Download Construction of bacterial artificial chromosome library

FEMS Microbiology Letters 238 (2004) 65–70
www.fems-microbiology.org
Construction of bacterial artificial chromosome library from
electrochemical microorganisms
Jung Ho Back a,b, Man Su Kim a,b, Hyuk Cho a, In Seop Chang b, Jiyoung Lee
Kyung Sik Kim b,c, Byung Hong Kim c, Young In Park b, Ye Sun Han d,*
b,c
,
a
d
Biomedical Research Center, Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul, Korea
b
The Graduate School of Biotechnology, Korea University, Seoul, Korea
c
Water Environment and Remediation Research Center, Korea Institute of Science and Technology,
39-1 Hawalkok-dong, Sungbuk-ku, Seoul 136-791, South Korea
Department of Advanced Fusion Technology, Konkuk University, 1, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
Received 3 February 2004; received in revised form 12 July 2004; accepted 12 July 2004
First published online 21 July 2004
Abstract
A microbial fuel cell is a device that directly converts metabolic energy into electricity, using electrochemical technology. The
analysis of large genome fragments recovered directly from microbial communities represents one promising approach to characterizing uncultivated electrochemical microorganisms. To further assess the utility of this approach, we constructed large-insert
(140 kb) bacterial artificial chromosome (BAC) libraries from the genomic DNA of a microbial fuel cell, which had been operated
for three weeks using acetate media. We screened the expression of several ferric reductase activities in the Escherichia coli host, in
order to determine the extent of heterologous expression of metal-ion-reducing enzymes in the library. Phylogenetic analysis of 16S
rRNA gene sequences recovered from the BAC libraries indicates that they contain DNA from a wide diversity of microbial organisms. The constructed bacterial library proved a powerful tool for exploring metal-ion reductase activities, providing information on
the electron transport pathway of electrochemical microbial (ECM) organisms.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Electrochemical microbial organisms; Bacterial artificial chromosome library; Microbial fuel cell; Metal reductase
1. Introduction
Bacterial metal-ion reduction is a common respiratory process in anaerobic environments. A large number of bacterial species have been identified that are
capable of metal-ion reduction. The proteobacteria,
Shewanella and Geobacter, are the most extensively
studied of the metal-ion reducing microorganisms [1–
4]. Previous studies have suggested that these bacteria
*
Corresponding author. Tel.: +82-2-958-5933; fax: +82-2-9585909.
E-mail address: [email protected] (Y.S. Han).
contain multi-component, branched electron transport
systems that include cytochromes, quinones, dehydrogenases and iron–sulfur proteins [5–9]. At neutral
pH, iron minerals are barely soluble, and the mechanisms of electron transfer to or from iron minerals
are still poorly understood [10]. Electron transfer from
microbial cells to metal-ions is facilitated by mediators, such as neutral red, methyl viologen and humic
acid [11,12]. In their natural habitat, humic substances
may act as electron carriers for metal-ion reducing
bacteria [10]. It has been shown that a metal-ion reducer, Shewanella putrefaciens, is electrochemically active and can be used as a catalyst in mediator-less
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.07.018
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J.H. Back et al. / FEMS Microbiology Letters 238 (2004) 65–70
microbial fuel cells [13,14]. In this study, a mediatorless microbial fuel cell (MFC) was used to enrich electrochemically active bacteria, using acetate as the
electron donor and activated sludge as the bacterial
source [14]. A MFC consists of anode and cathode
compartments separated by a cation specific membrane. Microbes in the anode compartment oxidize
fuel (electron donor) generating electrons and protons.
Electrons are transferred to the cathode compartment
through the circuit, and protons are transferred
through the membrane. Electrons and protons are
consumed in the cathode compartment, reducing oxygen to water [14].
Genes involved in metal-ion reduction are tightly
clustered on bacterial artificial chromosome (BAC)
and, as a result, it has been possible to bring about
the cloning and heterologous expression of metal reductases from single continuous fragments of genomic
DNA [17]. In this paper, we describe: (i) the construction of a BAC library made with DNA isolated from
the MFC, (ii) the phylogenetic analysis of 16S rRNA
gene sequences recovered from the libraries and (iii) a
plate assay designed to facilitate the screening of an
array of ferric reduction clones. Our results suggest
that the BAC library provides a useful technique for
heterologously expressing metal-ion reductases in
Escherichia coli.
