CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity
Exhibits Manganese-Oxidase Activity. PLoS
ONE 8(4): e60573. doi:10.1371/journal.pone.0060573
CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity
Jianmei Su 0
Peng Bao 0
Tenglong Bai 0
Lin Deng 0
Hui Wu 0
Fan Liu 0
Jin He 0
Ligia M Saraiva, Instituto de Tecnologia Quimica e Biologica, Portugal
0 1 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University , Wuhan, Hubei , People's Republic of China, 2 Key Laboratory of Arable Land Conservation, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University , Wuhan, Hubei , People's Republic of China
Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53uC in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.8561.17 mM, 3.016102660.21 M?min21 and 0.3260.02 s21, respectively. Moreover, the Mn(II)oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mncontaining K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.
Funding: This work was supported by the Chinese National Natural Science Funds (grant 40830527), the National Basic Research Program of China (973 Program,
grant 2010CB126105), the National High Technology Research and Development Program of China (863 project, grant 2011AA10A205), and the Fundamental
Research Funds for Central Universities of China (grant 2011PY092). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Manganese is the second most abundant transition in the
Earths crust and the fifth most abundant element on the Earths
surface. Generally, manganese has three environmentally relevant
oxidation states, Mn(II), Mn(III) and Mn(IV) [1,2]. Among these,
Mn(II) is thermodynamically favored at low pH and Eh, whereas
Mn(III) and Mn(IV) oxides are stable at high pH and Eh . The
soluble form of Mn(II), serves as a crucial micronutrient for
organisms, while the insoluble form of Mn(III/IV) oxide, is a highly
reactive mineral phase that participates in a wide range of redox
and adsorptive reactions, playing a significant role in the
bioavailability and geochemical cycling of many essential or toxic
elements . In many environments, the chemical oxidation of
Mn(II) by O2 in the pH range of 6.08.5 is at a considerably low
level while in the presence of Mn(II)-oxidizing microorganisms,
including a variety of bacteria and fungi, the oxidation rate can be
accelerated by as much as five orders of magnitude [4,5].
Therefore, microbial processes are considered to be primarily
responsible for the formation of Mn oxides [1,6]. Although many
microorganisms capable of oxidizing Mn(II) have been isolated
and belong to diverse phyla, the biochemical mechanism of Mn(II)
oxidation is still enigmatic .
As new insights are gained regarding some proteins involved in
Mn(II) oxidation, several enzymes have been gradually identified
from some species of bacteria and most of them belong to a family
of multicopper oxidases (MCOs). MCOs are a class of copper
proteins that utilize copper as a cofactor to catalyze four
oneelectron oxidations of various substrates concomitantly with the
reduction of O2 to water [7,8]. MCOs have been found in a wide
range of organisms including bacteria, fungi (laccase), plants,
insects and vertebrates (ceruloplasmin) [7,9]. So far, three model
bacteria, Bacillus sp. strain SG-1 [6,10,11], Leptothrix discophora SS-1
 and Pedomicrobium sp. ACM3067 [13,14], have been
demonstrated to require the MCOs in Mn(II) oxidation by
disruption of the corresponding genes (mnxG, mofA and moxA,
respectively). The three MCOs identified as the putative Mn(II)
oxidases show little similarity to one another outside their
copperbinding motifs. In addition, the newest studies on the
Mn(II)oxidizing alpha-proteobacterium Aurantimonas manganoxydans SI85-9A1
and Erythrobacter sp. strain SD21 [4,15] have uncovered a second
class of enzymes involved in Mn(II) oxidation: heme-binding
peroxidase named MopA. Another putative MCO (CumA)
proposed in Pseudomonas putida GB-1 [16,17], however, has been
proven not to be a Mn(II) oxidase by in-frame deletion of cumA,
and instead reveals a complex two-component regulatory pathway
essential for Mn(II) oxidation in P. putida GB-1 .
To date, no bacterial Mn(II) oxidase has been purified to a large
quantity sufficient for detailed biochemical study. In addition, no
MCO gene thought to encode a Mn(II) oxidase has been
successfully expressed in a heterologous host to yield an active
enzyme [1,5,19], let alone the enzymological properties of the
Mn(II) oxidase. To conquer these unsettled problems, our
particular emphasis is placed on overexpression, purification and
biochemical characterization of plentiful recombinant MCOs in
Escherichia coli. Since most Mn(II)-oxidizing bacteria we have
previously isolated belong to Bacillus species, which naturally
strengthen our focus on the CotA (endospore coat protein A),
a previously reported MCO from B. subtilis , B. pumilus ATCC
7061  and B. licheniformis ATCC 14580 . CotA was
a classical bacterial laccase, which was able to oxidize
2,29-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS),
syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP) . To test
whether the CotA is responsible for Mn(II) oxidation from a highly
active Mn(II)-oxidizing strain B. pumilus WH4 isolated by our
collaborators [23,24], the cotA gene was cloned and the N-terminal
His-tagged CotA was overproduced and purified. Its Mn(II)
oxidase activity and enzymological properties provided the most
direct evidence between the MCO and the Mn(II) oxidation.
Furthermore, the Mn(II) oxidizing activities by the recombinant E.
coli strain cultured both in Mn-containing K liquid medium system
and on agar plates were also investigated comprehensively.
Materials and Methods
Taq DNA polymerase, restriction endonucleases and other
modifying enzymes were obtained from Takara Biotechnology
Co., Ltd. (Dalian, China) and Fermentas (St. Leon-Rot,
Germany). Ampicillin, kanamycin, isopropyl-b-D-thiogalactoside
(IPTG) and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
(HEPES) were from Amresco (Solon, Ohio, USA). Leucoberbelin
blue, ABTS, SGZ and 2,6-DMP were purchased from
SigmaAldrich (St. Louis, MO, USA). The Coomassie (Bradford) protein
assay kit was obtained from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). All other chemicals were of analytical
Bacterial Strains and Culture Conditions
B. pumilus WH4 was isolated from Fe-Mn nodules which were
collected at 40 cm depth from the subsoil horizon of a subacid
orthic agrudalf developed from quaternary siliceous and alluvial
sediments of the Hubei Province, Central China [23,24]. The
strain was deposited in China General Microbiological Culture
Collection (CGMCC No. 2089). It was grown at 28uC in modified
K medium [25,26] made with artificial seawater , and
supplemented with a mixture of sterile filtered 5 mM MnCl2
and 10 mM HEPES (pH 7.5). Control flasks without MnCl2 were
cultured simultaneously. E. coli strain M15 (Qiagen, Hilden,
Germany) was grown on Luria-Bertani (LB) medium at 37uC
containing antibiotics as needed. Antibiotics were added to the
following concentrations (mg ml21): ampicillin, 100; kanamycin,
Construction of a CotA-overexpressing Strain
The cotA gene (accession no. JQ797387) from B. pumilus WH4
was amplified by PCR with the primers
59-TTAGGATCCATGAACCTAGAAAAATTTGTTGACG-39 (forward) and
(reverse), the recognition sites for BamHI and HindIII endonucleases
are indicated in italics. The purified PCR product was digested
with BamHI and HindIII, and subsequently inserted into the
expression vector pQE-30 (Qiagen, Hilden, Germany) that had
been previously digested with the same enzymes. Consecutively,
the recombinant plasmid pQE-cotA was transformed into E. coli
strain M15 (Qiagen, Hilden, Germany) to obtain the recombinant
Purification of Recombinant Protein CotA
The overexpressing strain M15-pQE-cotA was cultivated
overnight and then inoculated into LB medium containing ampicillin
and kanamycin at 37uC and 180 rpm. When the optical density at
600 nm (OD600) reached approximately 0.6, the cells were
induced with 0.5 mM IPTG and supplemented with 0.25 mM
CuCl2. The temperature was changed to 25uC and agitation was
maintained for 20 h . Afterwards, cells were harvested by
centrifugation (8,000 6 g, 15 min, 4uC) and pellets were
suspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl,
and 20 mM imidazole (pH 8.0)) containing 1 mg ml21 lysozyme.
