Journal of Environmental Biotechnology
Vol. 19, No. 1, 73–80, 2019

Original paper (regular paper)

MerB3, an Organomercurial Lyase of Bacillus as an Antidote against
Organomercurial Poisoning
Mei-Fang Chien 1,2, Ying-Ning Ho1, Hui-Tzu Lin3, Kuo-Hsing Lin3,4,
Ginro Endo2 and Chieh-Chen Huang3*
Graduate School of Environmental Studies, Tohoku University, Miyagi 980–8579, Japan
2
Faculty of Engineering, Tohoku Gakuin University, Miyagi 985–8537, Japan3
Department of Life Sciences, National Chung Hsing University, Taichung 420, Taiwan
4
Cancer Metastasis Alert and Prevention Center, College of Chemistry, Fuzhou University, Fuzhou 350002, China
1* TEL: +886–4–2284–0416 ext. 405 FAX: +886–4–2287–4740* E-mail: cchuang@dragon.nchu.edu.tw(Received: 4 March, 2019/Accepted: 4 April, 2019)
The organomercurial lyase MerB has the unique ability to cleave carbon−Hg bonds. Three merB genes, merB1, merB2
and merB3, have been identified in the Bacillus megaterium strain MB1. To find out the similar mercury resistance geneclusters, the complete sequence of transposon TnMERI1 in MB1 strain was used as a query sequence gene cluster to blast
the bacterial sequences of Genbank database divisions, including 60,922 complete sequence loci and whole genome assembly
database of 2,510 Bacillus sp. strains. The results of genome mining showed that only few Bacillus strains harboredTnMERI1-like (three-merB type) mercury resistance gene clusters. However, the characterization of MerB3 protein for
carbon-metal bond cleavage has not been understood. In this study, we purified MerB3 of Bacillus megaterium MB1 andinvestigated its activity in vitro. The protonolysis of both alkyl- and aryl-organomercurials by MerB3 were confirmed andwere enhanced by thiol groups. High affinity of MerB3 to phenylmercury acetate showed the potential for using MerB3 as
an antidote against organomercurial poisoning.
Key words: organomercurial lyase, organomercurial poisoning, MerB, genome mining
Abbreviations: IPTG, isopropyl β-D-1-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride, protease inhibitor; LB
medium, Luria–Bertani medium; CVAAS, Cold vapor atomic absorption spectrometer; FPLC, fast performance liquid chromatography; PMA, phenyl mercury acetate; MMC, methyl mercury chloride; DTT, dithiothreitol; PHMB, p-hydroxymercuribenzoate

1.

Introduction

Mercury is a highly mobile and reactive element that is toxic
to organisms, while human activities facilitate its dissemination in the biosphere. Of all the mercury compounds,
some organomercurial compounds are extremely toxic andtend to be bioaccumulated in the living organisms, suchas methylmercury chloride (MMC), which has been identified as a causative agent of the Minamata disease6). Other
organomercurials, such as phenylmercury acetate (PMA)and ethylmercury, were commonly used as mercury fungicide,antiseptics or disinfectants while PMA has been consideredmore hazardous to human than MMC10). Most of theseuses have been discontinued, but small amounts of thesecompounds can still be found as preservatives in some medicines and threaten our daily life.
On the other hand, microbes have developed several defense
systems to overcome the toxicity of mercury compounds. The
most-studied mechanism is the enzymatic transformationthat degrades organomercurials to the ionic mercury, Hg2+,
and subsequently reduces the Hg2+ to the metallic form, Hg0.
This process depends on the mercury resistance operon (meroperon) which consists of linked genes encode organomercury
lyase (MerB), regulator (MerR), mercuric ion transporter(MerT, MerP) and mercuric ion reductase (MerA)1). Since
the protonolysis of carbon–mercury (C–Hg) bonds is the firststep in decomposition of organomercurials, MerB has been
known as the key enzyme to mediate biological organomercurial detoxification2). The decomposition of organomercurials
by MerB and subsequently mercury volatilization by MerA
has been interested for applying the resistance system tothe environmental mercury bioremediation and biomonitoring13,14). The most extensively characterized MerB was
identified from Escherichia coli (plasmid R831b) anddemonstrated that two highly conserved cysteines (Cys-96
and Cys-159 in R831b MerB numbering) were essential forcatalytic activity. However, the MerB identified enzymes so
far show low similarity and diverse substrate specificities5,8,15).

