Science of the Total Environment 806 (2022) 150911

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Transcriptome expression analysis of the gene regulation mechanism of

bacterial mineralization tolerance to high concentrations of Cd2+
Shanshan Huang a, Renlu Liu a,b, Menglin Sun a, Xiaofang Li a, Yong Guan c, Bin Lian a,⁎
a
School of Life Sciences, School of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China
b
School of Life Sciences, Key Laboratory of Agricultural Environmental Pollution Prevention and Control in Red Soil Hilly Region of Jiangxi Province, Jinggangshan University, Ji'an 343009, China
c
National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Under Cd2+ stress, bacteria can enhance

their resistance by inducing mineraliza-
tion.
• Some genes related to the mineral syn-
thesis regulation under Cd stress were
found.
• The finding provides a guidance for bio-
remediation of heavy metal contami-
nated sites.

a r t i c l e i n f o a b s t r a c t

Article history: Cadmium (Cd) pollution is a pressing environmental issue that must be addressed. In recent years, microbial miner-
Received 7 July 2021 alization biotechnology has been developed into an effective and eco-friendly heavy metal bioremediation solution. In
Received in revised form 23 September 2021 the present research, RNA-Seq technology was utilized to reveal the molecular mechanism through which Bacillus
Accepted 6 October 2021
velezensis LB002 induced the mineralization and Cd2+ fixation under high-concentration Cd2+ stress. The metabolic
Available online 12 October 2021
pathways involved in the genes that were significant differentially expressed in the process of bacterial mineralization
Editor: Xinbin Feng were also investigated. The results showed that the physiological response of bacteria to Cd2+ toxicity may include
bacterial chemotaxis, siderophore complexation, and transport across cell membranes. Bacteria subjected to high-
concentration Cd2+ stress can up-regulate genes of argH, argF, hutU, hutH, lpdA, and acnA related to arginine synthesis,
Keywords: histidine metabolism, and citric acid cycle metabolism pathways, inducing vaterite formation and Cd2+ fixation. Thus,
Cadmium the toxicity of Cd2+ was decreased and bacteria were allowed to grow. Real-time quantitative polymerase chain re-
Pollution action (RT-qPCR) results confirmed the data obtained by RNA-Seq, indicating that bacteria can reduce Cd2+ toxicity
Bacteria by regulating the expression of related genes to induce mineralization. A basic bioremediation strategy to deal with
RNA-Seq
high-concentration heavy-metal pollution was proposed from the perspective of gene regulation.
Biomineralization
© 2021 Elsevier B.V. All rights reserved.
Mechanism

1. Introduction

Cadmium (Cd) as a heavy metal often found in the environment

shows high toxicity and bio-mobility (Bolan et al., 2013). The discharge
⁎ Corresponding author at: Nanjing Normal University, Nanjing 210023, China. of industrial wastewater containing Cd, and its accumulation in the en-
E-mail address: bin2368@vip.163.com (B. Lian). vironment pose a severe threat to plants, animals, and human health

https://doi.org/10.1016/j.scitotenv.2021.150911
0048-9697/© 2021 Elsevier B.V. All rights reserved.
S. Huang, R. Liu, M. Sun et al.
(Pan et al., 2018; Sandhu et al., 2019; Shen et al., 2019). A variety of
chemical and biological methods were employed to treat Cd pollution
in industrial wastewater and soil (Ahluwalia and Goyal, 2007; Leštan,
2009), such as ion exchange, precipitation, adsorption, and chelation.
Microorganic diverse metabolic pathways enable them to tolerate a cer-
tain concentration of heavy-metal pollution, which have potential for
remediation of heavy metal pollution, and therefore have been widely
valued (Dua et al., 2002; Njoku et al., 2020).
