and hydroxylation reactions.
The major chemically active location for these reactions is the difuran
ring due to the presence of a double bond in conjugation with an oxygen
atom [[56]17]. AFO from Ar. tabescens and MnPs from the white-rot
fungus such as Phanerochaete sordida YK-624 could oxidize the furan
ring of AFB[1] to 8,9-epoxide formation, further forming
8,9-dihydrodiol through hydrolysis [[57]16,[58]18]. A conversion of
AFB[1] to AFQ[1] is also a common degradation pathway, which was found
in laccase of Lac2 from the white-rot fungus [[59]19], CotA laccase
from B. licheniformis [[60]20], and dye-decolorizing peroxidase type B
[[61]21]. The major targets of reductases are unsaturations in furan
and lactone rings and the α-β unsaturated carbonyl group [[62]17].
F[420]/H[2]-dependent reductases identified from M. smegmatis could
reduce α,β-unsaturated esters of AFB[1] [[63]9].
As determined by the structures of AFB[1] and degradation products,
hydrolysis, demethylation, demethoxylation, and decarbonylation
reactions are also involved in the degradation mechanisms [[64]17]. In
many AFB[1]-degrading strains, hydrolysis of the lactone ring has been
reported as a starting point [[65]17]. After hydrolysis, the presence
of the α-β unsaturated in the product increases its chemical activity,
leading to a series of degradation reactions, including decarboxylation
and the cleavage of the cyclopentenone ring, which convert AFB[1] to
AFD[1] and further to AFD[2] [[66]8]. Moreover, some demethylated,
demethoxylated, and decarbonylated products were also found in the
biodegraded products of T. versicolor [[67]14] and Tetragenococcus
halophilus CGMCC 3792 [[68]22]. However, the enzymes involved in the
above reactions have not been identified. Therefore, more
AFB[1]-degrading enzymes, especially of new types, need to be
identified. This will contribute to efficient AFB[1] degradation
through genetic engineering methods.
With the advancements of high-throughput sequencing and bioinformatics,
omics technologies offer a new in-depth insight into the response
mechanism, which will help identify more genes encoding degrading
enzymes [[69]23]. Xu et al. identified a gene encoding the novel
zearalenone degradation-associated thioesterase from B.
amyloliquefaciens H6 through transcriptomic analysis [[70]24]. On
investigating the detoxification mechanism of R. pyridinivorans GF3 in
response to thraquinone-2-sulfonate (ASA-2) through transcriptomic
analysis, Wang et al. found that cytochrome P450 and short-chain
dehydrogenase/reductase are involved in ASA-2 degradation [[71]25]. By
performing transcriptomic analysis, Wei et al. explored the response
mechanism of Cryptococcus podzolicus Y3 under ochratoxin A stress
[[72]26]. Protein processing in C. podzolicus Y3 was inhibited by
ochratoxin A, and C. podzolicus Y3 improved the excision repair pathway
to protect genetic information.
In this study, 12 AFB[1]-degrading strains were screened from moldy
maize, moldy rice, and strains stored in our laboratory (isolated from
Chinese traditional fermented foods). Among them, strain DDC-4
exhibited the highest degradation activity and was identified as B.
halotolerans through physiological, biochemical, and molecular methods.
The active component and its characteristics were explored, and
transcriptomic analysis was performed to explore the response
mechanisms of strain DDC-4 to AFB[1]. Several candidate
AFB[1]-degrading genes, especially the previously ignored alpha/beta
hydrolase (arylesterase) gene, were mined.
2. Results
2.1. Isolation and Identification of AFB[1]-Degrading Strains
The modified Hormisch medium, containing coumarin as the sole carbon
source, was used for obtaining potential AFB[1]-degrading strains
[[73]27]. Coumarin is the basic molecular structure of aflatoxin B[1]
with lower price and more secure. The strains that could grow on
modified Hormisch medium have the ability to utilize coumarin as their
carbon source indicating they might also be able to degrade aflatoxin
B[1] [[74]28]. In total, 12 strains were isolated from various sources,
namely 7 strains from moldy rice (i.e., ZYX1–ZYX7, respectively), 3
strains from moldy maize (i.e., DC-1, DC-3, and DC-5, respectively),
and 2 strains from among those stored in our lab (i.e., DDC-1 and
DDC-4, respectively). Of the strains isolated, strain DDC-4 exhibited
the highest AFB[1] degradation rate of 76.30% ± 2.18% after 72 h of
incubation at 37 °C, which was significantly higher than other strains
([75]Figure 1).
Figure 1.
[76]Figure 1
[77]Open in a new tab
The aflatoxin B[1] (AFB[1]) degradation rates of isolated strains
co-incubated with AFB[1] in the dark at 37 °C for 72 h with shaking.
Each value is presented as the mean ± SD (n = 3). Different letters
represent significant differences between species (p < 0.05).
During the physiological and biochemical tests, strain DDC-4, a
Gram-positive rod bacterium, displayed the typical characteristics of
Bacillus species ([78]Table 1). The strain could use glucose,
arabinose, xylose, mannitol, gelatin, starch, casein, citrate, and
malonate and reduce nitrate and grow at pH 5.7, but it could not grow
in the presence of lysozymes. Moreover, it exhibited certain
temperature adaptability (30 °C–50 °C) and salt resistance (up to 10%
(w/v) NaCl). Additionally, strain DDC-4 exhibited catalase activity but
no phenylalanine dehydrolase or tryptophanase activity. Meanwhile, an
approximately 1500 bp product was amplified from the genomic DNA of
DDC-4, and a neighbor-joining tree was constructed based on the results
of the 16S rRNA gene sequence analysis ([79]Figure 2). Compared with
the outgroup Metabacillus galliciensis, Bacillus species were grouped
together in a single cluster. Strain DDC-4 and B. halotolerans ATCC
25096^T were clustered into the same clade with 100% sequence
similarity. Thus, strain DDC-4 was recognized as B. halotolerans. To
the best of our knowledge, this study is the first to report B.
halotolerans as an AFB[1]-degrading strain.
Table 1.
Physiological and biochemical characteristics of strain DDC-4.
