AEM Accepted Manuscript Posted Online 28 August 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01814-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
cAMP-CRP regulates the heparosan production in Escherichia coli Nissle 1917
2 3
Huihui Yan1,2, Feifei Bao1,2, Liping Zhao3, Yanying Yu1,2, Jiaqin Tang1,2, Xianxuan Zhou1,2,*
4 5 6
1. Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus,
7
Xuancheng 242000, China
8
2. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009,
9
China
10
3. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027，
11
China
12 13
* Address correspondence to Xianxuan Zhou. E-mail: [email protected]
14 15 16 17 18 19 20 21 22 1
23
ABSTRACT
24
Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis
25
of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is
26
a significant concern for the cost-effective synthesis of bioengineered heparin. The
27
carbon source is known as the pivotal factor affecting heparosan production. However,
28
the mechanism by which carbon sources control the biosynthesis of heparosan is
29
unclear. In this study, we found that the biosynthesis of heparosan was influenced by
30
different carbon sources. Glucose inhibits the biosynthesis of heparosan while the
31
addition of either fructose or mannose increases the yield of heparosan. Further study
32
demonstrated that the cAMP-CRP complex binds to the upstream of region 3
33
promoter, and stimulates the transcription of the gene cluster for heparosan
34
biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its binding
35
capability of CRP and eliminated the stimulative effect on transcription. The 1H NMR
36
analysis was further performed to determine the Escherichia coli Nissle 1917 (EcN)
37
heparosan structure and quantify the extracellular heparosan production. Our results
38
add to the understanding of the regulation of heparosan biosynthesis and may
39
contribute to the study of other exopolysaccharide producing strains.
40 41 42 43 44 2
45
INTRODUCTION
46
Heparosan is the starting carbon backbone for the chemoenzymatic synthesis of
47
heparin. Heparin is a widely used clinical anticoagulant drug with a worldwide
48
production exceeding 100 tons/year (1). Pharmaceutical heparin is currently produced
49
from the porcine intestinal mucosa through a long supply chain that posts a potential
50
risk of contaminants and adulteration (2). The worldwide outbreak of heparin
51
contamination crisis in 2008 underscores the vulnerability of heparin supply chain.
52
The in vitro chemoenzymatic synthesis of bioengineered heparin-like polysaccharide
53
has shown promise as an alternative approach to producing heparin from non-animal
54
source (3-5). The chemoenzymatic synthesis of heparin-like polysaccharide starts
55
from heparosan, comprised of a [(→4) β-D-glucuronic acid (GlcA) (1→4)
56
N-acetyl-α-D-glucosamine (GlcNAc) (1→)]n repeating disaccharide unit (Fig. 1A).
57
The
58
N-deacetylase/N-sulfotransferase,
59
6-O-sulfotransferase and 3-O-sulfotransferase to produce the fully elaborated heparin.
60
As the starting carbon backbone for the cost-effective synthesis of
61
bioengineered heparin, the availability of heparosan is a big concern (6-9). Heparosan
62
is extracted from the capsular polysaccharide (CPS) of E. coli K5, Pasteurella
63
multicida, and EcN (8, 10, 11). The proteins encoded by the kps locus govern the
64
biosynthesis and export of heparosan (Fig 1B). The kps locus comprises a
65
serotype-specific region 2 (kfiABCD) flanked by two conserved regions (region 1 and
66
3) (12, 13). Region 1 includes the genes of the kpsFEDUCS cluster, and region 3
backbone
is
then
further
modified
by
C5-epimerase,
3
the
enzymes
including
2-O-sulfotransferase,
67
includes kpsMT. The gene products encoded by kfiABCD are responsible for the
68
biosynthesis of heparosan (14). Translocation across the cytoplasmic membrane is
69
mediated by the products of kpsC, kpsS, and kpsMT while translocation across the
70
periplasm and outer membrane involves the KpsD and KpsE proteins (15). The kpsU
71
gene encodes for a functional CMP-Kdo synthetase, and the specific activity levels of
72
CMP-Kdo synthetase elevate at capsule-permissive temperatures (16, 17). KpsF
73
catalyzes the conversion of the pentose pathway intermediate Ru5P (D-ribulose
74
5-phosphate) into A5P (D-arabinose 5-phosphate), the precursor of Kdo (18).
75
A complex regulatory network controls the expression of the kps locus (Fig. 1C).
76
The kps locus is temperature-regulated, being expressed at 37 oC but not at 20 oC (19).