Cells were disrupted through a French pressure cell (Aminco,
Silver Spring, Maryland, USA) at 15,000 psi, followed by
centrifugation (12,0006g, 30 min, 4uC) to remove cell debris.
The resulting soluble extract was filtered and loaded onto
nickelchelating nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen,
Chatsworth, CA). The N-terminal His-tagged recombinant
protein was purified following the general protocol of Ni-NTA
column. The eluted fractions containing CotA were pooled,
concentrated by ultrafiltration (molecular weight cutoff of 10 kDa)
and analyzed by SDS-PAGE.
Copper Content and Spectroscopic Properties
The protein concentration was determined based on the
instructions of a Coomassie (Bradford) protein assay kit (Jiancheng
Bioengineering Institute, Nanjing, China) using 0.563 g/l bovine
serum albumin as the standard. The total copper content of
purified CotA was determined by an Optima 8000 ICP-OES
(inductively coupled plasma optical emission spectrometry,
PerkinElmer, Norwalk, CT, USA) [28,29]. The UV-visible
absorption spectrum of purified CotA (,33 mM) was recorded
in the range of 300800 nm in 20 mM Tris-HCl buffer (pH 8.0)
at room temperature on a DU 800 UV-Vis spectrophotometer
(Beckman Coulter, Inc.,Fullerton, CA, USA) [22,30].
Determination of Laccase Activities
To assess the laccase activity of CotA, three canonical laccase
substrates ABTS, SGZ (dissolved in anhydrous ethanol) and
2,6DMP (dissolved in 10% ethanol) were measured in 100 mM
citrate-phosphate buffer (pH 4.0), 100 mM phosphate buffer
(pH 6.0) and 100 mM citrate-phosphate buffer (pH 5.0),
respectively . Oxidation of 0.5 mM ABTS was monitored at
420 nm (e = 36,000 M21cm21), of 0.6 mM SGZ at 525 nm
(e = 65,000 M21cm21) and of 2 mM 2,6-DMP at 468 nm
(e = 49,600 M21cm21) . Since CotA was evolved to accept
ABTS over SGZ , the following experiments were carried out
with ABTS to characterize the enzyme activities in detail. Unless
otherwise indicated, the standard reaction mixture (1 ml)
contained 100 mM citrate-phosphate buffer (pH 4.0), 0.5 mM ABTS
and 93 mg purified recombinant CotA. The reaction mixtures
were incubated at 37uC and 180 rpm for 5 min in a shaking
incubator. Thereafter, the reaction mixture was measured at
420 nm . Enzyme activity measurements were performed
either on a DU 800 UV/Vis spectrophotometer (Beckman
Coulter, Inc., USA) or on a Multiskan spectrum microplate
reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) with
a 96-well plate. All assays were performed in triplicate. One unit of
laccase activity was defined as the amount of enzyme that
produced 1 mmol of product per minute under the standard assay
The effect of pH on enzyme activity was determined at 37uC in
100 mM citrate-phosphate buffer (pH 3.08.0) for the ABTS. The
temperature optimum for the activity was performed at
temperatures ranging from 30 to 100uC by measuring ABTS oxidation.
The copper requirement was tested by adding CuCl2 (03 mM) to
the standard reaction mixtures .
Kinetic parameters for the purified recombinant CotA were
determined at room temperature by using different concentrations
of ABTS (20460 mM) . The initial rates, recorded within 3 to
10 min, were acquired from the linear portion of the reaction
curve. The kinetic parameters were calculated by non-linear
regression fitting of the data to the MichaelisMenten equation
using the software Graphpad Prism 5 (GraphPad Software, San
Mn(II) Oxidase Assays in Liquid Culture System
Mn(II) oxidation assays in liquid culture system were routinely
performed as follows: 1 ml of initial reaction mixture (10 mM
HEPES (pH 8.0) containing 5 mM MnCl2 and 0.093 mg ml21
purified recombinant CotA) was incubated at 37uC for about 24 h.
Mn(II) oxidation was monitored by the formation of brown or
black Mn oxides, and then checked by Leucoberbelin blue reagent
which was an acidified redox dye and could be specifically
oxidized by Mn with valences of +3 or higher, resulting in a blue
color product with an absorption maximum at 620 nm . For
the quantification of the biogenic Mn oxides, 50 ml of each sample
was reacted with 250 ml of Leucoberbelin blue solution, and then
the Mn (IV) oxide concentration was calculated by multiplying
with a factor of 2.5 according to the KMnO4 concentration in
standard curve. The measurement was performed either on a DU
800 UV-Vis spectrophotometer (Beckman Coulter, Inc., USA) or
on a Multiskan Spectrum spectrophotometer (Thermo Scientific,
Vantaa, Finland) with a 96-well plate. All assays were performed
The optimum pH value for Mn(II) oxidizing activity of CotA
was determined by varying the pH of reaction buffers. In this
assay, two different reaction buffers 10 mM HEPES and 100 mM
CHES, were used for pH ranges of 6.88.2 and 8.69.5,
respectively. Other reaction conditions were the same as described
above. To study the effect of temperature, the assay was carried
out at temperatures ranging from 28 to 85uC. The optimization of
Cu2+ concentration was evaluated by adding CuCl2 (0.05
1.4 mM) to the initial reaction mixtures. All assays were
performed in triplicate.
To investigate whether the Mn(II)-oxidizing activity of CotA
was solely depended on copper, each of 1 mM divalent metal
cations (Sr2+, Zn2+, Ca2+, Ba2+, Mg2+, Ni2+ and Hg2+) was
separately tested by the replacement of 1 mM Cu2+. After
individually pre-treating them with purified CotA for 1 h at
37uC, the metal-enzyme complexes were added into 10 mM
HEPES (pH 8.0) containing 5 mM MnCl2 at 37uC for 24 h and
the residual enzyme activities were measured, respectively. The
enzymatic activity assayed with CuCl2 was taken to be 100%. All
assays were performed in triplicate.
The effect of various organic compounds (1 mM EDTA, 1 mM
DTT, 1 mM guanidine-HCl, 1% SDS or 0.1 mM
o-phenanthroline) on the enzyme activity was also studied. It was performed by
pre-incubating the enzyme with each reagent at 37uC for 1 h
without substrate, then added to 10 mM HEPES (pH 8.0)
containing 5 mM MnCl2 and 0.8 mM CuCl2 to measure the
remaining Mn(II)-oxidizing activity. The activity assayed in the
absence of the reagent was taken to be 100%. All assays were
performed in triplicate.