Items DDC-4
Gram stain Gram-positive rod
Moveability +
Voges-Proskauer +
Oxidation of
glucose +
arabinose +
xylose +
mannitol +
Hydrolysis of
gelatin +
starch +
casein +
Growth on
citrate +
lysozyme −
5 °C −
10 °C −
30 °C +
40 °C +
50 °C +
55 °C −
65 °C −
NaCl (2%) +
NaCl (5%) +
NaCl (7%) +
NaCl (10%) +
pH 5.7 +
Phenylalanine dehydrolase −
Catalase activity +
Nitrate reduction +
Malonate +
Indole −
[80]Open in a new tab
“+” and “−” indicates that the result is positive and negative,
respectively.
Figure 2.
[81]Figure 2
[82]Open in a new tab
Neighbor-joining tree reconstructed using 16S rRNA gene sequences from
the EzBioCloud server by MEGA software version 6.0. Metabacillus
galliciensis was used as an outgroup. Numbers at branches indicate
bootstrap values (>50%) from 1000 replicates.
2.2. AFB[1] Degradation by the Active Component of Strain DDC-4 and Its
Characteristics
We here investigated whether the fermentation broth, cell-free
supernatant, cell suspension, and cell lysate can cause AFB[1]
degradation ([83]Figure 3A). Overall, the cell-free supernatant removed
55.04% ± 2.60% of AFB[1] after 72 h incubation, whereas the cell
suspension and cell lysate were almost unable to remove AFB[1], with
their degradation rates being −1.88% ± 8.46% and 4.44% ± 0.52%,
respectively, dramatically lower than that of the cell-free
supernatant. This indicated that AFB[1] removal by strain DDC-4
predominantly depended on degradation rather than on absorption. The
cell-free supernatant was the main active component of strain DDC-4
during degradation. These findings are consistent with those of
Bacillus species (e.g., B. licheniformis CFR1, B. subtilis UTBSP1, B.
velezensis DY3108, and B. amyloliquefaciens WF2020)
[[84]7,[85]29,[86]30,[87]31]. Although the cell-free supernatant had a
major role in degradation, the degradation rate with the supernatant
was significantly lower than that with the fermentation broth.
Therefore, AFB[1] could be speculated to exert an induction effect on
the degradation activity of strain DDC-4. In other words, the
expression level of genes encoding degradation-associated extracellular
metabolites might be augmented under AFB[1] stress to reduce
AFB[1]-induced damage. To verify this hypothesis, we evaluated the
induction effect of AFB[1]. The degradation rate of the cell-free
supernatant increased to 68.08% ± 4.11% after induction. This rate was
significantly higher than that of the noninduction group (50.49% ±
8.26%) and was almost the same as that of the fermentation broth
([88]Figure 3B).
Figure 3.
[89]Figure 3
[90]Open in a new tab
Degradation of AFB[1] by different components of strain DDC-4 in the
dark at 37 °C for 72 h (A), and AFB[1]-induced enhancement effect (B).
Each value is presented as the mean ± SD (n = 3). Different letters
represent significant differences between species (p < 0.05).
The degradation rate of the cell-free supernatant dramatically
decreased to 10.60% ± 8.61% and 39.37% ± 1.18% after SDS and proteinase
K pretreatments, respectively ([91]Figure 4A). This might be because
the structure of the protein in the supernatant was destroyed. By
contrast, the degradation rate exhibited no decrease but increased
slightly after heat treatment. The degradation rate increased more
significantly when the heat treatment was prolonged. We further
investigated the effect of incubation temperature on degradation
activity. Similarly, the degradation rate significantly increased at 30
°C–60 °C, from 18.51% ± 1.34% to 79.24% ± 3.67%, and remained stable
(approximately 80%) at 60 °C–90 °C ([92]Figure 4B). According to these
results, thermostable proteins or perhaps enzymes with a broad
temperature adaptability present in the supernatant were involved in
AFB[1] degradation, and their activities were activated by heat
treatment. Similar results have been observed in some AFB[1]-degrading
strains, such as B. velezensis DY3108 [[93]30], B. shackletonii L7
[[94]32], and P. aeruginosa N17-1 [[95]33], which have made them more
advantageous in industrial applications.
Figure 4.
[96]Figure 4
[97]Open in a new tab
The influences of different factors on AFB[1] degradation by the
cell-free supernatant of strain DDC-4. The influence of heat, SDS and
proteinase K treatments (A), temperature (B), pH (C), metal ions (D),
copper concentration (E), and incubation time (F) are shown. Each value
is presented as the mean ± SD (n = 3). Different letters represent
significant differences between species (p < 0.05).
Considering the enzyme activity loss during freeze–drying, although the
cell-free supernatant was pH sensitive, it could still degrade AFB[1]
within 5–10 pH ([98]Figure 4C). The maximum degradation rate of 45.11%
± 1.99% was observed at pH 7, while decreased significantly as the pH
increased or decreased because of the impaired activity of the enzymes
in the supernatant. Compared to acid, the supernatant had a stronger
tolerance to alkalis. The degradation rate decreased to 30.63% ± 2.75%
at pH 8 and decreased to 22.07% ± 4.22% at pH 6. The degradation rates
at pH 5, pH6, pH 9 and pH 10 were not significantly different.
Moreover, the activity of the cell-free supernatant was almost lost at
pH 4 and pH 11, with their degradation rates being 1.63% ± 0.09% and
6.33% ± 1.92%, respectively, which were significantly lower than
others.
Additionally, the effects of metal ions on degradation by the cell-free
supernatant were evaluated ([99]Figure 4D). Cu^2+ dramatically enhanced
the degradation rate to 95.88% ± 1.51%, whereas Zn^2+ and Fe^3+ exerted
no significant effect. However, Li^+, Ni^+, Mg^2+, and Ca^2+ inhibited
the degradation activity to a certain extent. Furthermore, the
influence of the copper concentration revealed that the degradation
rate increased sharply within the range of 0–10 mM Cu^2+ and decreased
slightly afterward ([100]Figure 4E). Thus, Cu^2+ might act as an
activator or membrane stabilizer or an electron transfer medium for
enzymes to stimulate AFB[1] degradation activity, comparatively similar
to the results obtained in other AFB[1]-degrading strains, including B.
amyloliquefaciens WF2020 [[101]31], B. velezensis DY3108 [[102]30], and
B. shackletonii L7 [[103]32]. When 10 mM Cu^2+ was added, 41.56% ±
2.02% AFB[1] was degraded in the initial 6 h and 95.45% ± 1.81% AFB[1]
was degraded after 48 h incubation ([104]Figure 4F). This indicated
that the supernatant caused relatively rapid degradation. Moreover, the
supernatant decreased the AFB[1] content in the moldy maize powder from
6.39 ± 0.43 μg/kg to 2.96 ± 0.92 μg/kg (the degradation rate was 53.77%
± 14.42%, [105]Table 2), which demonstrates that the cell-free
supernatant of strain DDC-4 can be a potential tool for handling moldy
grains.