The maximum allowable contaminant cadmium concentration
denoting the threshold at which a soil pollution risk is deemed to
arise in China is 0.8 mg/kg (GB15618-2018), beyond which, a state of
pollution is declared. Cd pollution is toxic to microorganisms because
they not only cause conformational changes of nucleic acids and
peptides, but also interfere with the oxidative phosphorylation and
osmotic balance of cells (Poole and Gadd, 1989). In order to tolerate
Cd, microorganisms used several possible mechanisms including:
pump extracellular diffusion, complex formation, redox reaction, intra-
cellular and extracellular chelation (Malik, 2004; Veglio et al., 1997;
Hynninen et al., 2009), which vary according to the strains and metal
evaluated. For example, Wang et al. (2015) sequenced the genome of
Cupriavidus gilardii CR3 (a bacterium with resistance to multiple metals)
and identified diverse operons that codify efflux pumps, such as czc
(linked to Cd2+ resistance). Staphylococcus aureus and Listeria
monocytogenes have Cd2+ transporting ATPase encoded by the cadA
gene, which can promote the efflux of Cd2+ in cells (Silver and Phung,
1996; Ashraf et al., 2017). A cysteine-rich bacterial metallothionein
(MTs) can bind to Cd2+ and reduce its toxicity (Olafson et al., 1979;
Higham et al., 1983; Blindauer, 2011). However, in certain extreme en-
vironments, such as in mine tailings and mine wastewater, the concen-
tration of heavy-metal ions far exceeds that of more commonplace
polluted environments. According to statistics pertaining to 17 lead‑zinc
mines in China (Xu et al., 2015), the average content of Cd in soils of
lead‑zinc mining areas in China reaches 23 mg/kg. In such a highly pol-
luted environment, the growth of bacteria may require some special
methods to maintain their own viability and safety. It is found from
our preliminary experiments and other reports (Yang et al., 2018; Hao
et al., 2019; Xia et al., 2020; Chen et al., 2021) that under the stimulation
of high concentrations of metal ions, microorganisms can reduce the
harm caused by metal ions by synthesizing carbonates. Therefore, we
hypothesized that microorganisms stimulated by high concentrations
of heavy metal ions can regulate their metabolic pathways, produce
an environment that is conducive to the calcium carbonate synthesis
and heavy metal ion fixation to reduce their toxicity, but the molecular
mechanism underpinning this action remains unclear.
Microbial mineralization is a common natural phenomenon (Porter,
2007; Benzerara et al., 2014; Lv et al., 2017), and the carbonates formed
by microorganisms have good capacity to retain heavy metal ions (Liu
et al., 2018; Liu and Lian, 2019a; 2019b). Biological calcium carbonate
was found to be extensively distributed (Rodriguez-Navarro et al., 2007;
Gower, 2009; Zhou et al., 2010), which leads to the concept of using micro-
organisms to induce carbonate synthesis to implement the bioremediation
of heavy-metal pollution. However, little information is available regard-
ing evaluation of bacterial mineralization tolerance to high concentrations
of Cd2+. Transcriptome sequencing (RNA-Seq) can provide a collection of
all transcription products (mRNA) in a cell under certain physiological
conditions, providing accurate digital signals for studying the regulation
of cell metabolism thereunder (Brian et al., 2010; Jacob et al., 2010).
RNA-Seq technology was used to elucidate the mechanism by which bac-
teria regulate the mineralization process to improve Cd2+ tolerance.
2. Materials and methods
2.1. Bacterial strain and cultivation
The Bacillus velezensis strain LB002 (GenBank: CP037417.1) isolated
and preserved in our laboratory was used as the experimental strain.
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Two or three loops of LB002 were inoculated into 200 mL of steril-
ized LB liquid medium (Tryptone 1% (m/v), yeast extract 0.5% (m/v),
NaCl 1% (m/v), 7.0 ≤ pH ≤ 7.3), and the specimens were placed in a
shaker (180 r/min at 30 °C for 10 h) to prepare a bacterial seed solution
((7.75 ± 1.19) × 107 cfu/mL).
To compare the transcriptions of LB002 under different conditions,
the following experimental approach could be designed:
Group A: 2 mL of the aforementioned bacterial seed-liquid was inoc-
ulated in 100 mL of LB medium, which was used as a control group.
Group B: 100 mL of LB medium was supplemented with Cd2+ at a
final concentration of 20 mg/L, and 2 mL of the aforementioned bac-
terial seed-liquid was inoculated for the analysis of bacterial tran-
scriptome under 20 mg/L Cd2+ stress.