Kinetic Parameters of Mn(II) Oxidizing Activity
The kinetic parameters were determined by measuring the
initial reaction velocity at various concentrations of MnCl2 ranging
from 2.5 to 50 mM in 10 mM HEPES (pH 8.0) containing
0.8 mM CuCl2 at 37uC. For each Mn(II) concentration, we
measured the production of Mn oxides at regular intervals and
then drew the curve. In order to get the accurate initial rate (V0) of
enzymatic reaction, the reaction time was chosen before 5% of the
Mn(II) was oxidized. The initial rates (V0) of different Mn(II)
concentrations was obtained from the slope of the Mn oxide
process curve. The Km and Vmax were calculated with GraphPad
Prism 5 (GraphPad Software, San Diego, CA), using standard
settings for non-linear regression curve fitting in Michaelis-Menten
modus. The kcat parameter was determined using the equation
kcat = Vmax/[E] ([E] = 1.5561027 M). One unit of
Mn(II)-oxidizing activity was defined as the amount of enzyme that produced
1 mmol of product per minute under the standard assay
conditions. All assays were performed in triplicate.
Mn(II) Oxidation in-gel Activity Assay
The purified recombinant protein was subjected to native
polyacrylamide gel electrophoresis (PAGE) using 12%
polyacrylamide gels and Tris glycine buffer [9,37]. Native PAGE was
conducted with two sets of samples running side by side without
bmercaptoethanol, SDS, and sample boiling. After electrophoresis,
the gel was sliced into two pieces. One half was stained with
Coomassie blue and the other half was assayed for Mn(II) oxidase
activity. For measuring the Mn(II) oxidation, the gel was first
immersed in prewash solution containing 10% glycerol and 0.5%
Triton X-100 for 30 min, and then replaced with 10 mM HEPES
buffer (pH 7.5) to incubate the gel at room temperature for 10 min
. Finally, the gel was incubated overnight in 10 mM HEPES
buffer (pH 7.5) with 5 mM MnCl2 and 0.8 mM CuCl2 at room
temprature. The Mn(II) oxidase activity could be determined by
forming a visible brown band, which was at the same position of
Coomassie blue-stained gel.
Mn(II) Oxidation Assay of the Recombinant E. coli Strain
The mother strain M15 and recombinant strain M15-pQE-cotA
were separately grown overnight at 37uC in K medium containing
corresponding antibiotics. A portion of the culture was then
inoculated into fresh K medium (1:100 dilution) with 10 mM
HEPES (pH 7.5) and antibiotics if needed. The Mn(II) oxidation
assays were performed in the presence of 5 mM MnCl2 and the
reaction media without MnCl2 were set as a negative control.
After growing to an OD600 of 0.60.8, IPTG and CuCl2 were
added to the final concentrations of 0.5 mM and 0.25 mM,
respectively. The cultures were then shaken at 28uC for 7 days and
100 ml aliquots were taken and measured every day: the cells were
harvested by centrifugation at 12,0006g for 5 min and were
resuspended in 10 mM HEPES (pH 7.5). The suspensions were
reacted with Leucoberbelin blue for 10 min and the absorbances
of the supernatants were measured at 620 nm . The biogenic
Mn oxides were calculated by the KMnO4 standard curve.
Besides, the supernatant of 7-day cultivated culture was also
measured by an ICP-OES (PerkinElmer, Norwalk, CT, USA) to
assess the residual Mn(II). Another aliquots (5 ml) of the overnight
cultures were then spotted onto K plates supplemented with 5 mM
MnCl2 and 0.5 mM IPTG. After incubation at 28uC for 7 days,
the plates were photographed before and after the addition of
Leucoberbelin blue to record the extent of Mn(II) oxidation. All
assays were performed in triplicate.
Scanning Electron Microscopy (SEM)
The E. coli strains M15 and M15-pQE-cotA cultivated with and
without Mn(II) were collected by centrifugation after 7 days of
cultivation and pretreated before SEM. The SEM samples were
washed with phosphate buffer (pH 7.2) for 3 times, and then fixed
with 2.5% (v/v) glutaraldehyde overnight at 4uC, followed by
dehydration with an ethanol series (30%, 50%, 70%, 80%, 90%,
and 100%; every step was performed twice and continued for
15 min). Subsequently, the samples were freeze-dried at 2100uC
for 24 h and stored in a desiccator before measurement. Cell
morphologies and the biogenic Mn oxides produced on the cell
surface, were examined under a JSM-6390/LV scanning electron
microscope (SEM; JEOL, Japan) with 20,000 V accelerating
The Sequence Characteristics of CotA
Until now, there was no complete and annotated genome
sequence available for B. pumilus WH4 strain. By searching the
nucleotide and protein databases, we have identfied a CotA
(YP_001485796.1) sequence in the published genome sequence of
B. pumilus SAFR-032. The identity in amino acid sequence is 98%
with the B. pumilus ATCC 7061 (ZP_03054403.1) , which was
recently demonstrated to be a laccase-like MCO with similar
properties as CotA of B. subtilis . 16 S rDNA sequences
alignment and phylogenetic tree analysis showed that the B.
pumilus WH4 strain was closely (.97%) related to B. pumilus
SAFR-032. So the primers were designed according to the most
similar cotA sequence of the B. pumilus SAFR-032. The cotA gene
was amplified by PCR using genomic DNA from B. pumilus WH4
as template and 1530 bp PCR product was obtained.
Subsequently, the gene was inserted into vector pQE-30 and
transformed to host strain E. coli M15.
Amino acid sequence analysis revealed that the CotA protein
contained four conserved copper-binding motifs (Figures 1 and
S1). Moreover, the copper-binding motifs of CotA shared
a significant identity with other MCOs, but the similarity of the
remainder of the proteins was quite poor (Figures 1 and S1).
On the other hand, CotA also shows some similarities as well as
differences in comparison with the well investigated Mn(II)
oxidases such as MnxG, MofA and MoxA (Figure S1). First of
all, CotA (509 aa) is much shorter than MnxG and MofA (1217 aa
and 1661 aa, respectively) which are both large proteins
containing over one thousand amino acids [11,12], but it is similar with
MoxA (476 aa) . Moreover, the copper-binding residues are
usually located at the N- and C-terminus for many MCOs. In this
gene, the four conserved copper-binding motifs, A, B, C and D,
are transcribed in a same order as MofA and MoxA: A and B
motifs are existed at the N-terminus, C and D motifs are present at
the C-terminus. However, this order feature is transcribed in
reverse in MnxG of Bacillus sp. SG-1 and related strains, where A
and B motifs are located at the C-terminus while C and D motifs
are located at the N-terminus . Furthermore, there is also no
copper-binding F motif in CotA, which is predicted to be the fifth
copper-binding motif other than the four copper-binding motifs,
even though the function of F motif is still unknown .
Purification and Biochemical Properties
The overexpressing strain M15-pQE-cotA was induced with
0.5 mM IPTG at 25uC with the addition of 0.25 mM Cu2+, which
provided an appropriate state for folding and yielding a fully
copper incorporated holoenzyme . After purification, the
concentration of the recombinant CotA can reach 0.78 mg ml21,
and a single protein band with a molecular weight of 60 kDa was
detected by SDS-PAGE (Figure S2). The UV-visible spectrum of
the purified CotA showed the traditional band at 607 nm
(Figure 2), and the ratio of copper atoms/molecule of CotA was
calculated to be 3.4960.05 by ICP-OES, indicating that it was
a typical blue copper oxidases.