77
The IHF protein is required for maximum transcription from the region 1 promoter at
78
37 oC and binds to a single site located 130 bp 3’ to the transcription start point (19,
79
20). Three additional regulators including SlyA, BipA, and H-NS play a crucial role
80
in the temperature regulation of region 1 promoter (19, 21). The region 2 promoters
81
are weak and generate low levels of expression, which in the absence of
82
RfaH-mediated readthrough transcription from the region 3 promoter are insufficient
83
for the synthesis of detectable heparosan (22). The region 3 promoter is located 741
84
base pairs 5’ of the kpsM gene (23). The transcription of the region 3 promoter
85
proceeds through region 2 with the aid of the transcription antitermination factor
86
RfaH (23). The RfaH function is dependent on a short sequence present in the region
87
3
mRNA
known
as
the
JUMPStart
4
element
(just
upstream
of
many
88
polysaccharide-associated gene starts) (24). Besides the region 1 promoter, the region
89
3 promoter is also temperature-regulated via both SlyA and H-NS (25).
90
As a nonpathogenic probiotic strain without known toxins, EcN can be used as a
91
safe source of heparosan preparation. EcN carries all the genes for heparosan
92
biosynthesis and export. Although the above-mentioned studies have not been
93
performed on the kps locus of EcN, it is possible to investigate the heparosan
94
production in EcN based on the knowledge of the studied strains. Recent studies have
95
indicated that carbon sources have differential impacts on heparosan production. The
96
glycerol-defined medium allows a 3-fold increase of heparosan production than that
97
of LB medium (26). Another report has demonstrated that the glucose-defined
98
medium compares favorably to the glycerol-defined medium in heparosan yield (27).
99
However, the mechanism by which carbon sources control the biosynthesis of
100
heparosan is unclear.
101
In this study, we have found that glucose, fructose, and mannose have
102
discriminative impacts on the biosynthesis of heparosan. Further studies have
103
demonstrated that the expression of the region 3 promoter is regulated by the complex
104
of cyclic AMP (cAMP) and cAMP receptor protein (CRP). Gel shift assay shows that
105
the cAMP-CRP complex binds to a CRP binding motif in region 3 promoter. The
106
deletion of crp, the deletion of cya, and the base substitutions of the CRP binding site
107
dramatically decrease the expression from the region 3 promoter. Furthermore, the
108
yield of heparosan is apparently lower when glucose is used as the sole carbon source
109
than that of fructose and mannose. 5
110
MATERIALS AND METHODS
111
Bacterial strains, plasmids and culture conditions. Strains and plasmids used in
112
this study were listed in Table 1. E. coli strains were grown in Luria-Bertani (LB)
113
medium or on LB plates containing 1.5% agar. Polymerase chain reactions (PCR)
114
were performed on the Arktik Thermal Cycler (Thermo Fisher Scientific Inc.,
115
Waltham, MA, USA). The minimal medium (MM) was containing 2% carbon source
116
(glucose, fructose or mannose), 0.24 g/L MgSO4, 0.01 g/L CaCl2, 6 g/L Na2HPO4, 3
117
g/L KH2PO4, 0.5 g/L NaCl, and 1 g/L NH4Cl. The MM was used to culture EcN for
118
heparosan preparation. 1H NMR was performed on an Agilent VNMRS 600MHz
119
NMR Spectrometer (Agilent Technologies, Inc., USA). Unless otherwise stated,
120
glucose and cAMP were utilized at 0.8% and 10 mM, respectively. Ampicillin (Ap),
121
kanamycin (Kan), and chloramphenicol (Cm) were added to 50 μg/ml, 50 μg/ml, and
122
25 μg/ml when necessary. The chemicals were provided by Sangon Co. Ltd.,
123
Shanghai, China.
124
Plasmid construction. The low-copy-number vector pFZY1 was used to
125
construct the promoter fusion plasmids in this study (28). The region 3 promoter was
126
amplified by the primers 0011/0012 and inserted into pAH125 at the Kpn I- EcoR I
127
site to create pAH125-KpsMP. Then pAH125-KpsMP was cleaved with BamH I. The
128
purified DNA fragment carrying the region 3 promoter was cloned into the plasmid
129
pFZY1 to create pFZY1-kpsMP. Site-directed mutagenesis was used to create
130
pFZY1-KpsMPm that carried a mutated CRP binding site. Briefly, the primer sets
131
0060 and 0061 were used to introduce the mutant base pairs with pFZY1-KpsMP as 6
132
the template. Then the PCR products were digested with the enzyme Dpn I, purified,
133
and transformed into competent cells. The crp gene was cloned by the primers 0009
134
and 0010 to create pET28a-crp. The purified PCR product was digested and inserted
135
into pET28a(+) at the Nde I - Xho I site. The constructed plasmids were sequenced to
136
verify their integrity.