Table 2.
The content of aflatoxin B[1] (AFB[1]) in moldy maize powder.
Sample Name The Content of AFB[1] (μg/kg)
Initial 6.76 ± 0.85 ^a
Sterilization 6.39 ± 0.43 ^a
Treatment 2.96 ± 0.92 ^b
[106]Open in a new tab
Initial: moldy maize powder without treatment; sterilization: moldy
maize powder was sterilized in an autoclave; treatment: moldy maize
powder was mixed with the cell-free supernatant of strain DDC-4 and 10
mM Cu^2+ for 48 h after sterilization.
Overall, the active components of strain DDC-4 were thermostable
extracellular proteins. AFB[1] induced the expression of genes encoding
these proteins, and Cu^2+ and heat treatment increased the activity of
these proteins.
2.3. GO Term and KEGG Pathway Enrichment Analyses
To reveal the molecular response of DDC-4 to AFB[1], transcriptomic
analysis was performed. Q20 and Q30 values for each sample were greater
than 98% and 95%, respectively ([107]Table S1). More than 95% of the
clean reads were mapped to the reference genome ([108]Table S2), which
indicated the reliability of the RNA sequencing results. The distance
between the treated and untreated samples was significant ([109]Figure
S1). In total, 165 upregulated and 284 downregulated differentially
expressed genes (DEGs; [110]Figure S2), mapped to 27 and 32 GO terms
([111]Figure S3), respectively, were identified after AFB[1] treatment.
The upregulated DEGs were significantly enriched in 10 GO terms,
including the histidine catabolic process, the histidine catabolic
process to glutamate and formamide, the histidine catabolic process to
glutamate and formate, and developmental process ([112]Figure 5A). All
the top three GO terms were related to the histidine catabolic process,
with all the rich factors (the ratio of the enriched DEGs to total
transcripts) being 1.00. Similar results were observed in the KEGG
pathway enrichment analysis. The upregulated DEGs were significantly
enriched in the histidine metabolism pathway ([113]Figure 6A).
Following AFB[1] treatment, the expression of genes encoding histidine
ammonia-lyase (EC: 4.3.1.3, encoded by the gene RS11215, HutH),
urocanate hydratase (EC: 4.2.1.49, encoded by the gene RS11210, HutU),
imidazolonepropionase (EC: 3.5.2.7, encoded by the gene RS11205),
formimidoylglutamase (EC: 3.5.3.8, encoded by the gene RS11200), and
aldehyde dehydrogenase DhaS (EC: 1.2.1.3, encoded by the gene RS20930,
DhaS) was significantly upregulated to varying degrees ([114]Figure 7
and [115]Figure 8A). Among them, gene RS20930 was the most highly
expressed, and the transcripts per million (TPM) values in the samples
untreated and treated with AFB[1] were 5215 and 11,948, respectively.
The expression level of gene RS11215 showed the most significant
difference between untreated and treated samples with the log2 fold
change being 2.11. The induced histidine metabolism-related genes
promoted the conversion of histidine to glutamate, a precursor for
glutathione synthesis. Glutathione possibly participates in AFB[1]
degradation by binding to AFB[1] or intermediate products, which is
consistent with the results of Qiu et al. [[116]34]. Furthermore, the
number of DEGs enriched in the developmental process was the highest,
as determined through the GO term enrichment analysis ([117]Figure 5A).
DEGs in this process were predominantly related to sporulation
([118]Table S3), possibly because sporulation in strain DDC-4 was
promoted under AFB[1]-induced stress.
Figure 5.
[119]Figure 5
[120]Open in a new tab
GO term enrichment analysis of upregulated (A) and downregulated (B)
differentially expressed genes (DEGs). The circle size indicates the
number of DEGs enriched in each pathway. The Q value indicates the
significance of enrichment, increasing from blue to red. Rich factor
represents the ratio of the enriched DEGs to total transcripts in this
pathway. *, represents the DEGs in significantly enriched pathways.
Figure 6.
[121]Figure 6
[122]Open in a new tab
KEGG pathway enrichment analysis of upregulated (A) and downregulated
(B) DEGs. The circle size indicates the number of DEGs enriched in each
pathway. The Q value indicates the significance of enrichment,
increasing from blue to red. Rich factor represents the ratio of the
enriched DEGs to total transcripts in this pathway. *, represents the
DEGs in significantly enriched pathways.
Figure 7.
[123]Figure 7
[124]Open in a new tab
The pathway of histidine metabolism. The red rectangle indicates the
enzyme-encoding gene induced by AFB[1.] The reaction substrates and
products of these enzymes are also shown.
Figure 8.
[125]Figure 8
[126]Open in a new tab
Expression patterns of DEGs in the pathway of histidine metabolism (A),
DEGs in the pathway of ‘de novo’ IMP biosynthetic process (B),
upregulated oxidoreductase encoding genes (C), and upregulated
hydrolase encoding genes (D). Different colors represent different
expression levels (increasing from green to red). U and T indicate
AFB[1]-untreated and -treated samples, respectively.
The downregulated DEGs were significantly enriched in 22 GO terms,
including de novo IMP biosynthesis, IMP biosynthesis, IMP metabolism,
purine nucleobase biosynthesis, purine nucleoside monophosphate
biosynthesis, purine ribonucleoside monophosphate biosynthesis, and
purine-containing compound biosynthesis ([127]Figure 5B). The top three
GO terms were all related to the IMP metabolic process, with all the
rich factors being >0.75. Nearly all genes associated with the de novo
IMP biosynthesis process (including genes RS06555, RS06560, RS06565,
RS06570, RS06575, RS06580, RS06585, RS06590, RS06595, RS06600, RS06605,
and RS06610) were inhibited to varying degrees ([128]Figure 8B and
[129]Table 3). Among them, the expression of gene RS06585, RS06610,
RS06590, RS06595, and RS06605 were dramatically inhibited by AFB[1],
with the log2 fold change being −2.63, −2.53, −2.39, −2.36, and −2.28,
respectively. IMP serves as a precursor of AMP and GMP during de novo
purine nucleobase biosynthesis. Although de novo pyrimidine nucleobase
biosynthesis was not significantly inhibited, the expression of genes
encoding the enzyme (carbamoyl phosphate synthase, encoded by the genes
RS03965 and RS03960) involved in the first-step reaction of this
process was dramatically downregulated, with the log2 fold change of
the expression level being approximately −5. The expression of genes
encoding hypoxanthine/guanine permease (encoded by the gene RS06640,
PbuG) and uracil permease (encoded by the gene RS01625, PyrP), which
might transport raw materials for the salvage pathway, was also
downregulated. Similarly, the top three KEGG pathways with the highest
number of enriched DEGs were purine metabolism, the two-component
system, and ABC transporters, respectively ([130]Figure 6B). These
results indicated that AFB[1] significantly inhibited nucleotide
synthesis in strain DDC-4.
