Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions.

Abstract

The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1. Sphingopyxis sp. C-1 can grow; at pH 11.0; however, the optimum pH for growth was pH 7.0. The microcystin degradation activity of the bacterium was the greatest between pH 6.52 and pH 8.45 but was also detected at pH 10.0. The mlrA homolog encoding the microcystin-degrading enzyme in the C-1 strain was conserved. We concluded that alkali-tolerant microcystin-degrading bacterium played a key role in triggering the rapid degradation of microcystin, leading to the disappearance of toxic water blooms in aquatic environments.

Full text

Page 1

Hindawi Publishing Corporation
Journal of Toxicology
Volume 2009, Article ID 954291, 8 pages
doi:10.1155/2009/954291
Research Article
Characteristicsof a Microcystin-Degrading Bacteriumunder
AlkalineEnvironmentalConditions
Kunihiro Okano,1,2Kazuya Shimizu,1Yukio Kawauchi,3Hideaki Maseda,4Motoo Utsumi,1
Zhenya Zhang,1Brett A.Neilan,5andNorio Sugiura1
1Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
2Graduate School of Bioresource Science, Akita Prefectural University, 241-438 Kaidobata-Nishi, Nakano Shimoshinjo,
Akita 010-0195, Japan
3Mitsubishi Chemical Analytech Co. Ltd., 8-5-1 Tyuuou, Ami, Inashikigun, Ibaraki 300-0332, Japan
4Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima, Tokushima 770-8506, Japan
5School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia
Correspondence should be addressed to Norio Sugiura, cyasugi@sakura.cc.tsukuba.ac.jp
Received 23 September 2009; Accepted 1 December 2009
Recommended by Michael Cunningham
The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously
only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and
characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S
rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1.
Sphingopyxis sp. C-1 can grow; at pH 11.0; however, the optimum pH for growth was pH 7.0. The microcystin degradation
activity of the bacterium was the greatest between pH 6.52 and pH 8.45 but was also detected at pH 10.0. The mlrA homolog
encoding the microcystin-degrading enzyme in the C-1 strain was conserved. We concluded that alkali-tolerant microcystin-
degrading bacterium played a key role in triggering the rapid degradation of microcystin, leading to the disappearance of toxic
water blooms in aquatic environments.
Copyright © 2009 Kunihiro Okano et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
1.Introduction
Toxic blooms of cyanobacteria frequently occur in eutrophic
lakes, ponds, and reservoirs throughout the world [1–7]. It
has been reported that up to 70% of these blooms are poten-
tiallytoxic [8].Cyanobacteria,suchasMicrocystis,Anabaena,
Planktothrix (Oscillatoria),Nostoc,andHapalosiphonspecies,
produce a family of cyclic heptapeptide hepatotoxins called
microcystins (MCs) [9, 10]. The most common MC is
microcystin-LR (MCLR) that has the structure cyclo (D-Ala-
L-Leu-D-MeAsp-L-Arg-Adda-D-Glu-Mdha), where MeAsp
stands for D-erythro-β-methylaspartic acid and Mdha, for
N-methyl-dehydroalanine.Addais2S,3S,8S,9S-3-amino-9-
methoxy-2, 6, 8-trimetyl-10-phenyldeca-4E, 6E-dienoic acid
[9].
MCsinhibitproteinserine/threoninephosphatases1and
2A and promote tumour growth [10–13]. An important
concern is that chronic exposure to low concentrations of
MCs in drinking water may promote tumour growth in the
human liver [11, 14]. Currently, drinking water supplies all
over the world are regularly contaminated with MCs, posing
a threat to public health. For example, in 1996, more than 50
haemodialysis patients in Caruaru, Brazil, died because the
water used for dialysis was contaminated with MCs [15, 16].
In the aquatic environment, the dynamics of MCs
typically depend on the population density of Microcystis sp.
[1, 2, 5–7]. Levels of MCs in water have been observed to
dramatically decrease with any reduction in the population
density of Microcystis that occurs during the disintegration
of blooms [1, 2, 5–7]. Under natural conditions, MCs are
resistant to physicochemical and biological stresses, such as
pH, temperature, sunlight, and certain enzymes [17–20].
Biodegradation trials of MCs have been conducted [21, 22];
however, Okano et al. reported that common enzymes,