2. Materials and methods
2.1. Microbial fuel cell system
Mediator-less microbial cells were enriched for over
three weeks. The anode and cathode compartments
(working volume of 25 ml each) were separated by a cation exchange membrane (Nafion, Dupont Co., USA).
Graphite felts (50 · 50 · 3 mm3, GF series, Electro-synthesis Co., USA) were used as the electrodes, with platinum wires connecting them through a resistance and
multimeter. Injection ports were installed in each compartment of the fuel cell. The anode compartment was
kept anoxic by purging it with nitrogen gas. Air was
purged into the cathode compartment, in order to supply the oxygen needed for the electrochemical reaction.
The MFCs were placed in a temperature-controlled
chamber. A resistance of 10 X was selected by means
of a resistance box.
2.2. Bacterial strains and plasmids
Escherichia coli strain, EPI300, and the BAC vector,
pCC1BAC11, were purchased from Epicentre (Madison,
WI, USA). The pGEM-T easy vector system I, T4 DNA
Ligase and buffer 10· for T4 DNA Ligase were supplied
in the form of a pGEM-T vector Kit (Promega, USA).
2.3. Extraction of DNA from graphite felt
The optimized protocol developed in this study was
based on previously described DNA extraction methods for Bacillus cereus [15]. In the basic procedure,
a 16 · 13 cm graphite felt was ground with liquid nitrogen using a mortar and pestle for about 5 min or
until a fine powder remained. The powdered felt was
removed by filtering it through a miracloth (Calbiochem, Germany) and the supernatants were harvested
by centrifugation. The pellet was resuspended in TE
buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA) and
mixed with an equal volume of molten 2% SeaPlaque
GTG agarose (FMC Rockland, ME, USA), pipetted
into molds, and allowed to cool. The plugs were incubated for 24 h at 37 C in lysis buffer A (50 mM Tris–
HCl pH 8.0, 100 mM EDTA pH 8.0, 100 mM NaCl,
0.5% S-laurylsarcosine, 5 mg/ml lysozyme). The plugs
were transferred to 50 ml of lysis buffer B (50 mM
Tris–HCl pH 8.0, 100 mM EDTA pH 8.0, 100 mM
NaCl, 0.5% S-laurylsarcosine, 0.2 mg/ml proteinase
K) and incubated for 24 h at 50 C. This step was repeated once. The plugs were washed extensively with
TE buffer, followed by inactivation of proteinase K
with PMSF. The plugs were stored at 20 C in
70% EtOH. DNA extractions were performed using
a modified version of a previously described method,
which is normally used for extracting DNA from soil
[16]. Glass beads were weighed into a sterile 15 ml
conical tube, and fragmented felt was added. 5 ml
of buffer (50 mM Tris–HCl, pH 8.0, 100 mM EDTA,
pH 8.0, 100 mM NaCl, 0.5% S-laurylsarcosine) was
pipetted directly into the tube. The tubes were centrifuged using a vortex machine (Scientific Industries,
NY, USA). The supernatant was transferred into a
fresh sterile 2 ml microtube. The supernatant was subjected to a final extraction with phenol–chloroform.
The aqueous phase was incubated for 1 h with isopropanol at 20 C. The DNA was pelleted by centrifugation at 16,000g for 30 min at 4 C. The pellets
were washed once with 70% ethanol and resuspended
in TE.
2.4. Partial digestion of the electrochemical microorganism’s genomic DNA
Large fragments of DNA from the plugs were partially digested by HindIII using limiting enzyme concentrations. The agarose plugs were incubated in TE
for 1 h on ice to remove the storage buffer components. This step was repeated three times. The plugs
were loaded onto a 1% SeaPlaque agarose gel, and
the DNA was size-fractionated by pulsed-field gel
electrophoresis. Gel slices containing DNA of the appropriate size were cut out and digested with GELase
before ligation.
