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Applied and Environmental Microbiology, September 2000, p. 4168-4171, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression of a Tetraheme Protein,
Desulfovibrio vulgaris Miyazaki F Cytochrome
c3, in Shewanella oneidensis
MR-1
Kiyoshi
Ozawa,1
Alexandre I.
Tsapin,2
Kenneth H.
Nealson,2,*
Michael A.
Cusanovich,3 and
Hideo
Akutsu1
Department of Chemistry and Biotechnology,
Faculty of Engineering, Yokohama National University, Hodogaya-ku,
Yokohama 240-8501, Japan1; Jet
Propulsion Laboratory, California Institute of Technology,
Pasadena, California 911092; and
Department of Biochemistry, University of Arizona, Tucson,
Arizona 857213
Received 9 March 2000/Accepted 6 June 2000
 |
ABSTRACT |
Cytochrome c3 from Desulfovibrio
vulgaris Miyazaki F was successfully expressed in the facultative
aerobe Shewanella oneidensis MR-1 under anaerobic,
microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg
of cytochrome/g of cells. A derivative of the broad-host-range plasmid
pRK415 containing the cytochrome c3 gene from
D. vulgaris Miyazaki F was used for transformation of
S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine
residue at the N terminus.
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TEXT |
Cytochromes
c3 are low-potential tetraheme proteins found
almost exclusively in anaerobic bacteria, including sulfate-reducing bacteria in the genus Desulfovibrio. These cytochromes
(either cell free or in vivo) have many potential uses, including
pollutant degradation (6) and bioelectronics (4,
9), so an efficient production system would be very useful. One
approach has been to take the cytochrome c3
(cyc) genes from Desulfovibrio vulgaris from
which it was cloned (5, 17) and express it in other organisms (1, 11, 16). Such an approach has met with
considerable difficulty, although some success has been obtained in
several systems, including Rhodobacter sphaeroides
(1) and another species of Desulfovibrio
(16). However, although expression was obtained, a
user-friendly system for protein production has not yet been achieved.
Because Shewanella oneidensis MR-1 is known to produce
cytochrome c3 (14), and because it is
a facultative aerobe that can be easily and rapidly grown to high cell
densities, we reasoned that it might be useful as an expression
vehicle, and we report here that the gene for cytochrome
c3 from D. vulgaris Miyazaki F can be
expressed under either aerobic or anaerobic conditions in MR-1.
Cell growth and reagents.
S. oneidensis MR-1 (formerly
called Shewanella putrefaciens MR-1 [15])
and its rifampin-resistant strain, TSP-C, were cultured aerobically
overnight at 30°C using Luria-Bertani (LB) liquid medium, and
rifampin was added at 10 µg/ml for the TSP-C strain. For anaerobic
cultures of MR-1, glass bottles with butyl rubber caps containing
degassed LB media with 30 mM sodium fumarate as the terminal electron
acceptor were used. All enzymes, as well as low- and
high-gelling-temperature agaroses, were obtained from TaKaRa Shuzo Co.,
Ltd. (Kyoto, Japan), while radioactive compounds were purchased from
ICN Biomedicals Inc. (Irvine, Calif.) and were used for
dideoxynucleotide sequencing. Molecular mass markers for
electrophoresis were obtained from Bio-Rad Laboratories (Richmond, Calif.) and Amersham Pharmacia Biotech (Uppsala, Sweden). Both polyvinylidene difluoride membranes (0.2-µm pore size) and
horseradish peroxidase color detection reagents (goat anti-rabbit
immunoglobulin G secondary antibody conjugated with horseradish
peroxidase, 4-chloro-1-naphthol, and hydrogen peroxide) used for
Western blotting analysis were obtained from Bio-Rad Laboratories.
Albumen was purchased from Seikagaku Corporation (Tokyo, Japan).
Columns (SP-Sepharose [2.6 by 10 cm] and Hiload Superdex 75 [2.6 by
60 cm]) were purchased from Amersham Pharmacia Biotech. All other
reagent-grade chemicals and antibiotics were obtained from Wako Pure
Chemical Industries, Ltd. (Tokyo, Japan).