Table 3.
Differentially expressed genes (DEGs) enriched in the ‘de novo’ purine
nucleobase biosynthetic process.
Gene id Gene Name Gene Description
RS06555 PurD phosphoribosylamine-glycine ligase
RS06560 PurH IMP cyclohydrolase
RS06565 PurN phosphoribosylglycinamide formyltransferase
RS06570 RS06570 phosphoribosylformylglycinamidine cyclo-ligase
RS06575 RS06575 amidophosphoribosyltransferase
RS06580 PurL phosphoribosylformylglycinamidine synthase subunit PurL
RS06585 PurQ phosphoribosylformylglycinamidine synthase subunit PurQ
RS06590 PurS phosphoribosylformylglycinamidine synthase subunit PurS
RS06595 RS06595 phosphoribosylaminoimidazolesuccinocarboxamide synthase
RS06600 PurB adenylosuccinate lyase
RS06605 PurK 5-(carboxyamino)imidazole ribonucleotide synthase
RS06610 PurE 5-(carboxyamino)imidazole ribonucleotide mutase
[131]Open in a new tab
2.4. Identification and Expression Analysis of Potential Degrading Genes
The reported AFB[1]-degrading enzymes were primarily oxidoreductases.
Meanwhile, hydrolase may be involved in AFB[1] degradation from the
degradation product perspective. According to our results, eight genes
encoding oxidoreductases and six genes encoding hydrolases were induced
following AFB[1] treatment ([132]Table 4). Among these genes, the gene
RS11000 (aldo/keto reductase-encoding gene) was the most highly
expressed ([133]Figure 8C,D), and the TPM values in the samples
untreated and treated with AFB[1] were 305 and 930, respectively.
Aldo/keto reductase, short-chain dehydrogenase/reductases (SDR) family
oxidoreductase, and alpha/beta hydrolase (arylesterase), encoded by the
genes RS11000, RS07845, and RS04140, respectively, possibly destroyed
the lactone ring within the coumarin ring of AFB[1] to decrease its
toxicity and mutagenicity [[134]35]. Moreover, other oxidoreductases
and hydrolases might be involved in AFB[1] degradation ([135]Table 4),
but their potential action sites need to be further investigated.
Table 4.
Genes encoding oxidoreductase and hydrolase induced by AFB[1] in strain
DDC-4.
Gene id Gene Name Gene Description
RS16210 RS16210 cytochrome ubiquinol oxidase subunit II
RS16215 RS16215 cytochrome ubiquinol oxidase subunit I
RS02280 AhpA biofilm-specific peroxidase AhpA
RS11000 RS11000 aldo/keto reductase
RS07845 RS07845 SDR family oxidoreductase
RS04960 RS04960 NAD(P)H-dependent oxidoreductase
RS05120 RS05120 NAD(P)H-dependent oxidoreductase
RS02275 YkuV thiol-disulfide oxidoreductase YkuV
RS03385 RS03385 NUDIX hydrolase
RS19530 RS19530 alpha/beta hydrolase (haloalkane dehalogenase)
RS05750 RS05750 amidohydrolase
RS04140 RS04140 alpha/beta hydrolase (arylesterase)
RS02520 RS02520 glycoside hydrolase family 18 protein
RS00295 RS00295 poly-gamma-glutamate hydrolase family protein
[136]Open in a new tab
Potential degrading genes are shown in red.
Genes RS11000, RS07845 and RS04140 were selected for the qRT-PCR
analysis ([137]Figure 9). The expression trend of these genes was
consistent with the RNA-seq results, which confirmed the credibility of
the transcriptomic analysis results.
Figure 9.
[138]Figure 9
[139]Open in a new tab
Relative expression levels of gene RS11000 (A), RS07845 (B), and
RS04140 (C) between AFB[1]-treated and -untreated samples based on
qRT-PCR analysis. U and T represent AFB[1]-treated and -untreated
samples, respectively. 16S rRNA was used as an internal control. Each
value is presented as the mean ± SD (n = 3). *, representssignificant
differences between species (p < 0.05).
3. Discussion
3.1. AFB[1]-Degrading Strains and Functional Genes
Many AFB[1]-degrading strains have been identified. Of them, the B.
subtilis group was more sought after by researchers because of its
potential probiotic characteristics and antibacterial action against
Aspergillus species [[140]30,[141]31]. To our best knowledge, although
several strains, including B. subtilis UTBSP1 [[142]7], B.
licheniformis CFR1 [[143]29], B. shackletonii L7 [[144]32], B.
velezensis DY3108 [[145]30], B. amyloliquefaciens WF2020 [[146]31], and
B. albus YUN5 [[147]1], degrade aflatoxins, this study is the first to
identify B. halotolerans to degrade AFB[1]. Consistent with the results
of most reports about the B. subtilis group, the extracellular proteins
of strain DDC-4 were chiefly responsible for AFB[1] degradation. When
activated with 10 mM Cu^2+, 95.45% AFB[1] (initial concentration: 1
μg/mL) was degraded by the extracellular proteins at 48 h, which was
comparable to the results obtained with B. velezensis DY3108 (initial
concentration: 0.5 μg/mL, >90%, 24 h) [[148]30], B. amyloliquefaciens
WF2020 (initial concentration: 2 μg/mL, ~100%, 48 h) [[149]31], B.
licheniformis CFR1 (initial concentration: 0.5 μg/mL, >90%, 24 h)
[[150]29], and B. subtilis UTBSP1 (initial concentration: 2.5 μg/mL,
78.39%, 72 h) [[151]7] and higher than those obtained with B.
shackletonii L7 [[152]32] and B. subtilis JSW-1 [[153]36].
