Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration

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1
3Heterologous expression and purification of a multiheme cytochrome
4from a Gram-positive bacterium capable of performing extracellular
5respiration
6
7
8N.L. Costa
a
, H.K. Carlson
b
, J.D. Coates
b,c
, R.O. Louro
a
, C.M. Paquete
a,
⇑
9
a
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República – EAN, 2780-157 Oeiras, Portugal
10
b
Energy Bioscience Institute, University of California, Berkeley, CA 94720, United States
11
c
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, United States
12
13
15
article info
16 Article history:
17 Received 14 January 2015
18 and in revised form 12 March 2015
19 Available online xxxx
20 Keywords:
21 Multiheme-cytochrome
22 Gram-positive bacteria
23 Microbial fuel cells
24 Thermincola
25 Extracellular electron transfer
26
27
abstract
28
Microbial electrochemical technologies are emerging as environmentally friendly biotechnological pro-
29
cesses. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial
30
fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were
31
implicated in the process of electricity production. In order to understand the molecular basis by which
32
Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be
33
characterized in detail. Towards this end, a chimeric gene containing the signal peptide from
34
Shewanella oneidensis MR-1 small tetraheme cytochrome c(STC) and the gene sequence of the target pro-
35
tein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric
36
gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and character-
37
ization. This methodology opens the possibility to study other multiheme cytochromes from Gram-posi-
38
tive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be
39
unraveled.
40
Ó2015 Published by Elsevier Inc.
41
42
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44
Introduction
45
Microorganisms capable of performing extracellular electron
46
transfer hold great potential for the development of environmen-
47
tally friendly biotechnological applications. These are generally
48
known as microbial electrochemical technologies of which micro-
49
bial fuel cells (MFCs)
1
are the best known example. These technolo-
50 gies have the potential to significantly improve the production of
51 sustainable and renewable energy, wastewater treatment processes,
52 and implementation of sustainable biorefinery processes [1,2]. Both
53 Gram-negative and Gram-positive bacteria are known to successfully
54 transfer electrons directly to a solid anode in an operating MFC [3].
55 Studies performed on these devices showed that thermophiles pro-
56 duce higher levels of current than mesophiles in the same reactor,
57 being often the prevalent electricity generating communities in the
58 anode [4–6]. Recently a thermophilic Gram-positive bacterium,
59 Thermincola potens JR, was isolated in an operating MFC [7].
60
Studies exploring electron transfer in MFCs have taken place
61
mainly on mesophilic Gram-negative bacteria, where the best
62
understood electron transfer pathway is from the gamma pro-
63
teobacterium Shewanella oneidensis MR-1. In this organism multi-
64
heme c-type cytochromes (MHCs) transfer electrons from
65
cytoplasmic and inner-membrane oxidizing enzymes towards
66
redox super-complexes at the cell surface that are responsible for
67
the reduction of solid phase electron acceptors [8]. When com-
68
pared with Gram-negative bacteria, Gram-positive bacteria lack
69
the outer membrane and present a thicker cell wall made of
70
peptidoglycan. Moreover, the width of the periplasmic space
71
between the membrane and the cell wall is smaller than the typical
72
periplasm of Gram-negative bacteria [5,9,10]. These differences
73
suggest a different electron transfer mechanism towards the cell
74
surface. Interestingly, like the well-studied Gram-negative bacteria
75
S. oneidensis MR-1 and Geobacter sulfurreducens [11], the genome of
76
T. potens JR contains 32 genes that code for putative c-type cyto-
77
chromes [12]. The physiological characterization of T. potens JR
78
revealed that it is able to reduce insoluble ferric compounds [7].
79
Furthermore, a recent study by Carlson and co-workers showed
80
biochemical and biophysical evidence that MHCs are implicated
81
in the reduction of insoluble ferric compounds [13]. However, to
http://dx.doi.org/10.1016/j.pep.2015.03.007
1046-5928/Ó2015 Published by Elsevier Inc.