Preparation of plasmids and transfer of the cyc genes
to MR-1.
Two plasmids (pRKM3F and pRKM
) were used in these
studies, giving essentially identical results. The former contained the wild-type cytochrome c3 gene from D. vulgaris Miyazaki F, while the latter contained the same gene
modified with regard to codon usage by R. sphaeroides (K. Ozawa et al., unpublished results). Both plasmids were derivatives of
plasmid pRK415 (3) and were constructed as follows.
Plasmid pKM300 containing the cyc gene from D. vulgaris Miyazaki F (900-bp AatII-SphI
fragment in the AatII-SphI site of pUC18) (5) was digested with AatII and SphI.
The resultant AatII-SphI fragment (900 bp) was
treated with T4 DNA polymerase to produce blunt ends and was ligated
into the SmaI site of plasmid pUC118. The plasmid containing
the cytochrome c3 gene in the opposite direction
to the lac promoter was selected. The constructed vector was
called pUKM300. Plasmid pUKM300 was then digested with XbaI and EcoRI, and the 900-bp fragment was isolated from the gel
and ligated into the vector pRK415. The ligation product was
transformed into Escherichia coli JM109 and plated onto LB
plates with 15 µg of tetracycline per ml, 40 µg of X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactoside) per ml, and
0.1 mM IPTG
(isopropyl--D-(
)-thiogalactopyranoside. Recombinant plasmids were isolated from white colonies and
analyzed by restriction digestion with XbaI and
EcoRI, resulting in excision of the cyc
gene as a 900-bp fragment. The recombinant plasmid, pRKM3F,
was then used for transformation of MR-1.
Both pRKM3F and pRKM
were transformed into
E. coli S17-1
(
13) and subsequently transferred to
S. oneidensis TSP-C by conjugation.
In order to confirm the presence
of the
cyc gene from
D. vulgaris Miyazaki F, the
soluble protein fractions of
S. oneidensis MR-1
and the
exconjugant
S. oneidensis TSP-C (pRKM
) were subjected
to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and stained with Coomassie brilliant blue R-250 (CBB) (Fig.
1A)
for proteins or
o-tolidine
dihydrochloride for hemes (Fig.
1B).
Comparison of lanes 4 and 6 in
Fig.
1A and B shows that
S. oneidensis TSP-C(pRKM
)
expressed the product of the
D. vulgaris Miyazaki
F
cytochrome
c3 gene as a
c-type
cytochrome of 14 kDa. The position
of the band of interest in lane 6 was identical to the
D. vulgaris Miyazaki F cytochrome
c3 marker (lanes 3 and 7) and clearly was
different from that of cytochrome
c3 from
S. oneidensis (lane
5). The 14-kDa band seen in lane 6 was
increased approximately
twofold when the concentration of tetracycline
in the culture
of
S. oneidensis TSP-C(pRKM
) was raised
from 15 to 30 µg/ml (data
not shown). Heme staining of the gel (Fig.
1B) revealed a variety
of
c-type cytochromes present in
S. oneidensis MR-1, consistent
with previous reports
(
7). Western blot analysis of these bands
was also performed
using anti-
D. vulgaris Miyazaki F cytochrome
c3 serum (Fig.
1C). Cross-reactivity was
revealed for the bands
in lanes 3, 6, and 7. The 14-kDa band in lane 6 indicates the
presence of
D. vulgaris Miyazaki F cytochrome
c3 in
S. oneidensis TSP-C, while the
absence of cross-reactivity in the negative controls
(Fig.
1C, lanes 4 and 5) indicates that the serum has no cross-reactivity
with
cytochromes produced by MR-1.
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FIG. 1.
SDS-PAGE analyses of cytochrome
c3 in S. oneidensis. Cell
preparations were loaded onto SDS-15% PAGE gels and electrophoresed.