137
Gene
disruption
and
complement.
The
Red-mediated
homologous
138
recombination system was used to construct the in-frame deletions (29). A kanamycin
139
resistance cassette was amplified using pKD4 as template and the primers 0007/0008
140
and used to delete the lacZ gene in EcN. The PCR products were treated by the
141
enzyme Dpn I and introduced by electroporation into EcN containing the pKD46
142
expressed Red recombinase. Transformants were selected on LB plates supplemented
143
with kanamycin. The helper plasmid pKD46 was later cured by incubation at 42 oC.
144
In orde to construct the strain YHH1302 (EcN ΔlacZ), the kanamycin resistance
145
cassette of the strain YHH1301 (EcN ΔlacZ::Kan) was eliminated using pCP20 as
146
previously described (29). To construct the strain YHH1303 (EcN ΔlacZ Δcrp::Cm), a
147
chloramphenicol cassette was amplified from pKD3 using the primers 0013/0014.
148
Then the chloramphenicol cassette was introduced into YHH1302 (EcN ΔlacZ )
149
containing the plasmid pKD46. The strain YHH1304 (ZK126 Δcrp::Cm) and
150
YHH1305 (ZK126 Δcya::Cm) was created similarly using the chloramphenicol
151
cassette amplified from pKD3 with the primers 0013/0014 and 0056/0072,
152
respectively. To create the complementary strains, the DNA fragments carrying the
153
crp and cya gene were amplified with the primers 0017/0018 and 0058/0059. Then 7
154
the DNA products were purified and introduced into YHH1304/pKD46 and
155
YHH1305/pKD46. The complements, which grew faster than the isogenic crp/cya
156
mutants, were selected on the LB plates without any antibiotics. All the deletions and
157
complements were verified by PCR test.
158
Expression and purification of CRP. E. coli BL21(DE3) carrying the plasmid
159
pET28a-crp was grown in LB at 37 oC to an optical density of 0.6 at 600 nm, then
160
induced with 0.2 mM IPTG overnight at 22 oC. All subsequent procedures were
161
performed at 4 oC. The cells were harvested and resuspended in 30 ml of solution I
162
(20mM Tris-HCl, pH 7.6, 200mM NaCl). After the addition of 100 μM PMSF, the
163
cells were lysed by sonication. The lysate was centrifuged at 12000 rpm for 20min,
164
and the supernatant was applied to a nickel-NTA column. The column was washed
165
with 30 ml solution II (20mM Tris-HCl, pH 7.6, 200mM NaCl, 50mM imidazole).
166
CRP was eluted with a gradient of 50 to 250 mM imidazole in solution I. The
167
His-tagged CRP were dialyzed against solution I and stored at -80 oC until use. The
168
purity of CRP was analyzed by SDS-PAGE.
169
β-Galactosidase Assay. E. coli ZK126 was used as the wild-type (WT) strain.
170
ZK126 and its derivatives were used in the β-Galactosidase activity assays. Overnight
171
cultures of E. coli were diluted 1:100 into fresh LB medium. The cultures were
172
incubated at 37 oC with shaking at 250 rpm. At different time points during cell
173
growth, aliquots were removed for the determination of OD600 and β-Galactosidase
174
activity as previously described (30). The β-Galactosidase activity was expressed in
8
175
Miller units. All assays were performed in triplicate. The error bars in the graphs
176
indicated the standard deviations.
177
Gel shift assay. The double-stranded region 3 promoter fragments containing
178
the WT/mutated CRP binding motif were produced by boiling and slowly cooling the
179
synthetic DNA oligonucleotides 0080/0081 and 0082/0083 (Table 2). The DIG gel
180
shift kit (Roche Ltd., Mannheim, Germany) was used for DNA labeling and signal
181
detection. A DNA fragment without the CRP binding motif was used as the
182
competitive probe (primers 0004/0084). The labeled DNA fragments (1.6 nM) were
183
incubated with various amounts of purified CRP at 37 oC for 10 min in the
184
CRP-binding buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1 mM EDTA; 1 mM
185
DTT ; 50 μg/ml BSA; 100μM cAMP; and 160 nM of the competitive DNA probe).