⇑
Corresponding author. Tel.: +351 214469309.
E-mail address: cpaquete@itqb.unl.pt (C.M. Paquete).
1
Abbreviations used: STC, small tetraheme cytochrome c; MFCs, multiheme c-type
cytochromes; N-region, N-terminal region; H-region, hydrophobic region; TB, Terrific
Broth; DEAE, diethylaminoethyl; DDM, n-Dodecyl-b-maltopyranoside.
Protein Expression and Purification xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
YPREP 4668 No. of Pages 5, Model 5G
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Please cite this article in press as: N.L. Costa et al., Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium
capable of performing extracellular respiration, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.007

82
explore the mechanisms by which Gram-positive bacteria perform
83
extracellular electron transfer it is necessary to characterize in
84
detail the proteins involved in this process.
85
Escherichia coli is by far the best studied heterologous expres-
86
sion system for recombinant proteins [14,15]. However the
87
heterologous expression of c-type cytochromes requires additional
88
care because it relies on specific cellular machinery to ensure the
89
covalent binding of the heme to the apo-cytochrome. This process
90
depends on the cytochrome cbiogenesis system that ensures the
91
translocation of the heme to the periplasmic space and the cova-
92
lent heme attachment to the apo-cytochrome [16,17]. The native
93
cytochrome cmaturation system, coded in E. coli by the gene clus-
94
ter ccmABCDEFGH [16–18] does not operate under aerobic condi-
95
tions [19]. Therefore, co-expression of this system with the target
96
c-type cytochrome is necessary for the proper maturation of the
97
recombinant c-type cytochrome under aerobic conditions [20–24].
98
In addition to the cytochrome cmaturation system, the success-
99
ful production of recombinant c-type cytochromes also depends on
100
the translocation of the apo-protein to the periplasmic space
101
where the heme assembly occurs [17,18]. The translocation to
102
the periplasmic space is possible due to the presence of a specific
103
signal peptide sequence. This signal sequence generally contains
104
15–30 residues and consists of three specific regions: the positively
105
charged N-terminal region (N-region), the hydrophobic region (H-
106
region) and the neutral C-region at the C-terminal end that is
107
recognized by the signal peptidases in the periplasmic space
108
[25,26]. Although for both Gram-positive and Gram-negative bac-
109
teria the machinery of the Sec pathway responsible for the recog-
110
nition of the hydrophobic N-terminal leader sequence and
111
translocation of the apo-protein are very similar, the cleavage of
112
this sequence by peptidases is quite distinct [25,27]. While in
113
Gram-negative bacteria the signal peptidase cleavage occurs three
114
to seven residues from the C-terminal end of H-region, in Gram-
115
positive bacteria this cleavage takes place preferentially from
116
seven to nine residues from the same position. Therefore, special
117
care has to be taken in the heterologous expression of Gram-posi-
118
tive c-type cytochromes in Gram-negative bacteria like E. coli.
119
In this paper, we present, for the first time, the heterologous
120
expression and purification of a multiheme c-type cytochrome
121
from a Gram-positive bacterium. In order to ensure the proper
122
cleavage of the target protein, the signal peptide of the periplasmic
123
decaheme protein ThrJR_0333 from T. potens JR was replaced with
124
the signal peptide from the small tetraheme cytochrome cfrom S.
125
oneidensis MR-1 (SO_2727), an abundant constitutively expressed
126
protein [28–30]. This approach enabled the over-expression of
127
the Gram-positive decaheme cytochrome TherJR_0333 in the
128
Gram-negative bacterium E. coli. This methodology will facilitate
129
studies aimed at elucidating the mechanisms of extracellular elec-
130
tron transfer performed by Gram-positive bacteria that colonize
131
anodes, and will contribute to the understanding and improve-
132
ment of microbial electrochemical technologies such as MFCs.