J.H. Back et al. / FEMS Microbiology Letters 238 (2004) 65–70
2.5. Phylogenetic analysis of 16S rRNA gene sequences
recovered from microbial fuel cell
67
2.5
2.6. Plate assay-based screening for ferric reductase
We used plate assay-based screening according to a
previously described procedure for ferric reductase
[17]. In the plate assay, the growth medium consisted
of Luria–Bertani media (Difco) supplemented with 10
mM ferric citrate. Fe(III) reducing clones were identified
using ferrozine to detect Fe(II) production. Briefly, ferrozine was sprayed onto the agar surface as described
previously [17]. The production of Fe(II) was detected
by the observation of a magenta color that formed immediately following ferrozine addition.
The ferric reductase clones were each tested for ferric
reduction activity in a liquid assay. Batch cultures were
grown under aerobic conditions in a 100 ml tube. The
growth medium consisted of an LB (NaCl 1%, Bacto
peptone 1%, Yeast extract 0.5%) medium supplemented
with chloramphenicol. The cells were harvested and
washed in 50 mM HEPES pH 7.0. Ferric reductase activity was measured using 10 mM ferric citrate, 50
mM HEPES pH 7.0, and 1 mM ferrozine. The concentration of the complex formed by the ferrous iron and
ferrozine was measured at 562 nm [18].
3. Results
3.1. Enrichment of acetate – using bacteria in microbial
fuel cell
The MFCs were inoculated with activated sludge,
and artificial wastewater containing 5 mM acetate was
continuously fed at a rate of 0.15 ml/min1 for the purpose of enriching the microbial consortium used to oxidize acetate with concomitant current generation. The
MFCs generated a stable current of around 1.5 mA
within three weeks of the inoculation (Fig. 1).
Current (mA)
2.0
PCR was run in a T-gradient thermocycler (Biometra,
Germany) in 0.2ml tubes using 20 ml reaction volumes.
The 5 0 - and 3 0 -primers used were 5 0 -AGAGTTTGATCMTGGCTCAG-3 0 and 5 0 -GGTTACCTTTGTTACGACTT-3 0 , targeting conserved regions 27–46 and
1473–1492 [18]. For the PCR amplification of the 16S
rRNA genes, we used 100 ng of genomic DNA, 10 pmol
of both of the primers, 2.5 mmol of each dNTP, 1 · reaction buffer and 1 U of Ex-Taq (Takara). Initial denaturation was performed at 95 C for 45 s, amplification was
carried out using 30 cycles including denaturation at 95
C for 30 s, annealing was done at 52–63 C for 30 s and
DNA extension at 72 C for 60 s; and the final extension
was performed at 72 C for 7 min. The phylogenetic tree
was constructed based on available 16S rRNA sequences.
1.5
1.0
0.5
0.0
0
5
10
15
20
25
30
35
Date (day)
Fig. 1. Typical current generation from a MFC during the enrichment
process, using acetate as the fuel.
3.2. Optimization of DNA purification methods
The quantity and fragment size of the DNA extracted
using the bead beating method were compared with
those of the DNA obtained by freezing in liquid nitrogen followed by grinding. The DNA yield obtained by
freezing in liquid nitrogen followed by grinding was
found to be higher than that of the bead beating
method. The maximum DNA yield of about 20 lg
(2 · 13 cm graphite felt size) was obtained from grinding. However, the DNA was also considerably sheared
when using this method. To avoid this fragmentation,
the DNA was embedded in an agarose matrix. The harvested cells on the powdered felt were immobilized in agarose, and lysis buffer was used to disrupt the cell walls
and remove cellular protein. Subsequently, the DNAcontaining agarose plug is manipulated in much the
same way as DNA in solution. The size range of the
DNA was above 300 kb (data not shown).
3.3. Construction of electrochemical microorganism bacterial artificial chromosome Library
To access genomic information from such a large a
pool of cultivable electrochemical microorganisms
(ECMs), we constructed a BAC library. The BAC library consisted of 10,000 clones, containing approximately 1400 Mb of electrochemical microorganism
DNA. Fourteen randomly picked clones having inserts
were analyzed by pulsed-field gel electrophoresis after
NotI digestion (Fig. 2). All lanes contained the 8.0 kb
vector band and the average insert size was 140 kb.