186
The formed DNA-protein complexes were separated by 8% PAGE in 1 × TBE buffer
187
containing 100 μM cAMP.
188
Preparation of heparosan from liquid cultures. The overnight cultures of
189
EcN and E. coli K5 were expanded into fresh medium, shaked at 250 rpm, 37 oC for
190
variable times. The supernatant of bacterial cultures was recovered by centrifugation,
191
filtered
192
vacuum evaporator. Heparosan in the supernatant was precipitated with 3 volumes of
193
ethanol at -20 oC overnight and pelleted by centrifugation at 12000 rpm for 15 min at
194
4 oC. After washing with 75% ethanol, the heparosan was resuspended in deionized
195
water. The phenol/chloroform extraction was performed to remove proteins. Then, the
196
heparosan sample was dialyzed using the Spectra/Por dialysis membrane (MWCO 10
through
a
0.45-µm
membrane,
9
and
concentrated
by
a
rotary
197
kDa) against Buffer A (20 mM NaAcO, 50 mM NaCl, pH4) and applied to
198
DEAE-Sepharose column (1.6 X 50 cm) purification. The sample was loaded into the
199
column at 1 ml/min. After loading of heparosan, the column was washed with 10
200
column volumes of Buffer A at 3 ml/min. Then heparosan was eluted at 3 ml/min
201
with Buffer B (20 mM NaAcO, 1 M NaCl, pH4). Fractions containing heparosan were
202
pooled, dialyzed against water, and freeze-dried. The purified heparosan was stored at
203
-80 oC for later use.
204
Preparation of heparosan from bacteria grown on the MM plates. The
205
extracellular capsular polysaccharide from EcN was purified using the method
206
described previously with modifications (31). The overnight cultures of EcN were
207
diluted with fresh medium, and the 100 μl of aliquots were plated onto the MM plates
208
supplemented with the specific carbon source. The plates were incubated at 37 oC for
209
24 h for the bacteria to biosynthesize and translocate heparosan. Then, cells were
210
harvested and resuspended in the PBS buffer. To restrain the cell breakage and release
211
of large amounts of chemical compounds, we gently extracted heparosan by shaking
212
the cell resuspension solution overnight at 100 rpm, 4 oC. Then, the cell suspension
213
was centrifuged at 12000rpm for 10min at 4 oC. Heparosan in the supernatant was
214
precipitated with 3 volumes of ethanol at -20 oC overnight and pelleted by
215
centrifugation at 12000 rpm for 15 min at 4 oC. After washing with 75% ethanol, the
216
heparosan was dried and resuspended in deionized water. DNA in the heparosan
217
suspension was degraded by the addition of 20 units/ml Dpn I. Then proteinase K was
218
added at a final concentration of 400 μg/ml and the suspension was incubated at 37 oC 10
219
overnight. Following the phenol/chloroform extractions, the CPS preparation was
220
dialyzed using the Spectra/Por dialysis membrane (MWCO 10 kDa) against deionized
221
water. Then the sample was freeze-dried and stored at -80 oC for later use.
222
PAGE analysis of the capsular polysaccharide. The isocratic large-slab
223
PAGE was cast as previously described (6). A total of 45 μl sample plus 5 μl loading
224
buffer were loaded into each well. The gel electrophoresis was performed at 120V for
225
5h. Following electrophoresis, the gel was gently shaken at room temperature in the
226
washing buffer (10% acetic acid and 25% ethanol) for 30min. After staining with a
227
solution of Alcian blue (0.5%) in acetic acid (2%) for 30min, the gel was destained in
228
the washing buffer until the gel background was transparent.
229
1
H NMR analysis. The
1
H NMR analysis was performed as described
230
previously with sodium terephthalate as an internal standard for the heparosan
231
quantification (7). The CPS preparations were lyophilized and dissolved in 0.5 ml
232
D2O, and this step was repeated two times. Then the lyophilized CPS was redissolved
233
in 0.5 ml of D2O containing 71 μg of sodium terephthalate before being transferred to
234
a 5-mm NMR tube. 1H NMR was performed on an Agilent VNMRS 600MHz NMR
235
Spectrometer (Agilent Technologies, Inc., USA), and acquisition of the spectra was
236
carried out using VnmrJ Rev. 3.2A software. The 1H NMR spectra were processed in
237
MestReNova software. The integration of the peaks was performed using the
238
“integration” function, with the peak area being selected manually.