133
Materials and methods
134
Bacterial strains and growth conditions
135
The sequence of the signal peptide derived from the small tetra-
136
heme cytochrome c(STC) from S. oneidensis MR-1 was used to
137
produce a chimeric gene with therJR_0333 gene from T. potens
138
[29], using the primers listed in Table 1 (NZYtech, Portugal). The
139
stcsp_FW primer was designed according to Shi et al. [31] to clone
140
efficiently the chimeric gene (named stcsp_therJR0333) into
141
pBAD202/D-TOPO vector (Invitrogen, USA). Cloning was performed
142
according to manufacturer specifications, and the final expression
143
vector (pCP01) was inserted into the E. coli strain JM109
144
(DE3) co-transformed with vector pEC86, which contains the
145
ccmABCDEFGH genes [32]. Positive transformants were used for
146
expression tests, where different media, induction times and indu-
147
cer concentrations were tested. Bug-buster reagent (Novagen, USA)
148
was used to check the best over-expression condition. The selected
149
condition was later used to over-express TherJR_0333. Briefly, the
150
transformants were grown in Terrific Broth (TB) with 50
l
gml
1
151
kanamycin and 34
l
gml
1
chloramphenicol in 5 L Erlenmeyer
152
flasks containing 2 L of medium and 1% of inoculum at 37 °C and
153
150 rpm. At mid-log phase (about 6 h of growth) protein expres-
154
sion was induced with 1 mM of arabinose, the temperature was
155
lowered to 30 °C and cells were allowed to grow for additional
156
16 h. Cells were harvested by centrifugation at 10 000gfor
157
10 min at 4 °C.
158
Protein purification
159
The cell pellet was suspended in an osmotic shock solution
160
(0.5 M sucrose, 0.2 M Tris–HCl, 0.5 mM EDTA and 100 mg L
1
lyso-
161
zyme, pH 7.6) containing protease inhibitor cocktail (Sigma) and
162
DNase I (Sigma) [33]. This mixture was incubated at 4 °C for
163
30 min with gentle stirring. The supernatant containing the
164
periplasmic fraction was cleared by ultracentrifugation at
165
20 000gfor 1 h at 4 °C, dialyzed overnight against 20 mM Tris–
166
HCl (pH7.6) and concentrated in an ultrafiltration cell with a
167
10 kDa cut-off membrane. The resulting sample was loaded onto
168
an ion exchange diethylaminoethyl (DEAE) column (GE
169
Healthcare) previously equilibrated with 10 mM Tris–HCl (pH
170
7.6). The resulting fractions were eluted with a salt gradient from
171
0 to 1 M KCl in the same buffer. The chromatographic fractions
172
were followed by UV–visible spectroscopy and SDS–page (12%)
173
to select those containing the protein of interest. The target pro-
174
tein, TherJR_0333, was eluted at 10 mM Tris–HCl. The fractions
175
containing TherJR_0333 were concentrated and analyzed by UV–
176
visible spectroscopy and SDS–PAGE (12% gel) to check for the pres-
177
ence of further contaminating proteins. Over time, the fraction
178
containing TherJR_0333 protein started to form a red precipitate.
179
Solubilization of this precipitate was achieved using 10 mM
180
potassium phosphate buffer (pH7.6) with 100 mM potassium
181
chloride and 0.05% of the non-ionic detergent n-Dodecyl-
182
b-maltopyranoside (DDM). Since the addition of the detergent
183
prevent further precipitation of the target protein, the later
184
characterization was performed with solubilized protein using
185
0.05% DDM. A single band on the SDS–Page gel confirmed the pur-
186
ity of the target protein, and the purity index of the sample was
187
defined by A
Soret peak
/A
280nm
ratio.