Page 2

2Journal of Toxicology
including proteases, cannot degrade MCs [20]. Therefore, it
was assumed that MCs can be degraded only by specific MC-
degrading bacteria.In 1994, Jones et al.isolated thefirstMC-
degrading bacterium Novosphingobium sp. (synonymous
with Sphingomonas sp.) [21]. Subsequently, ten strains of
MC-degrading bacteria have been isolated and studied in
detail [23–32]. Bourne et al. suggested an enzymatic pathway
for the degradation of MCLR that included the proteins
MlrA, MlrB, and MlrC, as well as identifying the gene cluster
encoding these enzymes (mlrA, mlrB, mlrC, and mlrD) in
an environmental isolate [33, 34]. It was also determined
that the mlrA gene codes for an enzyme responsible for the
hydrolytic cleavage of the cyclic structure of MCLR [33],
while the mlrD gene forms an operon with mlrA and encodes
a putative transporter protein of MC and its degradation
products [34]. In addition, Imanishi et al. also suggested an
enzymatic pathway for MC degradation in Sphingomonas sp.
strain B9 [35]. According to the results of these previous
studies, MlrA, MlrB, and MlrC probably catalyze the degra-
dation of MCLR, linear MC, and the tetrapeptide H-Adda-
Glu-Mdha-Ala-OH, respectively, with the release of Adda
and oligopeptides. No information, however, is available on
the mechanism underlying the dramatic degradation of MCs
in natural waters. Aquatic monitoring has revealed the high
pH (8.5–11.0) of the upper layer of natural waters during the
suddenproliferationofwaterbloomsasaconsequenceofthe
photosynthetic activity of cyanobacteria [3, 5–7]. Therefore,
in order to rapidly degrade MCs, a significant number of
MC-degrading bacteria must be able to proliferate and show
activity in the alkaline pH range. However, to date only
MC-degrading bacteria growing at a neutral pH have been
isolated. For example, Novosphingobium sp. strain MD-1
cannot grow under alkaline conditions [24].
Characterization of an MC-degrading bacterium that
can grow at high pH would contribute to a better under-
standing of MC dynamics in aquatic environments and may
also provide useful applications in the field of water quality
management and public health. In this study, we have
examined the characteristics of an alkaliphilic/alkali-tolerant
MC-degrading bacterium under high pH environmental
conditions.
2.MaterialsandMethods
2.1. Isolation of the MC-Degrading Bacterium. The bac-
terium was isolated from Hongfeng Lake in Guizhou
Province, China, where cyanobacterial blooms have reg-
ularly occurred. Water samples collected from the lake
water column were filtered using GF/C glass-fiber filters
(mean pore diameter, 1.2μm; Whatman, Japan). To this
filtered lake water, added MCLR was added to a final
concentration of 2,000μg/L (Wako Pure Chemical Indus-
tries Ltd., Japan) in 0.1M carbonate buffer (pH 9.5) and
an antibacterial-antimycotic mixed stock solution (Nacalai
Tesque Japan) containing final concentrations of penicillin
(10unit/mL), streptomycin (100μg/mL), and amphotericin
B (0.25μg/mL). The samples were then cultured at 25◦C for