Chien, et al.

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Therefore, the further studies in regarding the biochemicaland structural properties of MerBs, in addition to enzymaticengineering, are required to evolve MerB for the efficientdetoxification of organomercurials poisoning.
Previous study reported that in the mercury resistantbacterium, Bacillus megaterium MB1, three merB genes
are identified in its transposon, TnMERI1, while merB3
gene harboring recombinant confers the broadest substratespecificity (9). Matsui et al., 2016 used the PCR amplification transposon sequences of a total 65 spore-forming
mercury-resistant bacteria for investigating the horizontal
gene movement11). The results show that the TnMERI1-like
transposons can be wide horizontal dissemination acrossbacterial species and geographical barriers. In this study, we
try to use the TnMERI1 transposon sequence as a query
sequence gene cluster to blast and compare the bacterial
sequences of Genbank database divisions and selectedbacteria whole genome sequence databases for investigating
the similarity of merB genes in mercury resistance genecluster. In addition, we focus on the ability of the organomercury lyase MerB3 protein. In this study, MerB3 of B.
megaterium MB1 was purified and its enzymatic properties(enzyme activity assay, substrate specificity, and kinetic analysis) were investigated. This is the first study to investigate
the enzymatic properties of MerB3 in TnMERI1 transposon.

2.

Materials and methods

2.1 Genome mining of mercury resistance gene cluster
A total of 60,922 bacterial completed sequences/loci of
Genbank bacterial division (December 2018) were downloaded from National Center for Biotechnology Information(NCBI) (https://www.ncbi.nlm.nih.gov). The Genbank
annotated whole genome sequences excluding assembly levelof contig (the selected genus of Bacillus (2510 strains),
Paenibacillus (161 strains), Clostridium (516 strains)and Exiguobacterium (36 strains)) were also downloaded
from NCBI (at January, 2019) for constructing the selected
whole genome sequence database. Homologous clusters forsequence similarity at the level of the entire gene cluster
were analyzed using the MultiGeneBLAST program12). The
complete sequence of transposon TnMERI1 from Bacillus
megaterium strain MB1 (GenBank: LC152290.1) was usedas the query sequence against the constructed database witha 30% sequence identify cut-off and 250 blastp hits mapped
per query sequence. The gene clusters, which hit morethan 7 genes, were selected for comparing with transposon
TnMERI1.
2.2 Bacterial strains, recombinant plasmids and culture
conditions
Escherichia coli DH5α (Invitrogen, Carlsbad, CA., USA)was used for cloning. E. coli Rosetta (DE3) pLysS (Novagen,Madison, WI., USA) was used for protein expression. Plasmid
pET-MerB3 was constructed by PCR of merB3 from genome
DNA of B. megaterium MB1 by using primers merBsFrbs(5’-GGAGGACATATGAATCAAAATCATTTGAAT-3’)
and merB3R (5’-CACGAGTTACTTACCCTTAGAGCC-3’).
The PCR product was digested with restriction enzymes NdeI
and XhoI, and the merB3-contained NdeI/XhoI fragment
was ligated into the expression vector pET-21b (Novagen).
The MerB3 reading frame was expressed under control of
the T7 promoter in the vector pET21b, and the sequence ofmerB3 was confirmed by DNA sequencing. Bacteria weregrown in LB medium at 37°C with agitation at 120 rpm.
Ampicillin (100 μg/ml) and chloramphenicol (35 μg/ml)
were added as needed.
2.3 Protein expression and purification
An overnight culture of E. coli Rosetta/pET-MerB3 wasdiluted 1 : 100 with LB medium up to 2 L and subjected tofurther incubation at 37°C until the absorbance at 600 nm
reached 0.6. IPTG was added to the culture at a final concentration of 0.5 mM. After further incubation at 37°C for 4 h,
the bacterial cells (1 L) were harvested by centrifugation(3,000 rpm, 15 min). All purification steps were performed
at 4°C, and DTT-containing solutions were freshly prepared
before use. The buffers used were: (A) 20 mM Tris-HCl(pH 8.0), 1 mM EDTA, 1 mM DTT, 10% glycerol; (B)20 mM Sodium Phosphate (pH 7.5), 1 mM EDTA, 1 mM
DTT, 10% glycerol; (C) 50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 1 mM DTT, 200 mM NaCl. Harvested cells weresuspended in buffer A with 20 μl/10 ml PMSF. The cells werelysed using three passes through a French press at 16,000 psi.
Lysates were 3 times cleared by centrifugation at 12,000 g for
1 h. The supernatant was passed through a 0.22 μm filter andapplied to three columns (DEAE column, sepharose column
and gel filtration) for enzyme purification. The supernatant
was applied to a DEAE column equilibrated with buffer A.
After washing with 80 ml of buffer A, an elution program
was performed with a linear gradient of 0–15 mM NaCl in
the same buffer at a flow rate of 0.5 ml/min (10 ml fractions). The elution of protein was monitored by SDS-PAGE.
Fractions containing organomercurial lyase were pooled. The
enzyme from DEAE column was diluted to 0.1 fold by buffer
B, and was applied to a Q Sepharose column equilibrated
with buffer B. After washing with a bed volume of buffer B,
an elution program was performed with a linear gradient of
0–0.5 M NaCl in the same buffer at a flow rate of 0.5 ml/
min (10 ml fractions). The elution of protein was monitored
by SDS-PAGE, and the fractions containing organomercurial
lyase were pooled. The enzyme from Sepharose column was
dialyzed in buffer C and concentrated to 2 ml. The enzyme
was then exchanged by gel filtration into buffer C usinga Superdex 75 column (GE healthcare, Piscataway, NJ.,
USA). The quality of purified enzyme was confirmed by 14%
SDS-PAGE and N-terminal sequencing.
2.4 Enzyme assay and organomercurials
Organomercurial lyase activity was determined by measuring the mercury ion production from organomercurials
by cold vapor atomic absorption spectrometer (CVAAS).