3.4. Phylogenetic analysis of bacterial population in
bacterial artificial chromosome library
To link the physiological function and phylogenetic
analysis of the ECMs, we amplified and cloned 16S
rRNA gene sequences from a given pool of 10,000
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Fig. 2. Restriction digest analysis of clones from MFC libraries
digested with NotI. The arrow indicates the BAC vector.
BAC clones. A total of 200 colonies were selected and
analyzed by RFLP. We recovered 30 sequences from
the pool, which are listed in Fig. 3. The majority were
proteobacteria consisting of one a-proteobacteria, six
b-proteobacteria, four c-proteobacteria and four d-proteobacteria. Geobacter sulfurreducens, an electrochemical microbial species, and three uncultured bacteria
were also included.
3.5. Detection of the clones responsible for the Fe(III)
reductase activity
The plate assay is based on the observation that wildtype S. putrefaciens liquefied the agar support directly
below the colonies, when grown aerobically on a ferric
citrate plate [17]. After three days of aerobic growth
on the ferric citrate plate, clones expressing ferric reductase liquefied the agar. Approximately 10,000 colonies
were screened via the ferric citrate plate assay described
above. Three colonies displayed a positive phenotype
(Fig. 4(a)). The three putative clones were each tested
with LB medium and the terminal electron acceptor ferric citrate. The ferric reductase activities of the three
clones are presented in Fig. 4(b). Only the ferric reductase clone, A48, gave a positive result for the ferrozine
assay. Although two clones, which were isolated via
the ferric citrate plate assay (A44 and A60), liquefied
the agar on the ferric plate, they were unable to express
ferric reduction activity after liquid culture. It is possible
that E. coli possesses ferric reduction activity on the ferric citrate plate [19].
4. Discussion
Microbial fuel cells have great potential for use in
wastewater treatment since, in this process, organic contaminants are converted into electricity with a concomitant reduction in the amount of sludge produced. It has
been shown that a mediator-less MFC can be constructed using electrochemically active microbial enrichment culture [20]. ECMs have the ability to obtain the
energy required for growth via electron transfer to
the graphite felt electrode under anaerobic conditions.
The ECM genes required for extracellular electron transfer to the electrode are often clustered in one contiguous
segment on the chromosome of the producer organism.
Fig. 3. Phylogenetic analysis of 16S rRNA sequences obtained from the MFC.
J.H. Back et al. / FEMS Microbiology Letters 238 (2004) 65–70
69
BACs have several distinct advantages, which include
rapid and facile clone isolation and analysis, very low
chimerism and the stable maintenance of extremely large
genomic inserts. In this study, we showed that the preparation of large insert-containing BAC libraries from
MFCs is feasible, informative and allows the recovery
of very large genomic fragments from diverse and uncultivated microbial species. With this approach we have
now generated BAC libraries from MFCs that have average insert sizes greater than 140 kb. Approximately
10,000 colonies were screened, and one clone was found
which expresses ferric reductase. It is likely that most
heterologous ferric reductases are not properly expressed in E. coli.
Acknowledgements
This work was supported by Microbial Genomics
and Applications Center, M1-02-KK-01-0001-02-K1101-004-2-0 under the auspices of the Korean Ministry
of Science and Technology, Korea.
References
Fig. 4. Ferric-ion reduction activity of clones A44, A48 and A60. (a)
Plate assay Reduction of Fe(III) is detected by the presence of the
magenta color in the ferric citrate plate, as seen in A44, A48 and A60,
but not in the control BAC. (b) Fe(III) reduction by BAC (control)
and ferric-ion reduction clones (A44, A48, A60), ferric production was
monitored at 562 nm using the ferrozine assay.
Essential components of the electron transport pathway
to metal oxides in S. putrefaciens are the c-type cytochromes (7.9). It has been reported that the maturation
of c-type cytochromes, which involves the covalent
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of maturation genes. In spite of these limitations,
screening for gene clusters from genomic DNA libraries is a valuable approach, because it can provide information about enzymes present in organisms that
cannot be cultivated.
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