Group C: 100 mL of LB medium (containing 0.8 g CaCl2) was added
at a final concentration of 30 mg/L Cd2+, and 2 mL of the
aforementioned bacterial seed-liquid was inoculated for the analysis
of bacterial transcriptome during bacteria-induced vaterite forma-
tion under 30 mg/L Cd2+ stress.
Each group of experiments could be replicated to allow three parallel
trials and specimens were all cultured in the shaker at 30 °C at 180 r/min.
The cultures were mainly used for the following research: precipita-
tion morphology and chemical composition analysis, and transcriptome
analysis.
2.2. Morphology of the precipitate and its chemical composition
The culture solution was sampled at 24 h, 48 h, and 72 h, respec-
tively, centrifuged at 9000 r/min for 10 min, the supernatant was then
removed, and the sediments of groups A, B and C were divided into
two parts, respectively. One part was freeze-dried for observation by
scanning electronic microscope and energy spectrometer (SEM-EDS,
Zeiss, Germany). The other part was completely dried at 60 °C, then
the dried material was gently ground with an agate mortar, passed
through a 150-μm square aperture cloth mesh sieve, thrice-washed
with double-distilled water to remove surface soluble impurities, and
thoroughly dried again at 60 °C before X-ray powder diffractometer
(XRD-526, Olympus, USA, using Co as the cathode) and soft X-ray imag-
ing (BL07W, Hefei National Synchrotron Radiation Laboratory NSRL,
China) analysis. XRD was used to determine the types of minerals
contained in specimens. SEM-EDS and soft X-ray imaging were utilized
to investigate the microscopic morphology, crystal structure, and chem-
ical composition of the minerals induced by the bacteria.
2.3. Transcriptome analysis
The culture medium of each group was sampled at 36 h, centrifuged at
4 °C and 9000 r/min for 10 min, and the supernatant was then removed.
The cell pellet was then frozen in liquid nitrogen and stored at -80 °C.
Three biological replicates were established for each group. RNA was ex-
tracted from each sample using TrizolTM reagent (Invitrogen, CA, USA).
Genomic DNA was removed with DNase I (TaKara, Japan). The RNA qual-
ity was verified by using 2100 Bioanalyzer (Agilent, CA, USA) (OD260/
OD280 = 1.8 to 2.0; OD260/OD230 ≥ 2.0; 23S:16S ≥ 1.0). At least, 1 mg of
RNA from each sample was used for RNA-Seq. Ribosomal RNA removal,
cDNA library construction, sequencing (using the Illumina HiSeq X Ten
platform), and subsequent analyses were conducted by Majorbio Bio-
Pharm Technology Co., Ltd. (Shanghai, China). The primers employed in
the RT-qPCR experiments were also designed by Majorbio Bio-Pharm
Technology Co., Ltd. The raw data for analysis were deposited in SRA
(Sequence Read Archive, http://www.ncbi.nlm.nih.gov/Traces/sra).
The original images acquired by Illumina Hiseq sequencing were
converted into raw data through base calling and were stored in
FASTQ file format. To make the subsequent assembly more accurate,
clean data (reads) were obtained by trimming of the raw data and
S. Huang, R. Liu, M. Sun et al.
compared with the reference genome to obtain mapped data for subse-
quent analysis. The transcriptome data were compared with the ge-
nome of Bacillus velezensis strain LB002 (GenBank: CP037417.1) using
Bowtie. The comparison efficiency of reads between the obtained sam-
ples of groups A, B, and C and the reference genome was 87.62%–99.27%
(Table S1, Supplementary material). Gene expression data were ob-
tained and were quantified as transcripts per million reads. Genes
were considered to be significantly up-/down-regulated when the abso-
lute value of fold-change (relative to the control) was greater than 2,
with a false discovery rate (FRD) of less than 0.05. A gene enrichment
analysis of the KEGG pathway of the differentially expressed genes
may explain the differences between samples at the gene-pathway
level. An enrichment analysis was conducted using an R script and
Fisher's exact test. In order to control the false-positive rate of the
analysis, four multiple testing methods (Bonferroni, Holm, Šidák,
and false-discovery rate) were used for P-value correction. In
general, when the corrected P-value (FDR) was less than 0.05, the
enrichment of the KEGG pathway was found to be significantly
different.