The Laccase Activity of CotA
We next investigated the laccase activity by oxidizing three
classical substrates ABTS, SGZ and 2,6-DMP, respectively. Like
the other MCOs [20,28,31], CotA was able to oxidize these
specific substrates, indicating a robust laccase activity (Figure S3).
It was supported likewise by the fact that CotA appeared to have
a flexible lidlike region close to the substrate-binding site that may
mediate substrate accessibility . The maximum oxidizing
activity towards ABTS was observed at 73uC in 100 mM
citratephosphate buffer (pH 4.0) containing 1 mM CuCl2 (Figure S4), it
was approximately consistent with the studies in B. subtilis, B.
pumilus and B. licheniformis [21,22,34], suggesting that CotA was
a thermoactive and copper-dependent enzyme. The highest
specific activity of the CotA ([E] = 1.3061027 M) with the ABTS
substrate was 0.15 U mg21. Kinetic constants Km, Vmax and kcat
for ABTS were 35.2463.17 mM, 1.226102660.05 M?min21 and
0.1660.01 s21, the Km value was very similar to the previous
observations, while kcat value was much smaller then CotA
[21,32,34] from other Bacillus sp. strains. A possible explanation
for these differences was that the experiments were performed in
different enzymes and reaction systems.
Mn(II) Oxidase Activities Determined in Liquid Culture
System in vitro
Mn(II) oxidation by purified recombinant CotA in liquid culture
system in vitro was firstly established in this study. When CotA was
incubated at 37uC in optimum reaction mixtures for about 24 h, it
was easy to find that lots of brown or black precipitates were
formed in the solution after exposure to Mn(II), and these
precipitates were confirmed to be Mn oxides by the Leucoberbelin
blue assay (Figure 3).
It can be seen from Figure 4 that the enzyme activity was
strongly affected by pH. The optimum pH range for
Mn(II)oxidizing activity was from 7.5 to 8.0 without any autoxidation in
the control sample, while few Mn oxides were detected below
pH 7.0. This pH value was very similar to those observed in P.
putida GB-1 , Pedomicrobium sp. ACM 3067 , Bacillus sp.
SG-1  and L. discophora , which all displayed an optimum
Mn(II)-oxidizing activity at pH values from 7.0 to 8.0. Notably,
when pH value exceeded 8.5, the Mn(II)-oxidizing activity increased
simultaneously with elevated pH regardless of whether CotA was
added or not. It was principally a result of autoxidation [13,26] and
thus pH 8.0 was selected for the further study. Furthermore, the rate
of enzymatic reaction was extremely slow below 28uC, and
asymptotically approached its maximum at 53uC (Figure 5).
Figure 1. Amino acid sequence alignment of the four conserved copper-binding sites of MCOs from diverse strains. The
copperbinding regions A, B, C and D correspond to those shown in Figure S1. The copper-binding residues are designated T1, T2, T3a and T3b on the basis
of the types of copper which they potentially bind. MnxG, marine Bacillus sp. strain SG-1 MnxG (GenBank accession no. AAB06489.1); MofA, L.
discophora SS-1 MofA (GenBank accession no. CAA81037.2); MoxA, Pedomicrobium sp. ACM 3067 MoxA (GenBank accession no. CAJ19378.1); CotA, B.
pumilus WH4 CotA (this study, GenBank accession no. AFL56752.1). Conserved amino acids are shaded in black (90% conservation or more) or in grey
(70 to 90% conservation).
However, CotA showed a declined stability when it was incubated
at higher temperatures for 25 h (data not shown). To reach
a compromise between activity and stability of the enzyme, 37uC
was chosen as the moderate reaction temperature in the
subsequent experiments. Among all the metal ions that tested for their
effects on enzymatic activity (Table 1), only Cu2+ significantly
enhanced the Mn(II) oxidizing activity of CotA, and the highest
activity was obtained at 0.8 mM Cu2+ (Figure 6). It coincided
pretty well with the previous studies that copper indeed had
a profound stimulating effect on the oxidation of Mn(II) . This
positive role of Cu2+ might be interpreted by the possibility that
Cu2+ was the essential cofactor and enhanced the folding of CotA
(Figure S7) .
On the other hand, the addition of classical metal chelators such
as o-phenanthroline and EDTA both led to a complete loss of
Mn(II)-oxidizing activity (Table 2), pointing to involvement of
a metal cofactor in the Mn(II) oxidation process [26,40,43]. The
Mn(II)-oxidizing activity was also negatively affected by SDS 
or DTT (Table 2).
The specific activity of purified CotA towards Mn(II) was
0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were
14.8561.17 mM, 3.016102660.21 M?min21 and
0.3260.02 s21, respectively.
Figure 3. Mn(II) oxidase activity of purified recombinant CotA in liquid culture system. Tube 1, 10 mM HEPES (pH 8.0) plus 5 mM MnCl2
and 0.8 mM CuCl2 (reaction mixture); tube 2, aliquots (50 ml) of tube 1 reacting with 250 ml Leucoberbelin blue; tube 3, reaction mixture in Tube
1 plus CotA; tube 4, aliquots (50 ml) of tube 3 reacting with 250 ml Leucoberbelin blue.
In-gel Mn(II)-oxidizing Activity
Mn(II)-oxidizing activity analysis of CotA protein using native
PAGE in-gel activity assay was performed. The purified
recombinant CotA yielded a brown band which agreed well with
the corresponding Coomassie blue-stained band (Figure 7).
Moreover, the brown band turned blue when the Leucoberbelin
blue was added, conclusively indicating that Mn oxides just
deposited on the enzyme band.
Oxidation of Mn(II) by the Recombinant E. coli Strain
On K agar plates, E. coli strains M15 and M15-pQE-cotA were
grown with the addition of IPTG, which could induce the
overexpression of CotA. Strikingly, after 7 days of cultivation in
the presence of Mn(II), the recombinant strain M15-pQE-cotA
showed the characteristic brown color resulting from the
accumulation of Mn oxides on the bacterial surfaces (Figure
S5A2) and showed apparent blue after the detection by
Leucoberbelin blue (Figure S5B2). While in the absence of Mn(II),
the precipitates were not observed (Figure S5A1) and did not turn
blue after reaction with Leucoberbelin blue (Figure S5B1). As
Figure 4. Effect of pH on Mn(II)-oxidizing activity of CotA. & (black square) CotA plus 5 mM MnCl2 and 0.8 mM CuCl2 in HEPES buffer
(pH 6.88.2) at 37uC for about 24 h; % (red square) control test with 5 mM MnCl2 and 0.8 mM CuCl2 in HEPES buffer (pH 6.88.2) at 37uC for about
24 h; m (black triangle) CotA plus 5 mM MnCl2 and 0.8 mM CuCl2 in CHES buffer (pH 8.69.5) at 37uC for about 24 h; g (red triangle) control test with
5 mM MnCl2 and 0.8 mM CuCl2 in CHES buffer (pH 8.69.5) at 37uC for about 24 h. The values were means 6 standard deviations for triplicate assays.