(A) Protein staining with CBB; (B) heme staining with
o-tolidine dihydrochloride heme stain prepared as previously
described (2); and (C) antigenically active material via
Western blotting with antibody to cytochrome c3
from D. vulgaris Miyazaki F. Lanes 3, 5, and 7 were loaded
with ca. 0.005 mg of protein, and lanes 4 and 6 were loaded with ca. 1 mg of protein. Lane 1, high-molecular-mass markers (phosphorylase
b [97.4 kDa], bovine serum albumin [66.2 kDa], ovalbumin
[45 kDa], carbonic anhydrase [31 kDa], soybean trypsin inhibitor
[21.5 kDa], and lysozyme [14 kDa]); lanes 2 and 8, low-molecular-mass markers (globin [16.95 kDa], globins I and II
[14.4 kDa], globins I and III [10.7 kDa], and globin I [8.16
kDa]); lanes 3 and 7, wild-type cytochrome c3
from D. vulgaris Miyazaki F; lane 4, cell lysate from
S. oneidensis MR-1; lane 5, wild-type cytochrome
c3 from S. oneidensis MR-1; lane 6, cell lysate from S. oneidensis TSP-C(pRKM).
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|
Isolation and characterization of recombinant D. vulgaris Miyazaki F cytochrome c3 from
S. oneidensis.
Recombinant D. vulgaris Miyazaki F
cytochrome c3 was purified (Fig.
2, lane 5) from a supernatant obtained
after centrifugation of the broken-cell supernatant treated with
streptomycin sulfate (0.16 g per g of cells). Purification was carried
out at 4°C and pH 7.0. The recombinant cytochrome
c3 was purified in two steps. First, after
dialysis against 10 mM sodium phosphate buffer, the supernatant was
loaded onto an SP-Sepharose column (2.6 by 10 cm) previously
equilibrated with the same buffer. Under these conditions, D. vulgaris Miyazaki F cytochrome c3 (pI = 10.6) binds to the ion-exchange resin, while endogenous S. oneidensis cytochrome c3 (pI = 5.8) is
eluted together with other proteins. A gradient of 0 to 500 mM NaCl in
10 mM sodium phosphate buffer was then used to remove the D. vulgaris Miyazaki F cytochrome c3, which was eluted at 150 mM NaCl. Second, the eluted cytochrome
c3 fraction was further purified by gel
filtration on fast protein liquid chromatography system (Amersham
Pharmacia Biotech) using a Hiload Superdex 75 column (2.6 by 60 cm)
equilibrated with 50 mM NaCl-10 mM sodium phosphate buffer. Relative
purity was confirmed by the absence of other bands after SDS-15% PAGE
using CBB staining and a purity index
(A552red/A280ox of 
3.0.
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FIG. 2.
SDS-PAGE analyses of recombinant cytochrome
c3 in the purification process. Enzyme
preparations were loaded onto SDS-15% PAGE gels and electrophoresed.
Two different treatments are shown, CBB staining (A) and heme staining
(B). Lane 2 was loaded with ca. 1 mg of protein, lane 3 was loaded with
0.3 mg of protein, lanes 4, 5, and 7 were loaded with ca. 0.01 mg of
protein, and lane 6 was loaded with 0.005 mg of protein. Lanes 1 and 8, high-molecular-mass markers that are the same as those in lane 1 of
Fig. 1; lane 2, supernatant of S. oneidensis TSP-C(pRKM)
extract; lane 3, supernatant after dialysis; lane 4, cytochrome
c3 fraction after SP-Sepharose column
chromatography; lane 5, cytochrome c3 fraction
after Superdex 75 column chromatography; lane 6, S. oneidensis cytochrome c3 marker; lane 7, D. vulgaris Miyazaki F cytochrome c3
marker.
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|
The UV and visible spectra were recorded with a Shimadzu UV-2200
spectrophotometer at room temperature. The recombinant
D. vulgaris Miyazaki F cytochrome
c3 showed a
peak at 409 nm and
a broad band centered at 530 nm in the oxidized
state and peaks
at 552, 523, and 419 nm in the dithionite-reduced state
in 10
mM sodium phosphate buffer (Table
1), identical with those of
the
wild-type cytochrome
c3 isolated from
D. vulgaris Miyazaki
F (
18).
The sequence of the N-terminal 15 amino acid residues of the
recombinant
D. vulgaris Miyazaki F cytochrome
c3 purified from
S. oneidensis(pRKM
) was determined by sequential Edman
degradation.