188
TherJR_0333 identification by mass spectrometry
189
The data were acquired in positive linear MS mode using a
190
4800plus MALDI-TOF/TOF (AB Sciex) mass spectrometer and using
Table 1
Oligonucleotides used to construct the chimeric gene.
stcsp_FW CACCTAAGAAGGAGATATACATCCCGTGAGCAAAAAACTATTAAG
therJR0333_RV GTGTTAAAAAGGCTACATAAATTTTCCTTGAAAAAGGTTATG
stc_therJR0333_FW CAACCGCATTTGCCACTGCTCCCGAGAAG
stc_therJR0333_RV CTTCTCGGGAGCAGTGGCAAATGCGGTTG
2N.L. Costa et al./ Protein Expression and Purification xxx (2015) xxx–xxx
YPREP 4668 No. of Pages 5, Model 5G
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Please cite this article in press as: N.L. Costa et al., Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium
capable of performing extracellular respiration, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.007

191
4000 Series Explorer Software v.3.5.3 (Applied Biosystems).
192
External calibration was performed using Promix3 (Laser BioLabs).
193
TherJR_0333 spectroscopic characterization
194
UV–visible spectroscopy
195
UV–visible spectra of solubilized TherJR_0333 in 10 mM potas-
196
sium phosphate buffer (pH 7.6) with 100 mM KCl and 0.05% DDM
197
were acquire on a Shimadzu UV-1800 spectrophotometer in the
198
range of 250–800 nm at room temperature. Reduced spectra were
199
obtained by adding an excess of sodium dithionite (Sigma) to the
200
oxidized sample. Protein concentration was estimated using the
201
absorption coefficient of
e
409
= 125 000 M
1
cm
1
per heme for
202
the oxidized state of the protein.
203
NMR spectroscopy
204
10% (v/v) of D
2
O was added to the protein sample prepared sol-
205
ubilized in 10 mM potassium phosphate buffer (pH 7.6) with
206
100 mM KCl and 0.05%DDM before spectral acquisition. NMR
207
experiments were performed on a Bruker AVANCE II 500 MHz
208
spectrometer equipped with a TXI probe. The
1
H NMR spectrum
209
was acquired at 25 °C with spectral width of 80 kHz, and processed
210
in the Topspin 3.2 software from Bruker using an exponential
211
apodization function.
212
Results and discussion
213
Multiheme c-type cytochromes are major components of elec-
214
tron transfer networks in metal reducing bacteria, being essential
215
for the extracellular electron transfer processes performed by these
216
organisms [3,8,13]. The decaheme cytochrome TherJR_0333 from
217
the Gram-positive bacterium T. potens JR was identified as a par-
218
ticipant in extracellular electron transfer in this bacterium [13],
219
perhaps playing a similar role to the periplasmic decaheme cyto-
220
chrome MtrA from S. oneidensis MR-1 [34]. The heterologous
221
expression of this protein was successfully achieved in the Gram-
222
negative E. coli.
223
The production of c-type cytochromes requires specific cell
224
machinery, such as the cytochrome cmaturation systems and
225
Sec translocation system that are generally different between
226
Gram-negative and Gram-positive bacteria. Protein BLAST (NCBI
227
NIH) reveals that both E. coli and T. potens JR have the same cyto-
228
chrome cmaturation system, which is the Ccm system I.
229
However, while E. coli uses Sec B as the major translocation system,
230
T. potens JR uses Sec A [35]. Thus, to ensure that the target protein
231
TherJR_0333 will be recognized by the E. coli SecB translocation
232
system the signal peptide of STC (SO_2727) was used [29,28].
233
Synthetic oligonucleotides were designed to contain the signal
234
peptide of STC from S. oneidensis MR-1, that it is known to be con-
235
stitutively expressed in high levels [29,36]. Although the use of the
236
STC signal peptide was previously shown to be highly efficient for
237
over-expression of c-type cytochromes [28,29], this is the first time
238
that it was used to over-express a multiheme c-type cytochrome
239
from a Gram-positive bacterium in a Gram-negative bacterium.
240
Protein expression was assessed on SDS–page followed by
241
heme-staining coloration to confirm the protein-bound heme.