MerB3, an Organomercurial Lyase of Bacillus as an Antidote against Organomercurial Poisoning

The assay mixture consisted of organomercurials, MerB3,
and DTT was mixed and incubated at room temperature asneeded. One hundred micro liter of the mixture was removed
at various time intervals and analyzed for the mercury ion
by CVAAS. Each experiment was done in at least triplicate. CVAAS was calibrated with mercury chloride solution.
Three organomercurial compounds were used in this study;
methylmercury chloride (MMC) and phenyl mercury acetate(PMA) were purchased from Wako Pure Chemical (Osaka,
Japan). p-hydroxymercuribenzoate (PHMB) was purchasedfrom Sigma Chemical Co. (St. Louis, Mo, USA).

3.

Results and discussion

3.1 Genome mining of TnMERI1-like transposons inbacterial database
Nowadays, nucleotide sequence information was explosive
growth by the development of next generation sequencing
technology. Genbank is a public database of nucleotide
sequences and biological annotations. There are 60,922
complete sequences/loci in the bacterial division in Genbank(BCT) (the latest file: BCT566, December 2018). After
blasting entire mercury resistance gene cluster of TnMERI1
transposon against BCT database, we found only Bacillus
cereus RC607 and Bacillus sp. TW6 harboring three MerB
encoding genes (merB1, merB2, and merB3) without group
II intron in the mercury resistance gene cluster (Fig. 1A). The
similar transposon Tn6294 in Paenibacillus sp. EOA1 is lackof merB2 and merB3 genes and the mercury resistance genecluster in Clostridium butyricum is lack of MerB3, transposase, and resolvase genes (Fig. 1A). However, this process
cannot hit the mercury resistance gene cluster (Tn5085)
from Exiguobacterium sp. TC38-2b, which is harboring
MerB genes (merB1, merB2, and merB3)3). The probablereason is incomplete sequence information in Genbank
database. The public sequences of Exiguobacterium sp.
TC38-2b are partial transposon Tn5085 encoding tnpR and
tnpA genes (Y17750), partial transposase genes (Y17751,
Y17752), mer operon (X99457) and partial merB (Y08064).
So, we collected whole genome sequences of the genus of
Bacillus, Paenibacillus, Clostridium and Exiguobacteriumfor constructing the selected database. After blasting, wefound only four matched strains (Bacillus cereus #17, B.
cereus strains B4120, B4155 and B4079) in the selected
database. Bacillus cereus #17 (JYFW01000036) was isolated
from mouse gut4). This strain #17 has 100% identity of merBgenes (merB1, merB2 and merB3), shorter merP (98%
identity, 75% coverage) and merA genes (99% identity, 89%
coverage) by comparing with TnMERI1 (Fig. 1B). Bacillus
cereus B4120, B4155 and B4079) were isolated from water,
beef salad, and canned chocolate beverage, individually7).
These strains have similar gene cluster harboring merB genes(absent of merB1 in the strain B4079) and lack a mercurictransport protein, merT encoding gene (Fig. 1B). We could
use this method to find out potential mercury resistancestrains which harboring similar gene clusters. In addition,