Additionally, different from the heat-labile proteins of B.
licheniformis CFR1 [[154]29] and B. subtilis UTBSP1 [[155]7], the
active extracellular proteins of strain DDC-4 were thermostable.
Furthermore, the degradation rate remained at approximately 80% at 90
°C, which was higher than those of B. amyloliquefaciens WF2020
[[156]31] and B. shackletonii L7 [[157]32], but slightly lower than
that of B. velezensis DY3108 [[158]30]. This facilitated the proteins
in maintaining catalytic stability in a harsh industrial environment.
The active extracellular proteins could remove 53.77% AFB[1] from the
moldy maize powder and is thus a promising agent for handling
AFB[1]-contaminated food in the industry.
Although few proteins with a degradation ability have been isolated
from the B. subtilis group, the response mechanism of this group to
aflatoxins has not been completely reported. A 22-kDa heat-stable
unidentified extracellular protein was purified from the cell-free
supernatant of B. shackletonii L7 [[159]32]. CotA laccase from B.
licheniformis ANSB82 could transform AFB[1] to aflatoxin Q[1] and
epi-aflatoxin Q[1] [[160]20]. Bacilysin biosynthesis oxidoreductase
(BacC) from B. subtilis UTB1 was involved in AFB[1] degradation by
reducing the α,β-unsaturated ester between the lactone rings of AFB[1]
[[161]37]. However, mass spectrometry of degradation products revealed
that the difuran and lactone rings of AFB[1] were all destroyed. Six
and eight major degraded products were identified in the reaction
mixture of AFB[1] coincubated with B. albus YUN5 [[162]1] and B.
subtilis [[163]14], respectively. Four major degraded products were
detected in the B. sp. H16v8 and B. sp. HGD9229 cocultures [[164]38].
This suggests that in addition to oxidoreductase, other types of
enzymes, particularly esterase, are involved in AFB[1] degradation.
According to Pereyra et al., N-acyl-homoserine lactonase might
contribute to AFB[1] degradation [[165]35]. However, not all
AFB[1]-degrading strains of the B. subtilis group could produce this
enzyme. In the present study, the transcriptomic analysis was performed
to identify the previously neglected gene-encoding alpha/beta hydrolase
(arylesterase) as the candidate gene for AFB[1] degradation. This study
provides a novel insight about AFB[1]-degrading enzymes. Alpha/beta
hydrolase is a class of enzymes having similar structures and diverse
functions, including esterase, lipase, proteases, and other hydrolytic
enzymes [[166]39]. Among the enzymes, arylesterase possibly targets the
ester bond of AFB[1] and thus cleaves its lactone ring to reduce its
toxicity and mutagenicity.
The gene RS11000 encodes for aldo/keto reductase, which might destroy
the lactone ring in AFB[1] by reducing the keto group to the OH group.
The gene RS07845 encodes for the SDR family oxidoreductase that has a
broad substrate specificity. After cloning the CgSDR gene from Candida
guilliermondii, Xing et al. found that recombinase transformed patulin
into non-toxic E-ascladiol [[167]40]. Thus, the SDR family
oxidoreductase in this study was speculated to cleave the lactone ring
in AFB[1] following the reduction reaction catalyzed by aldo/keto
reductase. Similar to the results of Xu et al., Cu^2+ possibly serves
as an electron transfer medium in redox reactions that boosts
degradation activity [[168]32]. Furthermore, all the aforementioned
proteins of strain DDC-4 belonged to the general stress protein, which
could confer advantages to bacteria under stress, such as salt,
osmosis, oxidative damage, and freezing [[169]41]. In this study, the
expression of genes RS11000 and RS07845 was significantly upregulated
under AFB[1] stress, which might allow the strain to survive in the
presence of the toxicological effects of AFB[1] because these genes are
associated with AFB[1] degradation.
Glutathione exerted its detoxification effect on AFB[1] by binding to
it or its intermediate products, and this was first observed in
mammals. In a reaction mixture of AFB[1] coincubated with A. niger
FS10, Qiu et al. analyzed AFB[1] degradation products through triple
quadrupole-linear ion trap-mass spectrometry (Q-Trap-MS) coupled with
LightSight™ software (Version 2.2.1) [[170]34]. They found that
glutathione formed AFB[2]-GOH (C[27]H[31]N[3]O[13]) with AFB[1] to
modify the toxicity site of AFB[1]. As mentioned above, glutathione
might participate in the AFB[1] degradation of strain DDC-4. As
glutamate is a precursor for glutathione synthesis, the conversion of
histidine to glutamate was promoted in the AFB[1]-treated samples
([171]Figure 10).
Figure 10.
[172]Figure 10
[173]Open in a new tab
Proposed response mechanisms of strain DDC-4 to AFB[1]. Up- and
downregulated encoding genes were displayed in red and green fillings,
respectively.
Several potential mycotoxins degradation genes were also selected by
transcriptomic analysis due to their upregulated expression in the
present of mycotoxins, such as short-chain aryl-alcohol dehydrogenase
for patulin degradation [[174]42] and carboxypeptidase A4 for
ochratoxin A degradation [[175]26]. However, the specific function of
these genes in mycotoxins degradation still needs to be validated by
heterologous expression. The degradation mechanism will be revealed by
analysis of degradation products of recombinant protein expressed in
host strain. The encoding gene of an acyl coenzyme A thioester
hydrolase in B. amyloliquefaciens H6 was selected from upregulated
genes under zearalenone stress by transcriptomic analysis [[176]24].
The recombinant protein was expressed in Escherichia coli. The purified
recombinant protein could convert zearalenone to the less toxic
metabolites by cleaving the lactone bond and breaking down its
macrolide ring [[177]24]. More experiments will be carried out in our
future study.