The values were means 6 standard deviations for triplicate assays.
a control, the mother strain M15 cultivated in the presence of
Mn(II) displayed nearly whitish (Figure S5C2) and only changed to
a weak blue after the addition of Leucoberbelin blue (Figure
S5D2). In order to further explore the Mn oxidizing ability of M15
and M15-pQE-cotA, both stains were grown in K liquid culture
medium. After 7 days, the mother strain M15 showed a weak
Mn(II) oxidizing activity and only 1.08% of Mn oxides were
produced (Figure 8). While for the recombinant strain
M15-pQEcotA, the Mn(II) oxidizing activity was much stronger, and yielded
about 3.16% of Mn oxides. Additionally, the soluble Mn(II) of the
7-day cultivated supernatant were determined accurately by
ICPOES. As illustrated in Figure 9, 18.2% dissolved Mn(II) was
removed by M15-pQE-cotA, which appeared to be 32 times
greater than that of the mother strain (0.56%).
SEM Analysis of Cell Morphologies and Mn Oxides
From the SEM photographs, we can clearly observe that both
the E. coli strains M15 (Figure 10A) and M15-pQE-cotA
(Figure 10C) behave in physiologically normal fashions after
cultivated for 7 days without Mn(II). However, the addition of
Mn(II) directly influences the cell morphology, it leads to the
pronounced morphological irregularities because the Mn oxide is
occurred as a precipitate covering the cell surface (Figure 10B and
10D). The SEM images show that the accumulation of biogenic
Mn oxides located on recombinant strain M15-pQE-cotA is much
more when compared with the mother strain M15. On the cell
surfaces of M15, only a few precipitates can be observed, while
many Mn oxides are deposited outside the cells of M15-pQE-cotA,
and therefore the strains are heavily encrusted with Mn oxides.
These biogenic Mn oxides are aggregated particles with irregular
geometric shapes, indicating their poor crystallinity and very small
CotA from B. pumilus WH4 is Firstly a Bacterial Laccase
Previous studies demonstrated that CotA was able to oxidize
ABTS, SGZ and 2,6-DMP [22,34,44]. In this study, the
recombinant CotA oxidized the classical laccase substrates
similarly to the other Bacillus CotA enzymes.
Amino acid sequence alignment of the CotA from B. pumilus
WH4 with other CotA protein origins shows that the
copperbinding motifs are alike [33,45]. Moreover, the CotA from B.
pumilus WH4 (AFL56752.1) displays a very high sequence
identities (96% and 97%) with the recently reported CotA from
The values were means 6 standard deviations for triplicate assays.
B. pumilus ATCC 7061  and B. pumilus SAFR-032, respectively.
It also shows 68% and 61% identities with the CotA proteins from
B. subtilis 168 (NP_388511.1)  and B. licheniformis ATCC 14580
(YP_077905.1) , respectively (Figure S6). The homology
model of CotA from B. pumilus WH4 (Figure S7A) is constructed
using SWISS-MODEL program based on its homologous
template from B. subtilis (2WSD) . Three-dimensional
structure models of the CotA and the template protein are superposed
very well within the four copper-binding motifs. The residues
containing H103, H105, H151, H153, H419, H422, H424, H491,
(N) (red circle) were grown at 37uC in K liquid medium containing HEPES (pH 7.5), 0.5 mM IPTG, 0.25 mM CuCl2 and 5 mM MnCl2 for 7 days. The
Figure 8. Determination of the Mn(II) oxidation activity of E. coli strains every day. E. coli strains M15-pQE-cotA (&) (black square) and M15
values were means 6 standard deviations for triplicate assays.
Figure 9. The Mn(II) removal percentages from the supernatants by E. coli strains M15-pQE-cotA and M15. The residual Mn(II) of the
7day cultivated culture was measured by ICP-OES. The values were means 6 standard deviations for triplicate assays.
Figure 10. SEM photographs of E. coli cells and biogenic Mn oxides (620,000 with insert 610,000). (A) SEM image showing the
morphologies of the mother strain M15 cultivated without Mn(II); (B) SEM image of the mother strain M15 cultivated with Mn(II) and the associated
biogenic Mn oxides; (C) SEM image showing the morphologies of the recombinant strain M15-pQE-cotA cultivated without Mn(II); (D) SEM image of
the recombinant strain M15-pQE-cotA cultivated with Mn(II) and the aggregated biogenic Mn oxides.
C492, H493, H497, and M502 are involved in copper ion binding
(Figure S7B). The coordination bonds among the 4 copper ions
and the 12 conserved amino acid residues of CotA are shown in
plane (Figure S7C). Thus, copper ions and copper-binding motifs
constitute the active center and benefit the stability of the
The Purified Recombinant CotA Exhibits
Manganeseoxidase Activity in vitro
Experimental evidences have demonstrated that bacterial
Mn(II) oxidation is an enzymatic process , and many MCOs
are postulated to be directly involved. In none of these cases,
however, have the Mn(II)-oxidizing macromolecules been purified
to such an extent as to allow further studying of biochemical
characteristics . Thus, quick and efficient methods for
purifying enough Mn(II) oxidases are needed urgently and surely
have been gained much attention in recent years . In L.
discophora SS-1, a small amount of Mn(II)-oxidizing protein was
isolated from the polyacrylamide gel by electroelution .
Furthermore, a three-step purification strategy consisting of ion
exchange, hydrophobic interaction, and size exclusion
chromatographies, was used to separate the Mn oxidase from the loosely
bound outer membrane protein fraction in both A. manganoxydans
strain SI85-9A1 and Erythrobacter sp. strain SD-21 . However,
these methods are much more laborious and ineffective for the
purification of larger amounts of enzyme compared with
heterologous overexpression. To date, attempts to purify active
Mn(II) oxidases by genetic manipulation in E. coli strain have not
yet succeeded [6,12]. Neither MnxG, MofA, nor CumA is able to
oxidize Mn(II) when produced from an expression vector in E. coli
. As a result, elucidation of their roles in the biochemical
mechanism of Mn oxidation awaits the breakthrough in
purification of active Mn(II) oxidase .
In this study, we harvested enough recombinant CotA by
heterologous overexpression. Subsequently, we successfully
verified that CotA was directly involved in Mn(II) oxidation.
Previously, the kinetic parameters for the oxidation of Mn(II)
were obtained either from whole cells (spores) or cell extracts, and
none of the research had been conducted with the purified
recombinant enzyme. In the present study, kinetic constants Km,
Vmax and kcat values of purified CotA towards Mn(II) were
14.8561.17 mM, 3.016102660.21 M?min21 and
0.3260.02 s21, respectively. The apparent Km was much higher
when compared with the study carried out with the whole cells
from Pedomicrobium sp. ACM 3067 (26 mm) . On the other
hand, the results of Km and Vmax values were a bit similar to the
previous observations made by Douka  who used the cell
extracts of two bacterial strains to oxidize Mn(II) (3.3 mM and
2061026 M?min21, respectively). While for kcat value, little
information is available on Mn(II) oxidation till now.
For the native PAGE in-gel activity assays, the purified
recombinant CotA produced a brown Mn oxide band in line
with the relevant coomassie blue-stained band. These results,
combined with the asssy in liquid culture system, further
confirmed that CotA had the Mn(II) oxidase activity.
Consequently, we believe that our data are the first direct
observation of Mn(II) oxidation with the heterologously expressed
protein in vitro, thereby giving a thorough understanding of the
enzymological properties of CotA that relates to Mn(II) oxidation.
Our result also put forward an effective overproduction system
and a practical purification protocol for active Mn(II) oxidases,
which will open the way for spectroscopic and eventually
crystallographic characteristics of these putative MCOs and their
Mn oxides .