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this method could be used in discovering novel bacterial
mercury resistant systems which contain mercury resistancegene clusters different with known ones.
Through the genome mining, we could find more similar
gene clusters from different sources and different regions.
The results of genome mining show that the gene clusters of
three-merB type are very rare in bacteria (only 6 gene cluster
sequence loci in ~63,000 complete sequence loci and wholegenome databases of Bacillus, Paenibacillus, Clostridium
and Exiguobacterium) (Fig. 1). Since the structural andbiochemical characterization of MerB and MerB2 were investigated9,16,17) and E. coli with the plasmid that harbored
merB3 of B. megaterium MB1 can confer a broad specificity
to decompose various organomercurials5), we focused on
MerB3 of B. megaterium MB1 and purified MerB3 protein
by heterogeneous expression.
3.2 Purification of MerB3
The previous study by in vivo experiments has reported
the broad substrate specificity of MerB3. The over expression of MerB3 by IPTG induction in both pellet and soluble
fraction (supernatant) of bacterial lysate was confirmed by
14% SDS-PAGE (Fig. 2A, lane P, S, T with IPTG induction). The soluble fraction was then used for purification.
Our previous study corrected the original Genbank registeredsequence of merB3 gene and indicated that merB3 is a
729 bp-frame gene and encodes a 242-amino acid polypeptide5). This open reading frame was applied to the ExPASy
proteomics server (Expert Protein Analysis System: http://
au.expasy.org/). According to the prediction on ExPASy, themolecular weight of MerB3 is 27.4 kDa, and the pI of MerB3
is 4.37. Therefore, the weak anion exchanger DEAE column
suitable for purifying proteins from pH 2 to 9 was applied
as the first column. Each 10 ml of elution was collected as afraction and was analyzed by 14% SDS-PAGE. As shown in
Fig. 2B, the fraction 12 of DEAE column purified products
showed a major signal near 30 kDa with limited contamination proteins. Thus this fraction was chosen and applied to astrong anion exchanger, Q Sepharose column. Every 10 mlof elution was collected and analyzed by 14% SDS-PAGE.
Fig. 2C shows that the fractions 14 and 15 contain large
amount of protein in target size with few impurity. These two
fractions were then applied to gel filtration and the purified
protein was confirmed by 14% SDS-PAGE (Fig. 2D) and by
N-terminal sequencing as MerB3.
3.3 Enzyme assay
CVAAS was employed to measure the concentration of
mercury ion in the solution by reducing the mercury ion andmeasuring the transmittance of mercury vapor at 253.7 nm.
Since organomercurial lyase broke the carbon-mercury bondof organomercurials and yields mercury ion, CVAAS wasapplied to determine the activity of MerB3. Fig. 3A shows
the calibration curve of CVAAS by using mercury chloride
solution. MerB3, PMA and DTT were mixed in the molecular ratio of 0.01 : 1 : 2. The mixture was incubated at room

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Fig. 1. The selected blasting results of mer gene modules among bacteria by using MultiGeneBlast. (A) Results of BCT database (Genbank:bacterial division, December 2018). (B) Results of selected database (whole genome sequences of Bacillus, Paenibacillus, Clostridium, andExiguobacterium genus). The threshold of gene identity is 30% and the gene clusters, which hit more than 7 genes, were selected. Grayshaded boxes below genes show the regions of DNA sequence homology (the percent of identify by comparing with transposon TnMERI1).
Isolation sources, regions and GenBank accession numbers are listed following bacterial strains. tnpA: transposase; tnpR: resolvase; B3:
organomercurial lyase merB3; R: mercury-responsive transcriptional regulatory protein merR; E: mercury resistance protein merE; T:
mercury transport protein merT; P: metal binding protein merP; A: Mercuric reductase protein merA; R2: mercury-responsive transcriptional regulatory protein merR2; B2: organomercurial lyase merB2; B1: organomercurial lyase merB1.