7 days with shaking. This process was repeated several times
to isolate the MC-degrading bacterium. To confirm whether
a pure culture had been isolated, it was plated onto diluted
(1/20) peptone-yeast (PY) medium (peptone, 0.5g; yeast
extract, 0.25g; agar, 15g; per liter, pH 7.0). Subsequently,
the isolated bacterium was partially characterized by Gram-
staining and named C-1. After culturing in diluted PY liquid
medium for 3 days, 20mL aliquots were placed in 2mL
serum tubes and stored at −80◦C.
2.2. 16S rRNA Gene Analysis. The isolate was cultured on
diluted PY liquid medium and recovered by centrifugation
at 15000×g for 5 minutes before DNA extraction. The
PrepMan Method (Applied Biosystems, CA) was used for
genomic DNA extraction according to the manufacturer’s
instructions. PCR was performed using Ex Taq DNA
polymerase (Takara Bio Inc., Shiga, Japan) and the 27F-
1492R primer set for amplification. Cycle sequencing of the
amplified16SrRNAgenewasperformedusingtheABIPrism
BigDye Terminator v. 3.0 Ready Reaction Cycle Sequencing
Kit (Applied Biosystems), according to the manufacturer’s
protocol. Purification of amplified and cycle sequencing
PCR products utilized a MicroSeqFull 16S rRNA gene
bacterial sequencing kit (Applied Biosystems) according to
the manufacturer’s protocol.
Sequence data were assembled using the AutoAssembler
2.1(AppliedBiosystems)inordertodeterminethe16SrRNA
gene sequence (GenBank accession number AB161684)
and used in a BLAST search (http://www.ncbi.nlm.nih.gov/
BLAST) to retrieve homologous sequences from GenBank
(http://www.ncbi.nlm.nih.gov/Genbank). The 16S rRNA
gene sequences of related species obtained by the BLAST
search together with those of other MC-degrading bacteria
were aligned using Clustal X version 2.0 [36]. The data were
used for neighbor-joining analysiswith 1000 bootstrap repli-
cates, using the NJ plot program [37] and the phylogenetic
tree was reconstructed.
2.3. Identification of the mlrA Gene and Sequence Analysis. A
FastDNA kit (Qbiogene Inc., USA) was used to extract the
genomic DNA of strain C-1, according to the manufacturer’s
instructions. Presence of the mlrA gene was confirmed by
PCR amplification as per Saito et al. [38]. The PCR was car-
ried out using the MF (5?-GACCCGATGTTCAAGATGCT-
3?) and MR (5?-CTCCTCCCACAAATCAGGAC-3?) primers
and KOD Plus polymerase (Toyobo, Japan). The amplified
fragments (approximately 800bp) were purified using a
QIAquickPCRpurificationkit(QiagenKK,Japan),andthen
cycle sequencing was performed using the ABI Prism cycle
sequencing kit. The DNA sequence was edited and aligned
using GENETYX-MAC (Software Development, Japan),
and a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST)
was performed against homologous sequences in GenBank
(http://www.ncbi.nlm.nih.gov/Genbank). The sequence of
the mlrA-C-1 gene was registered in GenBank with the
accession number AB161685.
2.4. Effect of pH on Growth. After isolating the newly isolated
MC-degrading bacterium (strain C-1) along with Novosph-
ingobium sp. strain MD-1, isolated from Lake Kasumigaura,