Fig. 1. FESEM-EDS results for the bacterial culture precipitation in each group after 72 h (a to c correspond to groups A to C, respectively; d shows EDS data for sediment in group C).
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2.4. RT-qPCR
With reference to previous research on the mechanism of bacterial
Cd tolerance and mineralization and based on the enrichment analysis
of the differential gene KEGG pathway function, related genes involved
in bacterial flagellum assembly and movement, amino acid metabolism,
and citric acid cycle metabolic pathways that were closely related to the
Cd tolerance of LB002 were subjected to RT-qPCR assay. The descriptions
of the gene and the gene differential expression information in the tran-
scriptome are shown in Tables S2 to S4 (Supplementary material).
A 5-mL broth sample was taken and the supernatant was removed
by centrifugation at 9000 r/min for 10 min at 4 °C. 18 cDNA samples
were prepared by RNA extraction and reverse transcription according
to RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China) and
PrimeScript™RT reagent Kit (Takara Biotechnology Co., Ltd., Dalian,
China), separately. Real-time PCR was conducted using a LightCycler®
480 II Real-time PCR Instrument (Roche, Switzerland) with 10 μL of
PCR reaction mixture that included 1 μL of cDNA, 5 μL of 2 × Perfect
StartTM Green qPCR Super Mix, 0.2 μL of forward primer, 0.2 μL of
S. Huang, R. Liu, M. Sun et al.
reverse primer, and 3.6 μL of nuclease-free water. Reactions were incu-
bated in a 384-well optical plate (Roche, Switzerland) at 94 °C for 30 s,
followed by 45 cycles at 94 °C for 5 s, 60 °C for 30 s. Each sample was
run in triplicate for analysis. At the end of the PCR cycles, melting curve
analysis was performed to validate the specific generation of the expected
PCR product. The expression levels of mRNAs were normalized to (16S
rRNA) and were calculated using the 2-ΔΔCt method. Primers for each
gene are listed in Tables S2 to S4 (Supplementary material).
3. Results and discussion
3.1. Bacterial growth under different Cd concentrations and mineral phases
of precipitation
Fig. S1a-d (Supplementary material) shows the growth status of
B. velezensis LB002 under the three culture conditions (groups A to
C) for 72 h. The growth of the bacteria under the culture conditions of
group A is found to be turbid (Fig. S1a), indicating that the bacteria
grow normally. Under the culture conditions of group B (20 mg/L
Fig. 2. Soft X-ray images and XRD analysis of sediments at different sampling times in group C (a to c correspond to the soft X-ray images of sediments at 24 h, 48 h, and 72 h, respectively; d
represents the XRD pattern).
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Science of the Total Environment 806 (2022) 150911
Cd2+), the growth of the bacteria was stressed by Cd2+, and the turbid-
ity was lower than that in group A (Fig. S1b). Under the culture condi-
tions of group C (30 mg/L Cd2+) adding 0.8% CaCl2, the growth of the
bacteria occurred by precipitation (Fig. S1c), but if no CaCl2 was added
to group C, the growth of the culture of the bacteria was not visible
(Fig. S1d), indicating that the bacteria could not grow. A high concentra-
tion of Cd2+ was thus deemed toxic to bacterial growth, but the addition
of Ca2+ could induce a certain degree of detoxification in the culture.
The results of SEM-EDS indicated that the cell morphology of the bacte-
ria under the culture conditions of group B (20 mg/L Cd2+) was signifi-
cantly different from that of those in group A (Fig. 1a & b). Under the
culture conditions of group C (30 mg/L Cd2+), the bacteria induced
the formation of spherical mineral particles (Fig. 1c), combined with
EDS results, the spherical mineral particles formed by the bacteria
were found to retain Cd2+ (Fig. 1d). XRD results demonstrated that
the mineral formed by the bacteria at 72 h under the stress imposed
by high concentrations of Cd2+ with 0.8% CaCl2 addition was a
vaterite-type calcium carbonate, while the characteristic peaks of
vaterite were not detected at 24 h and 48 h (Fig. 2d). From the soft X-
S. Huang, R. Liu, M. Sun et al.
ray results, it is also found that there were spherical minerals in the pre-
cipitate at 72 h (Fig. 2c), but not at 24 h or 48 h (Fig. 2a & b). The results
are in line with the XRD results, indicating that the spherical mineral in-
duced by the bacteria is a vaterite-type calcium carbonate.