3.2. Toxicological Effect of AFB[1] on Nucleic Acid Synthesis
On measuring the incorporation of me-[^3H] thymidine and 6-[^14C]
orotic acid into DNA and RNA, respectively, Butler and Neal found that
AFB[1] inhibited nucleic acid synthesis [[178]43]. Numerous subsequent
studies have supported this viewpoint. However, the underlying
molecular mechanism remains unclear. We here conjectured that AFB[1]
inhibited nucleic acid synthesis in strain DDC-4 through two
hypothetical pathways ([179]Figure 10). First, AFB[1] inhibited
nucleotide synthesis in strain DDC-4. As shown previously, AFB[1]
significantly inhibited de novo nucleotide biosynthesis by suppressing
the expression level of genes encoding enzymes involved in this
process. Moreover, the salvage pathway might be inhibited by reducing
the transportation of raw materials. Second, AFB[1] inhibited DNA
replication in strain DDC-4. The process of DNA replication is divided
into three stages: initiation, extension, and termination. At the
beginning of extension, short RNA fragments (called primers), which are
synthesized by primase, are acted as a starting point for DNA
polymerase III. After termination, the primers are removed by
ribonuclease H and DNA polymerase I ([180]Figure S4). In this study,
the expression level of genes encoding ribonuclease H, including
ribonuclease HI (encoded by the gene RS20480) and ribonuclease HIII
(encoded by the gene RS17285), were inhibited by AFB[1]; thereby, DNA
replication was inhibited.
AFB[1] was bioactivated by cytochrome P450 to generate the intermediate
AFB[1]-8,9-epoxide [[181]44]. This intermediate product was then
attacked by N^7 of guanine to form
trans-8,9-dihydro-8-(N^7-guanyl)-9-hydroxyaflatoxin B[1]
(AFB[1]-N^7-Gua). This was considered as the main AFB[1]–DNA adduct
causing mutations ([182]Figure 10). Nucleotide excision repair (NER) is
a pivotal player in removing AFB[1]–DNA damage in both bacterial and
mammalian systems [[183]44]. In prokaryotes, the Uvr system is involved
in NER. In the present study, the expression level of the UvrD gene was
upregulated after AFB[1] treatment, whereas that of the UvrABC gene
remained almost unchanged.
Altogether, AFB[1] inhibited the synthesis of nucleotides, including
purines and pyrimidines, and DNA replication ([184]Figure 10).
Cytochrome P450-mediated mutations may increase following AFB[1]
treatment. In the case of the resistance and adaptation to AFB[1], the
expression of genes encoding the potential AFB[1]-degrading enzyme was
upregulated, and sporulation in strain DDC-4 was promoted.
Although several potential AFB[1]-degrading enzymes were selected,
further validation of their function is needed. In the future, we will
obtain the aforementioned enzymes through heterologous expression and
purification. Degradation activity of the enzymes will be verified, the
structures of the degradation products will be determined, and the
safety of degradation products will be evaluated.
4. Conclusions
In this study, a novel AFB[1]-degrading strain was isolated and
identified as B. halotolerans DDC-4 (belonging to the B. subtilis
group). The active components of this strain were thermostable
extracellular proteins or enzymes with a wide temperature adaptability.
More than 90% AFB[1] was degraded by the proteins or enzymes when Cu^2+
was added. Thus, after adequate purification, these enzymes or proteins
could serve as promising agents for AFB[1] biodegradation in the food
industry. To our best knowledge, this study is the first to explore
response mechanisms of the B. subtilis group to AFB[1] through
transcriptomic analysis. Inhibition of nucleic acid synthesis was the
primary toxicological effect of AFB[1] on strain DDC-4. To survive
under this stress, sporulation was promoted in the bacteria and the
expression of genes encoding these degradation-related enzymes were
induced. The genes encoding alpha/beta hydrolase (arylesterase),
aldo/keto reductase, and SDR family oxidoreductase were selected as
candidate genes for AFB[1] degradation. Our study will be helpful to
reveal the degradation mechanism of AFB[1] and provide more options for
handling AFB[1]-contaminated food.
5. Materials and Methods
5.1. Isolation of AFB[1]-Degrading Strains
First, 10 g moldy maize and 10 g moldy rice were separately diluted in
90 mL sterile distilled water and incubated in water bath shaker
(Guangdong Foheng Instrument Co., Ltd., Dongguan, China) at 37 °C with
continuous shaking (150 rpm) for 72 h. The samples were serially
diluted to 10^−7 with sterile distilled water. Aliquots (150 µL) of
each dilution or strains stored in our lab (isolated from Chinese
traditional fermented foods) were spread on plates containing modified
Hormisch medium (HM: 0.1% coumarin, 0.05% KNO[3], 0.05%
(NH[4])[2]SO[4], 0.025% KH[2]PO[4], 0.025% MgSO[4]·7H[2]O, 0.0005%
CaCl[2], 0.0003% FeCl[3]·6H[2]O, 2% agar) [[185]27]. Each plate was
cultured at 37 °C for 7 days. Visible single colonies were isolated and
transferred to fresh HM plates. The aforementioned process was repeated
3–5 times until pure isolates were obtained.
To test the AFB[1] degradation activity, each pure isolate was
inoculated in Luria-Bertani (LB) medium, cultivated overnight at 37 °C
with continuous shaking (150 rpm), and diluted to an optical density at
600 nm (OD[600]) of 0.4. The medium was modified to maintain neutrality
during fermentation. Then, 500 μL of each dilution was added to the
modified LB medium (1% peptone, 1% NaCl, 0.5% yeast extract, 0.1%
KH[2]PO[4]). Fermentation was carried out at 37 °C for 48 h by shaking.
Then, 960 μL of the fermentation broth was co-incubated with 40 μL of
25 μg/mL AFB[1] (Yuanye Bio-Technology Co., Ltd., Shanghai, China) in
the dark at 37 °C for 72 h with shaking (150 rpm). Sterile modified LB
medium containing AFB[1] was used as the control. The supernatant was
recovered through centrifugation at 4500 rpm for 10 min at room
temperature. Subsequently, 650 μL of the supernatant was mixed with 350
μL methanol, and residual AFB[1] was analyzed using the ELISA kit
(Youlong Biotech Co., Ltd., Shanghai, China). According to kit
instruction, the cross-reactivity ration with similar toxin AFB[2],
AFG[1], and AFG[2] was 13%, 1.9%, and 5.7%, respectively, indicating
the kit could specifically detect AFB[1]. The AFB[1] degradation rate
was calculated as follows:
[MATH: Y=X1−X2
/X1×100% :MATH]
(1)
where X[1] is the residual AFB[1] in the control, X[2] is the residual
AFB[1] in the sample, and Y is the AFB[1] degradation rate (%).