Page 3

Journal of Toxicology3
on 1.5% agar plates containing PY medium (peptone, 10g;
yeast extract, 5g; pH 7.0), the cells were precultured at
28◦C for approximately 24 hours in test tubes containing
liquid PY medium. A 1/10 volume of bacterial culture in the
late logarithmic growth phase was inoculated into a series
of liquid PY media with pHs ranging from 7.00 to 11.0,
(adjusted by using 1M NaOH). The optical density of the
solutions was measured at 600nm every 2 hours by using
the GeneQuant Pro spectrophotometer (GE Healthcare Bio-
Sciences AB, Sweden).
2.5. Effect of pH on MC Degradation Activity. Strain C-1 was
precultured in 150mL of diluted (1/20) PY medium (pH
9.5) in a 500mL flask and collected by centrifugation at
3000×g for 5 minutes, followed by washing the pellet with
sterilized milliQ water. This procedure was repeated three
times, and the resultant pellet was used as the sample. The
sample was inoculated into test tubes containing microcystin
medium (1000μg/L MCLR, 0.1M phosphate buffer; pH 7.0)
with an optical density adjusted to 0.5 at 600nm and then
cultured at 25◦C for 3 hours with shaking. The final volume
of the medium was 5mL, and the MCLR concentration was
measured at 0 hour and 3 hours. The pH of the MC medium
was varied from 6.29, 6.72, 7.06, 7.48, 7.92, 8.04, 8.45, 8.91,
9.5, and 10.2 by using either 0.1M phosphate buffer or 0.1M
borate buffer. The experiment was repeated three times, and
the standard deviations and means were evaluated for each
treatment.
2.6. Analysis of MC and Its Primary Degradation Products.
MC was quantitatively analyzed using the Shimadzu 10A
series HPLC system (Shimadzu Corporation, Japan) and
a Cosmosil C18 column (4.6 × 250mm; Nacalai Tesque).
Methanol (60%) diluted with 0.05M phosphate buffer (pH
3.0) was used as the mobile phase, and the flow rate was
set at 1.0mL/min. The analysis was conducted at a column
temperature of 40◦C and using a detection wavelength of
238nm.
The culture samples of strain C-1 were inoculated into
MC medium containing MCLR (3000μg/L), and the final
optical density of the medium was adjusted to be 5.0
at 600nm. The culture was then incubated at 25◦C with
shaking. Once the degradation of MC had been confirmed,
the bacterial culture was separated by centrifugation at
3000 ×g for 5 minutes and the resultant bacterial pellets
were collected. The cells were resuspended in phosphate
buffer, sonicated using the ultrasonicator (VC-70, Sonics &
Materials, Inc., USA), and separated by centrifugation at
15,000×gfor10minutes.Thesupernatantwasfilteredusing
0.2μm filters (HPLC Millex, Millipore, Japan) to obtain
the cell-free extract (CFE) of strain C-1. Four milliliters of
the CFE were mixed with MCLR to a final concentration
of 3000μg/L. The pH of the solution was adjusted to
7.5 using 50mM phosphate buffer, and the degradation
reaction was initiated at 25◦C. The reaction volume was
5mL, and 0.1% sodium azide was added for sterilization
of the solution. MCLR were treated with the CFE before
purification, using a Sep-Pak C18 plus cartridge (Waters,
USA).Phenylmethylsulfonylfluoride (10mM) and ethylene-
diaminetetraacetic acid (1mM) were added to the reaction
solution to inhibit serinegand metalloproteases since these
inhibitors do not affect MlrA [33]. The purified samples
were analyzed by high-performance liquid chromatography
(Agilent 1100 Series; Agilent Technologies Inc., CA) and
mass spectrometry (API300; Applied Biosystems) using an
Inertsil ODS-3 column (2.1 × 150mm; particle size, 3.0μm;
GL Sciences, Japan) at 40◦C. The mobile phase under
linear gradient conditions was a combination of methanol
(LC/MS grade; Wako Pure Chemical Industries) and 0.05%
trifluoroacetic acid (TFA) (LC grade; Wako Pure Chemical
Industries). The analytical time was 30min, and the linear
gradient was 0%–100% of methanol and 100%–0% of TFA
(0.05%). The flow rate was set at 0.2mL/min, and mass
spectrometry analysis was conducted at an infusion rate of
10μL/min. Formic acid was added to the samples to facilitate
ionization at the time of injection. Furthermore, a turbo
ion spray interfaceand positive mode electrospray ionization
(ESI) were used. The ESI conditions in positive ion mode
were as follows: the interface, turbo ion spray; Ionization,
electrospray ionization; the nebulizer gas and the flow rate,
N2 and 6L/min; the curtain gas and flow rate, N2 and
10L/min; the ion spray voltage, 5000V; the orifice voltage,
and 20V. MS data were analyzed using MacQuan software
version 1.6 (Sciex, Concord, Canada).
3.Results
3.1. Isolation and Characterization. The MC degrading bac-
terium, termed the C-1 strain, was isolated from Lake
Hongfeng in Guizhou Province, China, by enrichment
culture using MCLR as the major carbon and nitrogen
source. The C-1 strain is Gram negative, rod shaped, and
forms yellow colonies on solid PY media. Analysis of its 16S
rRNA gene sequence revealed that the bacterium belonged
to the genus Sphingopyxis, order Sphingomonadales of the
α-proteobacteria (Figure 1). The 16S rRNA gene of strain C-
1 was 99.4% similar to that of Sphingopyxis sp. strain KT-1
(JCM10459; accession number AB022601), which does not
possess MC-degradation activity. Only the LH21 strain of
Sphingopyxis spp. has been reported to degrade MC, while
other MC-degrading strains of bacteria belong to various
other genera.
3.2. Effect of pH on Growth and MC Degradation. Strain
C-1 was isolated from lake water which exhibited alkaline
conditions (pH 9.50). The effect of pH on the growth of
the MC degrading strains C-1 and MD-1 was compared in
a batch culture experiment. The growth of the MD-1 strain
was inhibited significantly under alkaline conditions with
an initial pH as low as 9.00 or 10.0, whereas the growth of
the C-1 strain was not inhibited at a pH of 11.0 (Figure 2).
However, the optimum pH for the growth of the C-1 strain
was determined to be 7.00 and, therefore, the C-1 strain
should be described as alkali tolerant.
ThepHofmostwaterbodieschangesdynamicallyduring
theoccurrenceofawaterbloomandbecomeshighlyalkaline.