242
Fig. 1A depicts the result of protein expression tests of E. coli
243
JM109 (DE3) co-transformed with pEC86 and TherJR_0333 with
244
native signal peptide (lanes 2 and 3) and TherJR_0333 with STC sig-
245
nal peptide (lanes 4 and 5) respectively. The appearance of a band
246
around 43 kDa, estimated to be the molecular weight of
247
TherJR_0333 with 10 hemes [13] was only observed when the
248
native signal peptide of TherJR_0333 was replaced by the STC sig-
249
nal peptide.
250
Pure protein was obtained by solubilization of the red precipi-
251
tate that was formed in the fraction eluted with 10 mM Tris–HCl
252
using 10 mM potassium phosphate buffer and 100 mM KCl. To pre-
253
vent protein precipitation 0.05% of DDM was added to the sol-
254
ubilized solution. Since the periplasmic space of Gram-positive
255
bacteria is narrower than that of Gram-negative bacteria and
256
crossed by several membrane bound polymers [10] it is likely that
257
the detergent mimics the native environment by creating micelles
258
that stabilize the target protein. In fact, little precipitate formation
259
was observed in the sample after the addition of DDM. Solubilized
260
recombinant protein gave a single heme-stained band on SDS–
261
PAGE gel between the 35 kDa and 48 kDa markers (Fig. 1B_lane
262
a). Coomassie blue confirmed the purity of sample by revealing
263
the absence of any other protein (Fig. 1B_lane b).
264
N-terminal sequencing (Fig. 2) confirms that Sec B from
265
E. coli recognizes and cleaves correctly the signal peptide of STC
Fig. 1. 12% SDS–Page of TherJR_0333. (A) Expression tests using TB medium: Lane 1 – E. coli JM109 with pEC86; Lane 2 – E. coli JM109 with pEC86 and JR0333_pBAD; Lane 3 –
E. coli JM109 with pEC86 and JR0333_pBAD with 1 mM arabinose; Lane 4 – E. coli JM109 with pEC86 and pCP01; Lane 5 – E. coli JM109 with pEC86 and pCP01 with 1 mM
arabinose (B) solubilized precipitate: Lane a – heme staining; Lane b – Comassie blue R. Arrows indicates the expressed TherJR_0333. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Diagram of heterologous protein containing the STC signal peptide and TherJR_0333 sequences. The cleavage site AFA-TA was confirmed by N-terminal analysis.
N.L. Costa et al./ Protein Expression and Purification xxx (2015) xxx–xxx 3
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Please cite this article in press as: N.L. Costa et al., Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium
capable of performing extracellular respiration, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.007

266
allowing the biogenesis of the Gram-positive c-type cytochrome
267
TherJR_0333 in the periplasmic space of a heterologous
268
Gram-negative expression system. Indeed, the expression of
269
TherJR_0333 using the native signal peptide (in JR0333_pBAD vec-
270
tor) did not yield the production of c-type cytochrome (Fig. 1A
271
_lanes 2 and 3). This result clearly indicates that the presence of a
272
recognizable signal peptide is essential for the heterologous expres-
273
sion of proteins when using identical translocation systems.
274
MALDI-TOF mass spectrometry analysis of the solubilized protein
275
revealed a molecular mass of 42 767.6 Da (Fig. 3). This value agrees
276
with the calculated molecular mass of 42.7 kDa considering the
277
incorporation of the 10 hemes and a molecular mass of 616.5 Da
278
per each heme.
279
The UV–visible spectra of solubilized recombinant TherJR_0333
280
are shown in Fig. 4. This protein exhibits the typical UV–visible
281
spectrum of a low spin hexacoordinated c-type cytochrome with
282
absorption peaks at 409 nm (Soret peak) and 530 nm in oxidized
283
form (solid line) and 416 nm (Soret), 525 nm (b-band) and
284
552 nm (
a
-band) in the reduced state (dashed lines) [37]. These
285
spectral changes confirm that the solubilized TherJR_0333 can
286
undergo oxidation/reduction processes. The pure solubilized pro-
287
tein displays a ratio of the Soret peak in the oxidized state with
288
the absorbance at 280 nm of 3.