temperature, and the concentration of mercury ion in the
mixture was measured by CVAAS. As shown in Fig. 3, there
was no mercury ion production detected from the solution
contains only PMA and without MerB3, but the mercury
ion production was detected from the mixture. The activityof MerB3 to all organomercurials used in this study was also
confirmed (data not shown). The result showed that the purified MerB3 has activity, and this facile spectrophotometricassay is workable on evaluating activity of MerB3.

MerB3, an Organomercurial Lyase of Bacillus as an Antidote against Organomercurial Poisoning

77

Fig. 2. Expression and purification of MerB3. (A) SDS-PAGE image of the recombinant MerB3 protein. M, protein molecular weight marker;
P, pellet of cell lysate; S, supernatant of cell lysate; T, total cell lysate. (B) SDS-PAGE image of fractions from DEAE column. M: protein
molecular weight marker; 12–15, the numbers of fractions. (C) SDS-PAGE image of fractions from Q-Sepharose column. M: protein molecular weight marker; 13–16, the numbers of fractions. (D) SDS-PAGE image of fraction 23 to 31 from Gel-filtration. M: protein molecular weight marker; 23–31, the numbers of fractions. The size of each band in the marker from up to bottom is 97, 66, 45, 30, 20.1,
14.4 kDa, respectively.

Fig. 3. Enzyme assay by CVAAS. (A) The calibration curve by using mercury chloride (black diamond) in the concentration of 0–5 ppb. Xaxis: the concentration of mercury chloride; Y-axis, the transmittance of the solutions at 253.7 nm. The equation of calibration and thedetermination coefficient (R2) were shown. (B) The enzyme activity of MerB3 to PMA within 24 min. X-axis, reaction time; Y-axis, thetransmittance of the reaction solutions contains PMA only (empty triangle) and PMA with MerBs (black triangle) at 253.7 nm.

3.4 Substrate specificity of MerB3
The substrate specificity and enzyme activity of MerB3
was then investigated. In these experiments, PMA and
MMC represented as the substrate of aryl- and alkylorganomercurials, respectively. The purified MerB2 ofTnMERI1 was also applied. The organomercurials, MerB
enzymes and DTT were mixed in the molecular ratio of

1 : 1 : 2, and after incubating at room temperature for 6hours, the mercury ion production was determined. As
shown in Fig. 4, MerB3 catalyzed the protonolysis of the
carbon-mercury bonds of both PMA and MMC, while
MerB2 only performed slightly activity to PMA and almost
no activity to MMC. These results show that MerB3 could
perform activity to both aryl- and alkyl-organomercurials.
It also showed that MerB3 performs broader substrate

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Chien, et al.

specificity than MerB2 does, and it was consistent with ourprevious study through in vivo experiments5,8). Though low
homology among those MerB enzymes have been showed,
the phylogenetical analysis and the secondary structure
predictions divided the MerB enzymes into three distinct
subfamilies15). Following this grouping, MerB3 and MerB2
of MB1 belong to the same secondary structural group.
However, since the sequence of merB3 has been corrected
and the quite different substrate specificity and enzyme
activity of MerB2 and MerB3 are confirmed in vivo and in

Fig. 4. The substrate specificity between MerB3 and MerB2 in B.
megaterium MB1. Y-axis: the concentration of mercury ion
production from the reaction solution.