239 240 11
241
RESULTS
242
The biosynthesis of heparosan is affected by catabolite repression. To evaluate the
243
impact of carbon sources on heparosan production, we have extracted heparosan from
244
EcN grown on the MM plates. The medium has been supplemented with 2% of
245
glucose, fructose or mannose as the sole carbon source. The overnight bacterial lawns
246
have been scraped off, and heparosan is extracted according to the method described
247
in Materials and Methods. The amount of heparosan is checked by PAGE
248
electrophoresis. As shown in Fig. 2A, the yields of heparosan from bacteria using
249
fructose and mannose versus glucose as the carbon source are obviously higher.
250
Glucose is well known to affect gene expression through the cAMP-CRP
251
complex (32, 33). Thus, we set out to check the sequence of the kps locus and find a
252
potential CRP binding site located in the region 3 promoter (Fig. 1C). To study
253
whether cAMP-CRP is involved in the regulation of heparosan biosynthesis, we have
254
constructed the Δcrp::Cm strain and a region 3 promoter-lacZ fusion plasmid. The
255
deletion of crp gene has dramatically dropped the expression from region 3 promoter
256
(Fig. 2B). On the other hand, the addition of 0.8% glucose to LB medium
257
significantly inhibits the expression from region 3 promoter, and the corresponding
258
β-galactosidase activity is halved. However, the addition of 0.8% glucose plus 10 mM
259
cAMP offsets the decreased β-galactosidase activity. These results suggest that the
260
expression of the region 3 promoter is downregulated by the addition of glucose and
261
stimulated by both cAMP and CRP.
12
262
The complementary crp and cya genes restore the expression from region 3
263
promoter. To further study the regulatory relationship between cAMP-CRP and the
264
region 3 promoter, the Δcrp::Cm, Δcya::Cm, and the corresponding complementary
265
strains are constructed. All the in-frame deletions and the complementary
266
manipulations are carried out with the help of the λ Red homologous recombination
267
system (29). The in-frame deletion of crp and cya gene results in a clearly decreased
268
growth ability (Fig. 3AB and Fig. 4AB). The crp and cya gene are amplified with
269
EcN chromosome as template and utilized to complement the in-frame deletions of
270
both the Δcrp::Cm and Δcya::Cm constructs. The complements with restored
271
doubling time exhibit as the bigger colonies on the LB plate than other ones in the
272
bacteria lawn. The complements are selected and repurified on the LB plate without
273
any antibiotics. The in-frame deletions and complementary strains are verified via
274
colony PCR analysis (Fig. 3C and 4C).
275
Complementary experiments are conducted further to verify the stimulation of
276
expression from region 3 promoter by the cAMP-CRP complex. The region 3
277
promoter-lacZ fusion plasmid is transformed into WT, Δcrp::Cm, Δcya::Cm, and the
278
complementary strains including Δcrp(comp) and Δcya(comp). The β-galactosidase
279
activity expressed in Miller Units is determined to evaluate the impacts of different
280
genotypes on the expression from region 3 promoter. As shown in Fig. 5A, the
281
expression from region 3 promoter is significantly inhibited in the Δcrp::Cm strain,
282
and that of the crp complementary strain recovers to the level of the WT strain.
283
In-frame deletion of cya will eliminate the intracellular cAMP, leading to the loss 13
284
function of the cAMP-CRP complex (33). Similar to the phenotype of the Δcrp::Cm
285
strain, the deletion of cya gene eliminates the transcription from region 3 promoter
286
(Fig. 5B). The β-galactosidase activity of the cya complementary strain restores to the
287
level of the WT strain.
288
The cAMP-CRP complex directly binds to a CRP binding motif in the
289
region 3 promoter and stimulates the expression of this operon. The cAMP-CRP
290
complex controls gene transcription via binding to the consensus CRP binding site (34,
291
35). The region 3 promoter is located 741 base pairs 5’ of the kpsM gene (23). In the
292
region 3 promoter, there is a potential CRP binding site (TGTGAtataaaTCACA)
293
located 487 base pairs 5’ of the kpsM gene (Fig. 6A). To determine whether the
294
cAMP-CRP complex directly modulates the expression of region 3 promoter via the
295
potential CRP binding site, we have performed gel shift assays. Our results reveal that
296
cAMP-CRP directly binds to the DNA fragment of region 3 promoter in a
297
dose-dependent manner. However, the DNA fragment with a mutant CRP binding site
298
(cGatctataaaTtcgc) loses the ability to form complex with cAMP-CRP (Fig. 6BC).