5.2. Identification of Strain DDC-4
Strain DDC-4 was identified through physiological and biochemical tests
and 16S rRNA gene sequencing. The physiological and biochemical tests
were conducted using the specified reagents (Haibo Biotechnology Co.,
Ltd., Qingdao, China). Meanwhile, genomic DNA was extracted using the
E.Z.N.A Bacterial DNA Kit (Omega Bio-tek. Inc., Norcross, GA, USA). The
16S rRNA-coding gene was amplified through PCR by using the universal
primer pair 16S-F and 16S-R ([186]Table S1) [[187]45], sequenced by
General Biosystems Co., Ltd. (Chuzhou, China), aligned with sequences
found on the EzBioCloud server [[188]46], and deposited in the NCBI
GenBank with accession number [189]OQ306542. A phylogenetic tree was
constructed with MEGA software (version 6.0) using the neighbor-joining
method [[190]47].
5.3. AFB[1] Degradation by the Cell-Free Supernatant, Cell Suspension, and
Cell Lysate
Strain DDC-4 was fermented as mentioned above. After fermentation for
48 h, 2 mL fermentation broth was centrifuged at 4500 rpm for 10 min at
room temperature to separate the cell-free supernatant and cells. The
cells were washed with 2 mL phosphate buffer saline (PBS: 137 mM NaCl,
2.7 mM KCl, 4.5 mM Na[2]HPO[4], 1.4 mM KH[2]PO[4]) twice and
resuspended in 2 mL PBS. Then, the solution was divided into two
fractions. One fraction was processed without any treatment (namely,
cell suspension). The other fraction was disintegrated through
ultrasonication (Sonics, Newtown, Connecticut, USA, 50% of maximum
amplitude, subjected to ultrasound for 3 min with a 5 s interval
between two 3 s processing) in the ice bath and centrifuged at 10,000
rpm for 2 min at 4 °C to obtain the supernatant (namely, cell lysate).
The obtained cell-free supernatant and cell lysate were separately
filtered through a 0.45 µm filter. Then, 960 μL each of the
fermentation broth, cell-free supernatant, cell suspension, and cell
lysate was separately coincubated with 40 μL of 25 μg/mL AFB[1]. The
sterile modified LB medium or PBS containing AFB[1] served as the
control. Residual AFB[1] in each sample was determined as described
previously. The AFB[1] degradation rate was calculated using the
aforementioned formula.
5.4. Induction Effect of Degradation by AFB[1]
Strain DDC-4 was fermented as mentioned above. The fermentation broth
was divided into four fractions, namely fraction A, fraction B,
fraction C, and fraction D. To investigate the induction effect of
AFB[1] on degradation by comparing the degradation rate of the
cell-free supernatant induced by AFB[1], the cell-free supernatant
uninduced by AFB[1], and the fermentation broth; the AFB[1] addition
concentration and incubation time were the same as those used while
determining the degradation rates of the fermentation broth.
Fraction A (induced by AFB[1]): 960 μL of the fermentation broth was
treated with 40 μL of 25 μg/mL AFB[1] in the dark at 37 °C for 72 h
with shaking (150 rpm). The supernatant was collected through
centrifugation at 4500 rpm for 10 min at room temperature, filtered
through a 0.45 µm filter, and coincubated with 40 μL of 25 μg/mL AFB[1]
in the dark at 37 °C for 72 h with shaking (150 rpm).
Fraction B (without induction): 960 μL of the fermentation broth was
treated with 40 μL sterile distilled water in the dark at 37 °C for 72
h with shaking (150 rpm). The supernatant was treated in the same
manner as fraction A.
Fraction C: 960 μL of the fermentation broth was treated with 40 μL of
25 μg/mL AFB[1] in the dark at 37 °C for 72 h with shaking (150 rpm).
Fraction D: 960 μL of the sterile modified LB medium was coincubated
with 40 μL of 25 μg/mL AFB[1] in the dark at 37 °C for 72 h with
shaking (150 rpm).
Residual AFB[1] in each fraction was determined as described above. The
AFB[1] degradation rate was calculated as follows:
[MATH:
Yi
=C+D−A
/C+D×100% :MATH]
(2)
[MATH:
Yu
=D−B/D×100% :MATH]
(3)
where A is the residual AFB[1] in Fraction A, B is the residual AFB[1]
in Fraction B, C is the residual AFB[1] in Fraction C, D is the
residual AFB[1] in Fraction D, Y[i] is the AFB[1] degradation rate of
the induction group (%), and Y[u] is the AFB[1] degradation rate of the
noninduction group (%).
5.5. Effects of Heat, SDS, and Proteinase K Treatments on AFB[1] Degradation
by the Cell-Free Supernatant
The cell-free supernatant was prepared as mentioned above. To
investigate the effects of heat, SDS, and proteinase K treatments, the
cell-free supernatant was treated with boiling water for 10 and 30 min,
1% SDS in the dark for 24 h, and 1 mg/mL proteinase K in the dark for
24 h, respectively. The degradation experiment was conducted as
mentioned above. The sterile modified LB medium containing AFB[1] was
used as the control.
5.6. Effects of Incubation Conditions on AFB[1] Degradation by the Cell-Free
Supernatant
The cell-free supernatant was prepared as mentioned above. To
demonstrate the effects of temperature, the supernatant containing
AFB[1] was incubated at different temperatures (30 °C, 40 °C, 50 °C, 60
°C, 70 °C, 80 °C, and 90 °C) without shaking in the dark for 72 h. In
the pH test, the supernatant was freeze-dried, redissolved in an equal
volume of different buffers (citrate buffer (pH 4 and 5), phosphate
buffer (pH 6, 7 and 8), and sodium carbonate/sodium bicarbonate buffer
(pH 9, 10, and 11)), and coincubated with AFB[1] in the dark at 37 °C
for 72 h with shaking. Regarding metal ions, the supernatant was added
to 10 mM each of Li^+ (LiCl), Ni^2+ (NiSO[4]), Cu^2+ (CuSO[4]), Mg^2+
(MgCl[2]), Ca^2+ (CaCl[2]), Zn^2+ (ZnSO[4]), Mn^2+ (MnCl[2]), and Fe^3+
(FeCl[3]) and coincubated with AFB[1] in the dark at 37 °C for 72 h
with shaking. The influence of the copper concentration (1, 5, 10, and
15 mM) and that of incubation times (6, 12, 18, 24 h, 36, 48, and 72 h)
with 10 mM Cu^2+ on AFB[1] degradation were also determined. The
residual AFB[1] in each sample was determined as mentioned above, and
the sterile modified LB medium substituted the supernatant in the
control.