vitro5) (Fig. 4), the phylogenetical analysis of MerBs need tobe re-investigated.
3.5 Effect of thiol group on MerB3 catalyzed protonolysis
The alignment of MerB proteins identified so far has
shown that the well-conserved sequences of MerB locatedin the central region with 3 cysteines, which the part is themost likely protein ligand for Hg15). Previous study on themost extensively characterized MerB from plasmid R831b
presented that the high thiol requirement in MerB catalyzed
protonolysis is due to the stuck state intermediate, that canbe removed by extracellular thiol groups15). MerB3 identified
from B. megaterium MB1 contains 242 amino acids is longer
than that of MerB from R831b. And MerB3 contains other 6
cysteines in center and C-terminal other than the conserved 3
cysteines (Fig. 5A). In order to determine the effect of thiolgroup on MerB3 catalyzed protonolysis, organomercurials
degradation experiments were performed with or without
DTT addition. PMA or MMC was mixed to MerBs in themolecular ratio of 1 : 1, and the mercury ion production wasdetermined after 6 hours incubation at room temperature.
As shown in Fig. 5B, with DTT addition, both the mercury
ion productions from MerB3 catalyzed PMA protonolysis
and MMC protonolysis increased. It suggested the existenceof the thiol group helps to remove the stuck state of MerB3.
Besides, the effect of DTT addition to MMC protonolysis

Fig. 5. (A) Alignment of amino acids between MerB3 from MB1 and MerB from R831b. The red triangles figure out the conserved 3 cysteines. (B) The effect of DTT addition on MerB3 catalyzed organomercurial degradation. Y-axis: the concentration of mercury ion production from the reaction solution.

MerB3, an Organomercurial Lyase of Bacillus as an Antidote against Organomercurial Poisoning

79

Fig. 6. Kinetic analysis of MerB3. (A) The degradation curve of MerB3 catalyzed PMA protonolysis. X-axis, reaction time (min); Y-axis,
transmittance of the mercury vapor (%). The concentration of PMA is as follows. Square, 0.58 μM; circle, 0.87 μM; triangle, 1.45 μM;
inverted triangle, 2.03 μM. The maximal slope was reached within 5 min in all for concentration. (B) The hyperbolic distribution of
MerB3 catalyzed PMA protonolysis. X-axis, the concentration of PMA reacted (mM); Y-axis: the specific reaction velocity (μM/min).

was more than 30% larger than the effect to PMA protonolysis (Fig. 5B). Since the molecular of MMC is smaller
than PMA, it is considered that MMC matches the active
site of MerB3 looser than PMA does. Therefore, it is easierfor MMC to accept the extracellular thiol groups and escapefrom the stuck state.

Acknowledgement
This work was supported by JSPS KAKENHI Grant-in-Aid
for Exploratory Research Number 18K19042 and Grant-inAid for JSPS Research Fellow Number 18F18393. We
thank Yu-Shin Huang and Shiao-Wei Zo (National Chung
Hsing University, Taiwan) for assistance during this study.

3.6 Kinetic analysis of MerB3
In order to further characterize the MerB3 catalyzed reaction, the mercury fungicide PMA was used as a substrate toperform the kinetic analysis. One micro gram of MerB3 wasreacted to PMA in the concentration gradient varied from0.58 to 29 μM. The results of PMA degrading kinetic ana­lyses at four different initial concentrations (0.58, 0.87, 1.45,
2.03 μM) were shown in Fig. 6A. The hyperbolic distribution of the organomercurials concentration and the reaction
velocity for PMA was shown in Fig. 6B. The Km of MerB3
to PMA was determined as 4.5×10–3 mM by applying thesedata to the Michaelis-Menten equation. The Km of another
MerB found from E. coli plasmid R831b was reported as
0.5 mM9) which was much bigger than the Km of MerB3
from B. megaterium MB1. The small Km of MerB3 fromstrain MB1 shows its strong substrate affinity.
In this study, MerB3 of B. megaterium MB1 was purified and its enzymatic properties were investigated. This
study not only provides the supportive evidence regarding
the substrate specificity of MerB3 but also shows that MerB3
catalyzes the protonolysis of the carbon-mercury bonds ofboth alkyl- and aryl-mercurials and that the existence of
thiol groups enhances the MerB3 catalyzed protonolysis.
Besides, the kinetic parameters of MerB3 determined byquantitative spectrophotometric assays show strong substrateaffinity of MerB3. Further biochemical characterization andstructural studies of MerB3 currently in progress will provide
a fundamental understanding of this biotransformation oforganomercurials and provide a basis for developing strategies to apply MerB3 as an antidote in detoxification oforganomercurials.

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