The sequence was identical to that of wild-type cytochrome
c3 (
12) isolated from
D. vulgaris Miyazaki F, except for the addition
of an extra alanine
at the N terminus, and distinctly different
from that reported for
cytochrome
c3 from
S. oneidensis MR-1
(Table
1).
For nuclear magnetic resonance (NMR) studies, the sample was
lyophilized three times with 99.9%
2H
2O and
resuspended in deuterated 10 mM sodium phosphate buffer,
pH 7.0. One-dimensional
1H NMR spectra at 600 MHz were recorded at
303 K on a Bruker DRX-600
NMR spectrometer. The NMR spectra of the
oxidized form of wild-type
and recombinant cytochromes
c3 in the low-field region (10 to
40 ppm, i.e.,
the region most commonly used to detect chemical
or physical changes in
cytochromes) were virtually identical to,
but easily distinguishable
from, that of the cytochrome
c3 from
S. oneidensis (data not shown). The macroscopic redox potentials
of
the recombinant cytochrome
c3 were identical to
those of the
wild-type
D. vulgaris Miyazaki F cytochrome
c3 (
10). Furthermore,
the recombinant
cytochrome
c3 easily could be reduced by
hydrogen
in the presence of
D. vulgaris Miyazaki F
hydrogenase, just like
the wild-type cytochrome
c3. On the basis of these data, we conclude
that
the expressed protein is fully functional and identical to
the
wild-type
D. vulgaris Miyazaki F cytochrome
c3, except for
the additional alanine residue at
the N
terminus.
Yield of recombinant D. vulgaris Miyazaki F cytochrome
c3.
Cells were grown under three different
conditions: (i) aerobic, with strong aeration in a 5-liter fermentor;
(ii) microaerobic, with intermediate aeration (2 liters of culture in a
3-liter Erlenmeyer flask); and (iii) anaerobic, with fumarate as the
terminal electron acceptor. Yields of cytochrome
c3 were compared with those obtained during
anaerobic growth of D. vulgaris (Table
2). On a per-weight basis (milligrams of
cytochrome per gram [wet weight] of cells), D. vulgaris
yielded 0.15 mg, while S. oneidensis(pRKM) yielded 0.29 mg under aerobic conditions and 0.5 mg under microaerobic or anaerobic
conditions.
Per liter of culture,
D. vulgaris yielded 0.3 mg of
cytochrome
c3. In comparison,
S. oneidensis(pRKM
) yielded 1.9 mg aerobically
and 1 mg for
microaerobic and anaerobic culture. These results
reflect the major
differences obtained in growth yield under these
different conditions.
Because of the ease of aerobic growth, high
cell yield, and good
production of cytochrome
c3, this system
offers
an easy and efficient vehicle for cytochrome
c3 production.
The system may also have utility for the expression of other multiheme
protein genes, because
Shewanella produces a variety
of
c-type multiheme cytochromes of its own (
7,
14),
suggesting
that it has a very good heme ligase system. MR-1 is capable
of
reduction of both elemental sulfur and thiosulate (
8), an
unusual
ability even for many anaerobes, and mutants lacking
cytochromes
c are unable to reduce these compounds.
Furthermore, even though
D. vulgaris Miyazaki F and
S. oneidensis belong to different groups
of the
Proteobacteria (delta and gamma, respectively) based on
16S
rRNA analysis, MR-1 was able to transcribe the genes efficiently,
with
or without codon modification (data not
shown).
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ACKNOWLEDGMENTS |
We thank Kin-ichiro Miura at Gakushuin University and Izumi Kumagai
at Tohoku University for helpful discussions. K.H.N. thanks the
exobiology and astrobiology programs at NASA for support of this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: California
Institute of Technology, Jet Propulsion Laboratory, Mail Stop 183-301, 4800 Oak Grove Dr., Pasadena, CA 91109. Phone: (818) 354-9219. Fax: (818) 393-4445. E-mail: knealson{at}jpl.nasa.gov.
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Applied and Environmental Microbiology, September 2000, p. 4168-4171, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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