3.2. Transcriptome analysis
3.2.1. Comparative transcriptome analysis between groups A and B
Transcriptome analysis of bacterial samples in groups A and B was
performed to investigate the metabolic regulation process of
B. velezensis LB002 in the process of tolerance to higher concentrations
of Cd2+ (which can grow). Compared with group A, there were 790 sig-
nificantly different up-regulated genes and 981 down-regulated genes
with a significant difference, as shown in Fig. 3a, the three most signifi-
cantly enriched pathways were flagellum assembly, bacterial chemo-
taxis, and biosynthesis of siderophore group non-ribosomal peptides
(Fig. 3b). The bacterial flagellum plays an essential role in their own
movement and the formation of biofilms (Moens and Vanderleyden,
1996; Berg, 2003; Prüß and Birgit, 2017), which can help bacteria
achieve chemotactic movement or escape from harmful substances
Fig. 3. Comparative transcriptome analysis of groups A and B (a. differential genes volcano map; b. significantly up-regulated differential genes in KEGG enrichment).
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Science of the Total Environment 806 (2022) 150911
(Nakamura and Minamino, 2019; Nord and Pedaci, 2020; Suchanek
et al., 2020). The present results (Figs. 3 & 5) show that flagellar move-
ment under the stress of higher concentrations of Cd2+ is one of the
ways to help bacteria escape the toxicity of Cd2+. In addition, the results
in Fig. 3 illustrate that siderophores affect the migration of Cd2+ in the
culture medium through chelation, which helps to reduce the toxicity
of Cd2+ to bacterial cells. Heavy metals were found to stimulate the
siderophore biosynthesis of various bacteria (Neubauer et al., 2010). A
siderophore is an organic compound produced by microorganisms
that have super-complexing power on iron. Its function is to provide
iron nutrients to microbial cells, especially in a low-iron environment
(Neubauer et al., 2010; Hussein and Joo, 2014). Furthermore,
siderophores also affect the bioavailability of other metals including
Cd through complexation (Dimkpa et al., 2009; Schalk et al., 2011),
thereby reducing their toxicity.
3.2.2. Comparative transcriptome analysis between groups B and C
A transcriptome comparative analysis was conducted on the bacte-
rial samples in groups B and C explore the metabolic regulation process
of B. velezensis LB002 in the process of calcium carbonate formation
S. Huang, R. Liu, M. Sun et al.
when adding 0.8% CaCl2 under high concentrations of Cd2+ (to lethal
stress). As a result, 159 up-regulated genes with significant differences
were screened, and 57 genes with significant differences were down-
regulated (Fig. 4a). Significantly enriched metabolic pathways include
histidine metabolism, arginine biosynthesis, alanine, aspartic acid and
glutamic acid metabolism, degradation of valine, leucine and isoleucine,
and citric acid cycle metabolism, phosphoinositide metabolism, ribofla-
vin metabolism and propionate metabolism pathways (Fig. 4b). The re-
sults in Fig. S1c, Figs. 2 and 4b show that the bacteria can enhance their
tolerance to Cd2+ by inducing vaterite formation under high concentra-
tions of Cd2+ (to lethal stress). This stress-resistance process is related
to bacterial histidine metabolism, arginine biosynthesis, and the citric
acid cycle metabolic processes. The bacterially-induced formation of
minerals requires a suitable alkaline environment (Dhami et al.,
2013). The histidine metabolic pathway is correlated with the produc-
tion of ammonia, bacteria can convert histidine into ammonia through
the histidine enzyme. Furthermore, the synthesis of arginine is associ-
ated with the urea cycle, in which the action of arginase hydrolyzes
Fig. 4. Comparative transcriptome analysis of groups B and C (a. differential genes volcano map; b. significantly up-regulated differential genes in KEGG enrichment).