289
The NMR spectrum of solubilized recombinant protein
290
TherJR_0333 shown in Fig. 5 exhibits the typical NMR signals of
291
a low spin c-type cytochrome, where peaks from the methyl
292
groups of the hemes are shifted to the paramagnetic field are seen
293
from 15 ppm to 40 ppm [38,39]. The polypeptide sequence of
294
TherJR_0333 reveals ten canonical heme binding motif CXXCH
295
and nine spare histidine residues, which means that not all the
296
ten hemes are bis-histidine coordinated. Since the aminoacid
297
sequence of TherJR_0333 also shows the presence of three
298
methionine, NMR experiments with reduced sample were per-
299
formed showing the characteristic fingerprint for His-Met coordi-
300
nated hemes with a peak around 3 ppm that corresponds to the
301
e
-CH3 protons of the iron coordinated methionine [40]. The NMR
302
spectrum obtained for the sample purified by precipitation is iden-
303
tical to the spectrum obtained for the soluble fraction (data not
304
shown).
305
Conclusion
306
Previously, only monoheme c-type cytochromes from Gram-
307
positive bacteria were heterologously expressed in a Gram-nega-
308
tive expression system using specific plasmids that contain the
309
pelB leader sequence [41,42]. In the present study, a different
310
approach was used, where the signal peptide of a constitutively
311
expressed periplasmic multiheme c-type cytochrome from
312
S. oneidensis MR-1, STC, was added to the N-terminus of the
313
decaheme periplasmic protein TherJR_0333 from the Gram-
314
positive T. potens JR. Using this approach we were able to over-
315
express a multiheme cytochrome from a Gram-positive bacterium
316
in a Gram-negative bacterium for the first time. This construct is
317
also highly versatile since it allows the over-expression of the
318
heterologous protein in both E. coli and S. oneidensis MR-1 [31].
319
This methodology creates the opportunity to study other c-type
320
cytochromes from Gram-positive bacteria, paving the way to study
321
electron transport conduits across bacterial cell walls of Gram-
322
positive bacteria.
323
Acknowledgments
324
The authors thank Bruno M. Fonseca and Isabel Pacheco for
325
helpful discussions during the performance of this work and
326
Ryan A. Melnyk for help with preparation of genomic DNA and
327
early expression trials. This work was supported by Fundação da
328
Ciência e Tecnologia (FCT) Portugal [Grants SFRH/BD/88664/2012
329
to Nazua Costa and SFRH/BPD/96952/2013 to Catarina Paquete].
330
The NMR spectrometers are part of The National NMR Facility, sup-
331
ported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/
Fig. 3. MALDI-TOF/MS spectrum of recombinant TherJR_0333.
Fig. 4. Reduced (dashed line) and oxidized (solid line) UV–visible spectra of
TherJR_0333. The sample concentration was 100
l
M in 10 mM potassium phos-
phate buffer (pH7.6) with 100 mM KCL and 0.05% DDM.
Fig. 5.
1
H NMR spectrum of TherJR_0333 at 25 °C in the oxidized form in 10 mM
phosphate buffer (pH7.6) with 100 mM KCl and 0.05% DDM at 25 °C.
4N.L. Costa et al./ Protein Expression and Purification xxx (2015) xxx–xxx
YPREP 4668 No. of Pages 5, Model 5G
19 March 2015
Please cite this article in press as: N.L. Costa et al., Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium
capable of performing extracellular respiration, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.007

332
0230/2012). Mass spectrometry data were obtained by the Mass
333
Spectrometry Laboratory, Analytical Services Unit, Instituto de
334
Tecnologia Química e Biológica. N-terminal sequencing was
335
obtained by the Analytical Laboratory, Analytical Services Unit,
336
Instituto de Tecnologia Química e Biológica, Universidade Nova
337
de Lisboa.
338
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Please cite this article in press as: N.L. Costa et al., Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium
capable of performing extracellular respiration, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.007