5.7. Application of the Cell-Free Supernatant to Remove AFB[1] from the Moldy
Maize Powder
After the moldy maize powder was sterilized, 5 g of the powder was
mixed with 10 mL cell-free supernatant of strain DDC-4 and 10 mM Cu^2+
and incubated for 48 h. The AFB[1] content was analyzed using the ELISA
kit.
5.8. RNA Extraction and Sequencing
First, 960 μL of the fermentation broth was treated separately with 40
μL of 25 μg/mL AFB[1] and 40 μL sterile distilled water in the dark at
37 °C for 72 h with shaking. Three independent biological replicates
were used for each treatment. The cells were obtained through
centrifugation at 5000× g for 10 min. Total RNA was extracted using the
Total RNA Extractor Kit (Sangon Biotech Co., Ltd., Shanghai, China).
RNA quality and integrity was detected through 1% agarose gel
electrophoresis, and the RNA concentration was determined using the
NanoDrop (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The
concentration and quality of RNA met the requirements for libraries
construction ([191]Table S5). rRNAs were removed using the Ribo-off
rRNA Depletion kit (Vazyme Biotech Co., Ltd., Nanjing, China). cDNA
libraries were constructed using the VAHTS™ Stranded mRNA-seq Library
Prep Kit for Illumina^® (Vazyme Biotech Co., Ltd., China). Library
quality was examined through 8% polyacrylamide gel electrophoresis. The
libraries were sequenced on the DNBseq-T7 (BGI Genomics Co., Ltd.,
Shenzhen, China) platform (Sangon Biotech Co., Ltd., China) to obtain
raw reads. The clean reads were acquired using the Trimmomatic program
(version 0.36) for data processing. After the reads were evaluated for
quality, the clean reads were mapped to the reference genome of B.
halotolerans ZB201702 from the NCBI database
([192]https://www.ncbi.nlm.nih.gov/assembly/GCF_004006435.1/?shouldredi
rect=false, accessed on 9 January 2019) using the Bowtie2 program
(version 2.3.2). The transcriptome sequencing data were stored in the
Sequence Read Archive
([193]https://dataview.ncbi.nlm.nih.gov/object/PRJNA917813?reviewer=jk3
r3v6iq68bb7bch1o9esdkn9, created on 4 January 2023).
5.9. GO Term and KEGG Pathway Enrichment Analyses
Heatmaps were constructed using the gplots package in R to present the
distance between the samples. Transcripts per million (TPM) values were
calculated using the featureCounts program (version 1.6.0) to reflect
the gene expression level. Differentially expressed genes (DEGs)
between the samples untreated and treated with AFB[1] were selected
using the DESeq2 (version 1.12.4) package in R while considering |log2
fold change| > 1 and q value < 0.05 as the filtering criteria.
Functions of DEGs were annotated by referring to bioinformatics
databases, including the Nonredundant Protein, Gene Ontology (GO), the
Kyoto Encyclopedia of Genes and Genomes (KEGG), and Cluster of
Orthologous Groups of Proteins databases. GO term and KEGG pathway
enrichment analyses were performed using topGO (version 2.24.0) and the
clusterProfiler (version 3.0.5) package in R, respectively. The
significance level was determined using the q value (<0.05). The
expression of the selected genes was presented in the heatmaps
constructed using the pheatmap package in R.
5.10. Quantitative Real-Time PCR
To validate the RNA-seq results, genes RS11000, RS07845, and RS04140
were selected as target genes and examined through quantitative
real-time PCR (qRT-PCR). Primers were designed using Premier 6
([194]Table S1). Total RNA was transcribed into cDNA using the
PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China).
qRT-PCR was performed using SYBR^® Premix Ex TaqTM (Takara, Dalian,
China) on the 7500 Real-Time PCR System (ABI, Foster City, CA, USA).
Relative expression levels of the target genes were normalized by the
expression levels of the internal control gene (16S rRNA) and
quantified using the ΔΔCt method. Three independent biological
replicates were used.
5.11. Statistical Analysis
All assays were conducted in triplicate. The study results are
expressed as mean ± SD and analyzed conducting Duncan’s multiple
comparison test (p < 0.05) with SPSS software (version 22.0.0.0).
Supplementary Materials
The following supporting information can be downloaded at:
[195]https://www.mdpi.com/article/10.3390/toxins16060256/s1, Table S1:
Statistics of the read alignments in the RNA-Seq study, Table S2: The
reads mapped to the reference genome, Table S3: DEGs enriched in
developmental process, Table S4: Primers used in this study, Table S5:
The concentration and quality of RNA, Figure S1: Heatmap of distance
between samples, Figure S2: Volcano plot of DEGs, Figure S3: GO term
enrichment classification of DEGs, Figure S4: DNA replication.
[196]toxins-16-00256-s001.zip^ (1.7MB, zip)
Author Contributions
Conceptualization, J.G.; methodology, J.G.; formal analysis, J.G. and
H.Z.; investigation, H.Z., Y.Z., X.H., and Y.L.; resources, R.W.;
writing—original draft preparation, J.G.; writing—review and editing,
S.L. and R.W.; visualization, J.G. and H.Z.; project administration,
J.G.; funding acquisition, J.G. and S.L. All authors have read and
agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in the study are included in the
article and [197]Supplementary Material, further inquiries can be
directed to the corresponding authors.
Conflicts of Interest
Author Hanlu Zhang was employed by the company Greens SCI. & TECH.
Development 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.
Key Contribution
In this study, a novel AFB[1]-degrading strain was identified as
Bacillus halotolerans DDC-4, and the active components of this strain
were thermostable extracellular proteins. Transcriptomic analysis
indicated that the alpha/beta hydrolase-encoding gene might act as a
novel candidate gene for AFB[1] degradation, and inhibition of nucleic
acid synthesis was the main toxicological effect of AFB[1].
Funding Statement
This study was supported by a grant (LJKZ0654) from The Educational
Department of Liaoning Province, a grant (22-322-3-39) from the
Shenyang Bureau of Science and Technology and the Scientific Research
Project of Shenyang Agricultural University (880418067).
Footnotes
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referred to in the content.
References