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Science of the Total Environment 806 (2022) 150911
arginine into ornithine and urea, and the action of urease can decom-
pose urea into ammonia and CO2, and then increase the pH value. The
governing chemical equation is demonstrated as follows:
NH3 þ H2 O ⇄ NH4 þ þ OH− :
These bacteria can provide a suitable alkaline environment for the
formation of calcium carbonate through a series of physiological meta-
bolic regulatory processes under the stress of high concentrations of
Cd2+. The results of transcriptome analysis also showed that the up-
regulated differential genes were significantly enriched in the citrate
cycle (TCA cycle) pathway (Fig. 4), and the TCA cycle continued to pro-
duce energy to meet the large demand for ATP in the formation of min-
erals such as small molecules and macromolecules. The TCA cycle was
the final metabolic pathway of the three major nutrients (carbohy-
drates, lipids, and amino acids), and was also the hub of the metabolism
of carbohydrates, lipids, and amino acids (Fernie et al., 2004). Acetyl
CoA thus entered a cyclic system that consisted of a series of
S. Huang, R. Liu, M. Sun et al.
biochemical reactions, and was finally oxidized to H2O and CO2 (Bott,
2007). The resulting CO2 could undergo hydration catalyzed by the
ubiquitous carbonic anhydrase to produce carbonate and bicarbonate,
and then combine with Ca2+ in the solution to generate carbonate
minerals. During the formation and growth of carbonate minerals
(and some amorphous), Cd2+ in the environment could be effectively
captured, thereby greatly enhancing the tolerance of bacteria to Cd2+.
3.2.3. Comparative transcriptome analysis between groups A and C
A transcriptome comparative analysis of bacterial samples in groups
A and C was undertaken to characterize the metabolic regulation
process of B. velezensis LB002 during precipitation formation when
adding 0.8% CaCl2 under high concentrations of Cd2+ (to lethal stress).
The results indicated that 757 up-regulated and significantly
different genes were screened, and 759 down-regulated significantly
different genes were found (Fig. 5a). The two most significant pathways
for enrichment were flagellum assembly and bacterial chemotaxis
(Fig. 5b).
Fig. 5. Comparative transcriptome analysis of groups A and C (a. differential genes volcano map; b. significantly up-regulated differential genes in KEGG enrichment).
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3.3. RT-qPCR
Cd-transporting P-type ATPase is an enzyme that pumps Cd out of
the cell and plays an essential role in Cd resistance (Silver and Phung,
1996; Ashraf et al., 2017), so this enzyme gene-cadA was selected for
RT-qPCR verification. The results showed that compared with group A,
the cadA gene in group B was up-regulated by 22.43 times (Fig. 6a);
compared with group A, the cadA gene in group C was up-regulated
67.57 times (Fig. 6c). These results are consistent with the aforemen-
tioned literature.
Combined with the analysis of KEGG pathway data, bacterial flagel-
lum assembly and bacterial chemotaxis metabolic pathways were
found to be significantly enriched during the process of the bacterial re-
sistance to Cd2+ stress, so the genes involved in this process were se-
lected for further verification using RT-qPCR. The results are illustrated
in Fig. 6. Compared to group A, flagellum assembly and bacterial
chemotaxis-related genes fliJ, flgD, flgG, and fliG in group B were up-
regulated by 31.83, 21.17, 21.22, and 11.74 times, respectively
S. Huang, R. Liu, M. Sun et al. Science of the Total Environment 806 (2022) 150911

Fig. 6. RT-qPCR analysis for those up-regulated significantly differential genes in each group. (a. up-regulated significantly differential genes of group A vs group B at 36 h; b. up-regulated
significantly differential genes of group B vs group C at 36 h; c. up-regulated significantly differential genes of group A vs group C at 36 h).

(Fig. 6a); flagellum assembly and bacterial chemotaxis-related genes
fliJ, flgD, flgG, and fliG in group C were up-regulated by 19.08, 10.58,
12.11, and 7.43 times, respectively (Fig. 6c). The results of RT-qPCR
analysis were also in agreement with the data pertaining to the KEGG
pathway.