Page 4

4Journal of Toxicology
7CY (AB076083)
B9 (AB159609)
MDB2 (AB219940)
Y2 (AB084247)
440MDB3 (AB219941)
Sphingopyxis baekryungensis (AY608604)
969
760
MG-15 (personal communication)
MG-22 (personal communication)
Sphingopyxis terrae (D13727)
Sphingopyxis witflariensis (AJ416410)
LH21 (DQ112242)
Sphingopyxis alaskensis (AF337601)
KT-1 (AB022601)
C-1 (AB161684)
Sphingopyxis macrogoltabida (D84530)
MD-1 (AB110635)
CBA4 (AY920497)
MJ-PV (AF411072)
Novosphingobium subterraneum (AB025014)
Novosphingobium tardaugens (AB070237)
Novosphingobium stygia (AB0252013)
Novosphingobium aromaticivorans (AB025012)
Rhodospirillum rubrum (D30778)
999
969
574
1000
345
422
792
419
973
632
957
792
739
434
286
256
875
0.01
Figure 1: Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences indicating the position of isolate C-1 (in bold type) in
relation to other related species, including previously isolated MC-degrading bacteria (underlined). Numerical values represent bootstrap
support. Scale bar represents expected changes per site.
05 10
1
2
C-1
O.D.600
Time (h)
(a)
05 10
1
2
MD-1
O.D.600
Time (h)
(b)
Figure 2: Effect of pH on growth of MC-degrading bacteria. (a) Sphingopyxis sp. strain C-1, (b) Novosphingobium sp. strain MD-1. Values
presented are the means of 3 replicates. ?: pH 7.00, ?: pH 8.00, ?: pH 9.00, ?: pH, 10.0, ×: pH11.0.
Hence, it was important to examine the effect of pH on
the degradation activity of MC-degrading bacteria. Figure 3
shows that the MCLR degradation activity of strain C-1 was
high,especiallybetweenpH6.72and8.45.Inaddition,theC-
1strain retained 60% of its MCLRdegradation activityatpH
8.91. In contrast, the degradation activity of the MD-1 strain
was reduced by 80% at pH 8.50 [24]. The growth rate of the
C-1 strain at pH 10.0 was comparable to its growth rate at
near neutral pH although the optimum pH for degradation
was around neutral (pH 6.72 to pH 8.45). Therefore,