The Recombinant E. coli Strain has Higher
To testify this crucial role in vivo of cotA, the recombinant strain
M15-pQE-cotA was cultured both in Mn(II)-containing K liquid
medium and on the K agar plates. The results clearly indicated
that the colony color of M15-pQE-cotA was shifted from whitish to
brownish , while the mother strain M15 remained nearly
whitish. No change in colony color was observed when
M15-pQEcotA was grown in the absence of Mn(II). In addition, the results of
Mn removal efficiency and Mn oxide production also
demonstrated that the recombinant strain M15-pQE-cotA had greater
Mn(II) oxidation activity than the mother strain M15.
Most importantly, a clear impression could be gotten from the
SEM photographs that more Mn oxides were accumulated on the
recombinant strain M15-pQE-cotA than the mother strain M15.
Therefore, all these convincing proofs helped to verify our initial
hypothesis that CotA from B. pumilus WH4 was responsible for the
Mn(II) oxidation, which firstly established the immediate linkage
between the bacterial MCOs and Mn(II) oxidation, raising a new
and intriguing question about the fundamental role of other
MCOs played in those different Mn(II)-oxidizing bacteria.
Whats the Mechanisms of Bacterial Mn(II) Oxidation?
Until now, the structures and compositions of biogenic Mn
oxides as well as the molecular mechanisms of bacterial Mn(II)
oxidation, have remained largely a mystery. The available data
only suggests that the oxidation of Mn(II) may involve a unique
MCO system that contains three types of copper binding sites with
different spectroscopic and functional properties . The
mononuclear Type 1 blue copper (T1) site is the primary electron
acceptor from the substrate via two histidine and one cysteine
residue (Figure S7C). Electrons are then transferred to a trinuclear
cluster consisting of one type 2 (T2) and two type 3 copper (T3a/
T3b), which serves as the oxygen binding and reduction site
(Figure S7C), and from there four electrons (from four substrate
molecules) ultimately reduce O2 to 2H2O [3,6]. Nevertheless, in
a chemical reaction, this process of MnO2 production requires
a two-electron oxidation of Mn(II). Why are MCOs only known to
engage one-electron transfers from substrate to O2? Bioinorganic
chemists and microbiologists have long been interested in this
process, and finally they have trapped the one-electron oxidation
product-Mn(III) in experiments with the exosporium of a marine
Bacillus sp. strain SG-1 [48,49]. It has been demonstrated that
enzymatic Mn(II) oxidation proceeds via two one-electron steps: 1)
oxidation of Mn(II) to Mn(III) and 2) oxidation of Mn(III) to
Mn(IV). Both oxidation steps are catalyzed by the same enzyme
and the Mn(II) to Mn(III) step is the rate-limiting step. Mn(III),
which occurs as a transient intermediate, can be captured and
stabilized by the organic or inorganic ligands (L) such as
pyrophosphate, siderophore, small endogenous molecules and
polypeptides, to form a soluble Mn(III) complex (Mn(III)L). This
complex is either stable in solution or undergo oxidation or
disproportionation to Mn(IV) oxides . It is probable that the
Mn(II) oxidation mechanism interpreted by CotA in vitro, will
proceed along similar lines just like the study in Bacillus sp. strain
SG-1, producing an enzyme-bound Mn(III) intermediate.
However, the nature of the enzyme-Mn(III) intermediate and how the
MCO catalyzes both oxidation steps, are still unknown. Thus,
using purified enzymes to investigate this biochemically unique
process will be much easier and helpful.
CotA is an Appropriate Candidate for Biotechnological
Applications and Needs Further Study
Although the present study has given the ultimate confirmation
that the purified recombinant CotA protein possess the ability to
oxidize Mn(II), more available evidences still need to verify its
function and clarify the underlying Mn(II) oxidation mechanism.
Whether it is the only Mn(II) oxidase in B. pumilus WH4? The
answer is unknown, which demands us to put more efforts in the
future research. Recently, two Mn(II) oxidases, McoA (belong to
the bilirubin oxidase MCO superfamily) and MnxG, have been
demonstrate to be necessary for Mn(II) oxidation in P. putida GB-1
. In-frame deletions of either loci resulted in strains that
retained some ability to oxidize Mn(II) or Mn(III), loss of oxidation
was only attained upon deletion of both genes. If there are multiple
Mn(II) oxidase enzymes in B. pumilus WH4 as well, it can be
readily explained that why the deletion of cotA had no effect on the
Mn (II)-oxidizing activity in B. subtilis , possibly because the
independent two or more MCO enzymes dominate under
different growth conditions and the loss of Mn(II) oxidation may
be complemented by the residual Mn(II) oxidase. Therefore, in
order to prove this hypothesis on the co-existence of MCOs, a lot
of molecular manipulations should be carried out, involving the
conventional gene knockout approach.
On the other hand, as a bacterial laccase, CotA might offer
great potentials as biocatalysts in biotechnological and industrial
applications, such as the decolorization of textile dyes and
oxidation of a variety of organic and inorganic substrates.
Additionally, CotA showed a markedly higher affinity for bilirubin
than conventional bilirubin oxidase and catalyzed the oxidation of
bilirubin to biliverdin , which could be used clinically to
determine the levels of total and conjugated bilirubin in serum
CotA could also play a particular role in the economically
favorable removal of Mn(II) from groundwaters. Moreover, the
biogenic Mn oxide particulates or spores that produced by CotA
protein or B. pumilus WH4 could be acted as effective heavy metal
adsorbents [38,52]. The Cr(III) or Cd oxidation capacities of
biogenic Mn oxides were 0.24 mmol g21 and 2.04 mmol g21,
respectively, which even higher than the chemically synthesized
Mn oxides in the aquatic environment [38,52]. Such studies would
be greatly helpful in the feasibility and designing of industrial-scale
bioreactors for treating heavy metals contaminated wastewater,
and highlight the potential for the application of this
bioremediation friendly system, as products could be removed from
effluents in the form of a precipitate .
Figure S1 A diagrammatic representation of the operon
structure for MCO genes from various strains. Depicted
as white arrows are the genes that encode the putative Mn(II)
oxidase of Bacillus sp. strain SG-1 , L. discophora SS-1 ,
Pedomicrobium sp. ACM 3067  and B. pumilus WH4 (this study).
While neighboring genes are shown by grey arrows. Gene names,
when available, are listed above the genes and the putative
functions of the non-MCO genes are below. Cu2+ binding sites are
marked with black rectangles and are lettered according to
Figure S2 SDS-PAGE analysis of CotA expression and
purification. Lanes 1 and 2, whole cell protein fractions of
M15pQE-cotA induced without and with IPTG; lanes 3 and 4, the
soluble extract and the precipitate after disruption with a French
pressure cell; lane 5, the uncombined soluble extract after loading
onto Ni-NTA agarose column; lane 6, the last effluent liquid with
wash buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and
80 mM imidazole); M, molecular size markers; lanes 711, first
five tubes of CotA eluates (1 ml each); lane 12, the elution fractions
were dialyzed against a buffer containing 50 mM Tris-HCl
(pH 7.9) and 500 mM NaCl.