The analysis of KEGG pathway data shows that during the process of
the bacterial resistance to Cd2+ stress to induce the formation of
minerals, the metabolic pathways that are significantly enriched include
histidine metabolism, arginine biosynthesis, alanine, aspartic acid and
glutamic acid metabolism, degradation of valine, leucine and isoleucine,
phosphoinositide metabolism, riboflavin metabolism, propionate
metabolism and citric acid cycle metabolic pathway (Fig. 4b). The
genes involved in these processes were selected for further analysis
and verification using RT-qPCR. The results showed that, compared
with group B, the amino acid metabolism and citric acid cycle
metabolism-related genes argH, argF, glnA, gltB, gltD, gabT, lpdA, acnA,
CS, hutU, and hutH in group C were up-regulated by 305.26, 270.21,
6.21, 8.76, 9.07, 6.95, 13.92, 2.63, 5.20, 6.70, and 3.82 times, respectively
(Fig. 6b). Compared with group A, the amino acid metabolism and citric
acid cycle metabolism-related genes argH, argF, glnA, gltB, gltD, gabT,

8
S. Huang, R. Liu, M. Sun et al.
Fig. 7. Overview of bacterial tolerance to high concentrations of Cd2+: (i) flagellar motility; (ii) binding of Cd2+ by siderophores; (iii) efflux of Cd2+ by CadA transporter (P-type ATPase);
(iv) bacterially-induced mineralization.
lpdA, acnA, CS, hutU, and hutH in group C were increased by 7.54, 12.45,
2.65, 23.17, 25.33, 3.45, 7.12, 2.31, 2.70, 6.92, and 4.61 times, respec-
tively (Fig. 6c). The RT-qPCR analysis again validated the results of the
KEGG analysis.
In summary, under the conditions of this study, in response to the
stress of high concentrations of Cd2+, the bacteria may withstand the
toxicity of Cd2+ the following methods of metabolic regulation: escape
through flagellar motility; siderophore banding of Cd2+; over-
expression of the gene cadA to promote Cd2+ efflux. When the Cd2+
concentration reaches an excessive, or even lethal, concentration
(such as 30 mg/L), the bacteria may change the environmental pH
value by regulating amino acid metabolism and citric acid cycle meta-
bolic pathways that induce the formation of calcium carbonate to fix
Cd2+. Fig. 7 illustrates a schematic representation of the molecular reg-
ulation mechanism of bacterial resistance to high concentrations of
Cd2+.
4. Conclusion
The results of transcriptome analysis showed that the bacteria may
tolerate Cd2+ by regulating metabolic processes such as bacterial flagel-
lar motility, siderophore complexation, and transmembrane transport.
Under high concentrations of Cd2+ (to lethal stress), the bacteria may
regulate the high expression of genes argH, argF, hutU, hutH, lpdA, and
acnA, which are involved in the metabolism of acid metabolism and
the citric acid cycle. This provides an alkaline environment and the car-
bonate ions necessary for the formation of calcium carbonate, which can
cause the calcium carbonate to fix Cd2+, thus reducing its toxicity. The
bacteria respond to the harm of excessively high concentrations of
Cd2+ and enhance the tolerance to Cd2+ by regulating the expression
of genes related to induction of mineralization. This constitutes a differ-
ent basic bioremediation strategy, which is provides a theoretical basis
for the microbial remediation of soil contaminated by high concentra-
tions of Cd2+.
Data statement
Illumina sequencing raw data was uploaded to the NCBI Data Center
database SRA (Sequence Read Archive, http://www.ncbi.nlm.nih.gov/
Traces/sra) with the dataset identifier SRP268873. All datasets obtained
for this study are included in the manuscript/Supplementary material.
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Science of the Total Environment 806 (2022) 150911
Funding
This work was supported by the National Natural Science Founda-
tion of China (grant number 41772360), and the Priority Academic
Program Development of Jiangsu Higher Education Institutions (PAPD).
CRediT authorship contribution statement
LB conceived and supervised the project. HS conducted experiments,
analyzed the data and wrote original draft. LB, LR, SM, LX and GY partic-
ipated in the discussion and revisions of the manuscript. All authors
read and approved the final manuscript.
Ethical statement
This article does not contain any studies with human participants or
animals performed by any of the authors.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.150911.
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