Page 5

Journal of Toxicology5
6
0
810
50
100
Percentage of microcytin
degradation activity
pH
Figure 3: Effect of pH on microcystin-LR degradation activity
of Sphingopyxis sp. strain C-1. MC degradation activity was
determined as the MCLR concentration at 0 hour and 3 hours. The
error bars indicate standard deviation (n = 3).
degradation may depend on the optimum pH for enzyme
activity as opposed to the growth rate of the microorganism.
The C-1 strain was also shown to degrade both MCLR and
microcystin-RR (MCRR). HPLC chromatograms of MCLR
and MCRR degradation were the same for strains C-1 and
MD-1 at neutral pH (data not shown).
3.3. Analysis of the Primary Degradation Product. In this
study, we identified an mlrA homolog in strain C-1 (acce-
ssion number AB161685). We confirmed that the sequence
of this mlrA homolog was highly similar to that of previ-
ously characterized mlrA genes from other MC-degrading
bacteria. By using the LALIGN program (http://www
.eng.uiowa.edu/∼tscheetz/sequence-analysis/examples/LAL-
IGN/lalign-guess.html), the predicted amino acid sequence
of MlrA from strain C-1 was found to be 94.3%, 93.9%,
88.2%, and 95.9% similar to MlrA-MJ-PV (ACM 3962;
AF411068), MlrA-MD-1 (AB114202), MlrA-Y2 (JCM
13185T;AB114203), and
respectively. It was evident from these data that mlrA is
highly conserved in all known MC-degrading bacteria
from a variety of genera. Furthermore, the HEXXH motif,
required for the activation of zinc protease, was also found
to be highly conserved [34]. On the basis of these findings,
we propose that the function of MlrA in the C-1 strain
was similar to its function in the MJ-PV strain [33] and,
therefore, the primary degradation products of MCLR were
analyzedbymassspectrometry.Themassspectrumobtained
was highly similar to that reported by Bourne et al. [33]
(Figure 4). The parent and daughter ions of the degradation
products are shown in Table 1. The primary degradation
product was found to be linearized MCLR (Adda-Glu-
Mdha-Ala-Leu-Masp-Arg-OH),
MlrA-LH21 (DQ112243),
the sameintermediate
0
20
40
60
80
100
0100 200 300400 500 600
m/z
700 800 900 1000
133.2
175.5
213.3 304.4
571.4
1013.6
Relational intensity (%)
Figure 4: Mass spectrum of the primary degradation product of
Sphingopyxis sp. strain C-1. The raw data obtained using mass
spectrometry was treated by MacQuan and the figure shows only
the main peaks.
Table 1: Parent and daughter ions of the primary degradation
product.
m/z
1013.6
571.4
304.4
213.3
175.5
132.2
Identity
M + H (Adda-Glu-Mdha-Ala-Leu-Masp-Arg-OH + 2H)
Mdha-Ala-Leu-Masp-Arg-OH + 2H
Masp-Arg-OH + 2H
Glu-Mdha + H
Arg-OH + 2H
PhCH2CHOCH
∗Adda is 3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic
acid. Mdha is N-methyldehydroalanine, and Masp is D-β-methylaspartic
acid.
product mentioned in previous reports of the enzymatic
degradation of MC by heterotrophic bacteria isolated from
water blooms [25, 33] (Figure 5).
4.Discussion
A dynamic change in the pH of aquatic environments
from neutral to alkaline is associated with the occurrence
of the blooms of cyanobacteria in water bodies [3, 5–7].
However, all currently known MC-degrading bacteria have
been isolated under neutral pH conditions [21, 23–31].
Hence, the mechanism which accounts for the rapid degra-
dation of MCs in aquatic environments remains unclear.
In this study, it was assumed that alkali tolerant and/or
alkaliphilic MC-degrading bacteria contribute significantly
in the rapid degradation of MCs in natural waters, leading to
the disappearance of water blooms and their toxic metabo-
lites. Therefore, an alkali-tolerant MC-degrading bacterium,
namedC-1,wasselectivelyisolatedunderalkalineconditions
from a water bloom sample (Figure 2). The C-1 strain
could effectively degrade MCs even in high pH to neutral
conditions (Figure 3). From these results, it was considered
that C-1 strain might be actively involved in the dramatic
degradation of MCs at the disappearance of water blooms.
Recently, Fujimoto et al. isolated strains MG-15 and MG-
22 of Monas guttula [30] that predate on the MC-producing