Figure S3 The laccase activity assays of purified CotA
by oxidizing three different substrates. (A) The ABTS test
was performed in 100 mM citrate-phosphate buffer (pH 4.0) with
(tube 1) and without (tube 2) CotA. (B) The SGZ test was
performed in 100 mM phosphate buffer (pH 6.0) with (tube 1) and
without (tube 2) CotA. (C) The 2,6-DMP test was performed in
100 mM citrate-phosphate buffer (pH 5.0) with (tube 1) and
without (tube 2) CotA.
Figure S4 The optimal parameters for the oxidation of
ABTS by CotA. (A) The pH-dependent activity profile. The assay
was determined at 37uC in 100 mM citrate-phosphate buffer
(pH 3.08.0) supplemented with 0.5 mM ABTS and CotA. (B)
Effect of temperature on the ABTS oxidizing activity. The
optimum temperature was performed in 100 mM
citrate-phosphate buffer (pH 4.0) supplemented with 0.5 mM ABTS and
CotA at temperatures ranging from 30 to 100uC. (C) The optimal
cooper concentration. The experiment was tested by adding
CuCl2 (03 mM) to the 100 mM citrate-phosphate buffer (pH 4.0)
supplemented with 0.5 mM ABTS and CotA at 37uC. The values
were means 6 standard deviations for triplicate assays.
Figure S5 Mn(II) adsorption and oxidation on K plates
by IPTG induced E. coli strains. (A) The recombinant strain
M15-pQE-cotA cultured with (plate 2) and without (plate 1) 5 mM
Mn(II). (B) LBB test (plate 12) for the production of Mn oxides
corresponds to plate 12 of panel A, respectively. (C) The mother
strain M15 cultured with (plate 2) and without (plate 1) 5 mM
Mn(II). (D) LBB test (plate 12) for the production of Mn oxides
corresponds to plate 12 of panel C, respectively.
Figure S6 Multiple amino acid sequence alignments of
CotA proteins from B. pumilus WH4 (B.p.WH4), B.
pumilus ATCC 7061 (B.p.ATCC7061), B. subtilis 168
(B.s.168) and B. licheniformis ATCC 14580
(B.l.ATCC14580) using Clustal Omega software. Highly
conserved regions are boxed. Within those, invariant residues are
represented against a red background. The copper-binding regions
A, B, C and D are represented in blue color, and the conserved
copper-binding residues are marked with asterisks (w).
Figure S7 Three-dimensional structure model of CotA
from B. pumilus WH4. (A) The homology model of CotA. It is
constructed using SWISS-MODEL program based on its
homologous template CotA from B. subtilis (2WSD). a-helix
(red), b-sheet (yellow), loop (blue) as well as 4 copper ions (cyan)
are shown in the structure. (B) Residues which are involved in
copper ion (cyan) binding (H103, H105, H151, H153, H419,
H422, H424, H491, C492, H493, H497 and M502) are shown as
gray sticks. (C) The coordination bonds among the 4 copper atoms
and the 12 conserved amino acid residues (H103, H105, H151,
H153, H419, H422, H424, H491, C492, H493, H497 and M502)
of the CotA (see Figure 1) are shown in plane (the diagram was
constructed by the method described in reference .
We give thanks to Dr. Jieping Wang, Liwei Liu and Kanwal Maria for their
invaluable advices and stimulating discussions.
Conceived and designed the experiments: FL JH. Performed the
experiments: JS PB TB LD HW. Analyzed the data: JS PB TB LD.
Contributed reagents/materials/analysis tools: JH. Wrote the paper: JS FL
1. Tebo BM , Bargar JR , Clement BG , Dick GJ , Murray KJ , et al. ( 2004 ) Biogenic manganese oxides: properties and mechanisms of formation . Annu Rev Earth Planet Sci 32 : 287 - 328 .
2. Tebo BM , Clement BG , Dick GJ ( 2007 ) Biotransformations of manganese . In: Hurst CJ, Crawford RL , Garland JL , Lipson DA , Mills AL , Stetzenbach LD (eds) Manual of Environmental Microbiology . 1223 - 1238 .
3. Tebo BM , Geszvain K , Lee S-W ( 2010 ) The molecular geomicrobiology of bacterial manganese(II) oxidation . In: Barton LL et al ., eds. Geomicrobiology: Molecular and Environmental Perspective . 285 - 308 .
4. Anderson CR , Davis RE , Bandolin NS , Baptista AM , Tebo BM ( 2011 ) Analysis of in situ manganese(II) oxidation in the Columbia River and offshore plume: linking Aurantimonas and the associated microbial community to an active biogeochemical cycle . Environ Microbiol 13 : 1561 - 1576 .
5. Tebo BM , Johnson HA , McCarthy JK , Templeton AS ( 2005 ) Geomicrobiology of manganese(II) oxidation . Trends Microbiol 13 : 421 - 428 .
6. Dick GJ , Torpey JW , Beveridge TJ , Tebo BM ( 2008 ) Direct identification of a bacterial manganese(II) oxidase, the multicopper oxidase MnxG, from spores of several different marine Bacillus species . Appl Environ Microbiol 74 : 1527 - 1534 .
7. Brouwers GJ , Vijgenboom E , Corstjens PLAM , de Vrind JPM , de Vrind-de Jong EW ( 2000a ) Bacterial Mn2+ oxidizing systems and multicopper oxidases: an overview of mechanisms and functions . Geomicrobiol J 17 : 1 - 24 .
8. Sujith PP , Loka Bharathi PA ( 2011 ) Manganese oxidation by bacteria: biogeochemical aspects . In: Mu ller WEG, ed. Molecular Biomineralization, Progress in Molecular and Subcellular Biology 52 . 49 - 76 .
9. Lang M , Kanost MR , Gorman MJ ( 2012 ) Multicopper oxidase-3 is a laccase associated with the peritrophic matrix of Anopheles gambiae . PLoS One 7 : e33985 .
10. Francis CA , Casciotti KL , Tebo BM ( 2002 ) Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp . strain SG-1. Arch Microbiol 178 : 450 - 456 .
11. van Waasbergen LG , Hildebrand M , Tebo BM ( 1996 ) Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp . strain SG-1. J Bacteriol 178 : 3517 - 3530 .
12. Corstjens PLAM , de Vrind JPM , Goosen T , de Vrind-de Jong EW ( 1997 ) Identification and molecular analysis of the Leptothrix discophora SS-1 mofA gene, a gene putatively encoding a manganese-oxidizing protein with copper domains . Geomicrobiol J 14 : 91 - 108 .
13. Larsen EI , Sly LI , McEwan AG ( 1999 ) Manganese(II) adsorption and oxidation by whole cells and a membrane fraction of Pedomicrobium sp . ACM 3067. Arch Microbiol 171 : 257 - 264 .
14. Ridge JP , Lin M , Larsen EI , Fegan M , McEwan AG , et al. ( 2007 ) A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp . ACM 3067. Environ Microbiol 9 : 944 - 953 .
15. Anderson CR , Johnson HA , Caputo N , Davis RE , Torpey JW , et al. ( 2009 ) Mn(II) oxidation is catalyzed by heme peroxidases in Aurantimonas manganoxydans strain SI85-9A1 and Erythrobacter sp . strain SD-21. Appl Environ Microbiol 75 : 4130 - 4138 .
16. Brouwers GJ , de Vrind JPM , Corstjens PLAM , Cornelis P , Baysse C , et al. ( 1999 ) cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1 . Appl Environ Microbiol 65 : 1762 - 1768 .
17. Francis CA , Tebo BM ( 2001 ) cumA multicopper oxidase genes from diverse Mn(II)-oxidizing and non-Mn(II)-oxidizing Pseudomonas strains . Appl Environ Microbiol 67 : 4272 - 4278 .
18. Geszvain K , Tebo BM ( 2010 ) Identification of a two-component regulatory pathway essential for Mn(II) oxidation in Pseudomonas putida GB-1 . Appl Environ Microbiol 76 : 1224 - 1231 .
19. El Gheriany IA , Bocioaga D , Hay AG , Ghiorse WC , Shuler ML , et al. ( 2009 ) Iron requirement for Mn(II) oxidation by Leptothrix discophora SS-1 . Appl Environ Microbiol 75 : 1229 - 1235 .
20. Hullo M -F, Moszer I , Danchin A , Martin-Verstraete I ( 2001 ) CotA of Bacillus subtilis is a copper-dependent laccase . J Bacteriol 183 : 5426 - 5430 .
21. Reiss R , Ihssen J , Thony-Meyer L ( 2011 ) Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum . BMC Biotechnol 11 : 9 .
22. Koschorreck K , Richter SM , Ene AB , Roduner E , Schmid RD , et al. ( 2008 ) Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids . Appl Microbiol Biotechnol 79 : 217 - 224 .
23. He JZ , Zhang LM , Jin SS , Zhu YG , Liu F ( 2008 ) Bacterial communities inside and surrounding soil iron-manganese nodules . Geomicrobiol J 25 : 14 - 24 .
24. Zhang LM , Liu F , Tan WF , Feng XH , Zhu YG , et al. ( 2008 ) Microbial DNA extraction and analyses of soil iron-manganese nodules . Soil Biol Biochem 40 : 1364 - 1369 .
25. Boogerd F , de Vrind JPM ( 1987 ) Manganese oxidation by Leptothrix discophora . J Bacteriol 169 : 489 - 494 .
26. Rosson RA , Nealson KH ( 1982 ) Manganese binding and oxidation by spores of a marine Bacillus . J Bacteriol 151 : 1027 - 1034 .
27. Durao P , Chen Z , Fernandes AT , Hildebrandt P , Murgida DH , et al. ( 2008 ) Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes . J Biol Inorg Chem 13 : 183 - 193 .
28. Grass G , Rensing C ( 2001 ) CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli . Biochem Biophys Res Commun 286 : 902 - 908 .
29. Hall SJ , Hitchcock A , Butler CS , Kelly DJ ( 2008 ) A multicopper oxidase (Cj1516) and a copA homologue (Cj1161) are major components of the copper homeostasis system of Campylobacter jejuni . J Bacteriol 190 : 8075 - 8085 .
30. Mohammadian M , Fathi-Roudsari M , Mollania N , Badoei-Dalfard A , Khajeh K ( 2010 ). Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization . J Ind Microbiol Biotechnol 37 : 863 - 869 .
31. Johannes C , Majcherczyk A ( 2000 ) Laccase activity tests and laccase inhibitors . J Biotechnol 78 : 193 - 199 .
32. Durao P , Bento I , Fernandes AT , Melo EP , Lindley PF , et al. ( 2006 ) Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies . J Biol Inorg Chem 11 : 514 - 526 .
33. Gupta N , Farinas ET ( 2010 ) Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores . Protein Eng Des Sel 23 : 679 - 682 .
34. Martins LO , Soares CM , Pereira MM , Teixeira M , Costa T , et al. ( 2002 ) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat . J Biol Chem 277 : 18849 - 18859 .
35. Koschorreck K , Schmid RD , Urlacher VB ( 2009 ) Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis . BMC Biotechnol 9 : 12 - 21 .
36. Krumbein WE , Altmann HJ ( 1973 ) A new method for the detection and enumeration of manganese oxidizing and reducing microorganisms . Helgolander Wiss Meer 25 : 347 - 356 .
37. Schagger H, von Jagow G ( 1987 ) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa . Anal Biochem 166 : 368 - 379 .
38. Meng YT , Zheng YM , Zhang LM , He JZ ( 2009 ) Biogenic Mn oxides for effective adsorption of Cd from aquatic environment . Environ Pollut 157 : 2577 - 2583 .
39. Enguita FJ , Martins LO , Henriques AO , Carrondo MA ( 2003 ) Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties . J Biol Chem 278 : 19416 - 19425 .
40. Okazaki M , Sugita T , Shimizu M , Ohode Y , Iwamoto K , et al. ( 1997 ) Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1 . Appl Environ Microbiol 63 : 4793 - 4799 .
41. Brouwers GJ , Corstjens PLAM , de Vrind JPM , Verkamman A , de Kuyper M , et al. ( 2000b ) Stimulation of Mn2+ oxidation in Leptothrix discophora SS-1 by Cu2+ and sequence analysis of the region flanking the gene encoding putative multicopper oxidase MofA . Geomicrobiol J 17 : 25 - 33 .
42. El Gheriany IA , Bocioaga D , Hay AG , Ghiorse WC , Shuler ML , et al. ( 2011 ) An uncertain role for Cu(II) in stimulating Mn(II) oxidation by Leptothrix discophora SS-1. Arch Microbiol 193 : 89 - 93 .
43. Francis CA , Tebo BM ( 2002 ) Enzymatic manganese(II) oxidation by metabolically dormant spores of diverse Bacillus species . Appl Environ Microbiol 68 : 874 - 880 .
44. Enguita FJ , Marcal D , Martins LO , Grenha R , Henriques AO , et al. ( 2004 ) Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis . J Biol Chem 279 : 23472 - 23476 .
45. Ausec L , Zakrzewski M , Goesmann A , Schl uter A, Mandic-Mulec I ( 2011 ) Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes . PLoS One 6 : e25724 .
46. Douka CE ( 1980 ) Kinetics of manganese oxidation by cell-free extracts of bacteria isolated from manganese concretions from soil . Appl Environ Microbiol 39 : 74 - 80 .
47. Wang X , Wiens M , Divekar M , Grebenjuk VA , Schroder HC , et al. ( 2011 ) Isolation and characterization of a Mn (II)-oxidizing Bacillus strain from the demosponge Suberites domuncula . Mar Drugs 9 : 1 - 28 .
48. Toner B , Fakra S , Villalobos M , Warwick T , Sposito G ( 2005 ) Spatially Resolved Characterization of Biogenic Manganese Oxide Production within a Bacterial Biofilm . Appl Environ Microbiol 71 : 1300 - 1310 .
49. Webb SM , Dick GJ , Bargar JR , Tebo BM ( 2005 ) Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II) . Proc Natl Acad Sci USA 102 : 5558 - 5563 .
50. Geszvain K , McCarthy JK , Tebo BM ( 2012 ) A double knockout of two putative multicopper oxidase genes in Pseudomonas putida GB-1 eliminates manganese (II, III) oxidation . Am Soc Microbiol . doi:10.1128/AEM. 01850 - 12
51. Sakasegawa S- i, Ishikawa H, Imamura S , Sakuraba H , Goda S , et al. ( 2006 ) Bilirubin Oxidase Activity of Bacillus subtilis CotA . Appl Environ Microbiol 72 : 972 - 975 .
52. He JZ , Meng YT , Zheng YM , Zhang LM ( 2010 ) Cr(III) oxidation coupled with Mn(II) bacterial oxidation in the environment . J Soils Sediments 10 : 767 - 773 .