299
Further studies are performed to verify the essentiality of the CRP binding site
300
for the expression from the region 3 promoter. The CRP binding motif of region 3
301
promoter is base-substituted, and the β-galactosidase activity is determined. As shown
302
in Fig. 6D, the region 3 promoter carrying a mutated CRP binding site fails to express
303
LacZ and exhibits no substantial β-galactosidase activity when compared to that of
304
the WT region 3 promoter. This result is consistent with the deficiency of
305
β-galactosidase activities in the Δcrp::Cm and Δcya::Cm strains that carry a WT 14
306
region 3 promoter-lacZ fusion plasmid. These results indicate a requirement for the
307
CRP binding site in the cAMP-CRP mediated activation.
308
The substitution of selective hexose for glucose increases the yield of
309
heparosan. As mentioned above, our data has clearly demonstrated that both the
310
consensus CRP binding site and the cAMP-CRP complex are essential for the
311
expression of genes regulated by the region 3 promoter. To further confirm the impact
312
of the global regulator CRP on the biosynthesis of heparosan, the CPS is extracted
313
from the wild-type EcN and its isogenic crp mutant grown on glucose-defined MM
314
plates. Both the PAGE electrophoresis and 1H NMR are performed to analyze the
315
extracted heparosan. As shown by the PAGE electrophoresis, there is a substantial
316
amount of heparosan purified from the wild-type EcN, but the heparosan extracted
317
from the isogenic crp mutant is not detectable (Fig. 7A). Furthermore, the 1H NMR
318
spectrum for the wild type EcN heparosan is similar to the previously published
319
spectra for K5 polysaccharide (Fig. 7B) (7, 36, 37). The peak at 2.04 ppm,
320
corresponding to the methyl protons in N-acetyl groups of heparosan, is clearly shown
321
for the EcN heparosan. These results from both the PAGE electrophoresis and 1H
322
NMR analysis consolidate the conclusions that biosynthesis of heparosan is
323
stimulated by the cAMP-CRP complex.
324
To further address the impacts of different hexoses on the biosynthesis of
325
heparosan, shake flask experiments are performed. The liquid MM supplemented with
326
glucose, fructose, and mannose as the sole carbon source is used to culture the
327
wild-type EcN and E. coli BL21. The cultures are shaked at 250 rpm, 37 oC to an 15
328
optical density of 1.0 at 600 nm. The heparosan in the supernatant of bacterial cultures
329
is purified with a DEAE-Sepharose column. The supernatant of E. coli BL21 culture
330
is purified in the same way as that of EcN and used as a negative control. 1H NMR is
331
utilized to analyze the purified EcN heparosan (Fig. 8A). Heparosan purified from E.
332
coli K5 (50 μg, 100 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg) is used to develop the
333
standard curve (Fig. 8B). Sodium terephthalate is selected as a water-soluble, stable,
334
and nonreactive internal standard for the heparosan quantification as previously
335
described (7). The N-acetyl peak (2.04 ppm) area is selected and normalized to the
336
sodium terephthalate peak area. The yields of heparosan in the sugar-defined MM
337
cultures are determined (Fig. 8C). The addition of glucose results in 5 mg/L of
338
heparosan production, lower than that of fructose (13 mg/L)and mannose (10 mg/L )
339
(Fig. 8C).
340 341
DISCUSSION
342
Pharmaceutical heparin produced from porcine intestinal mucosa bears the potential
343
risk of contaminants and adulteration (2). The in vitro chemoenzymatic synthesis of
344
bioengineered heparin and oligosaccharides has shown promise as an alternative
345
approach to producing the anticoagulant drug from non-animal source (3-5). As the
346
starting material for the cost-effective synthesis of novel anticoagulant drugs, the
347
availability of heparosan is a significant concern (6-8). Recent studies show that
348
carbon sources have differential impacts on the yield of heparosan (26, 27). However,
349
the mechanism by which carbon sources control the biosynthesis of heparosan is 16
350
unclear. Furthermore, both the well-known probiotic (EcN) and urinary tract pathogen
351
(E. coli K5) share their extracellular CPS composed of heparosan as the molecular
352
camouflage for host colonization (38, 39). Understanding the regulation mechanism
353
of heparosan biosynthesis will also contribute to the study on the resistance of the host
354
to pathogens.
355
Our study shows that the yield of heparosan from EcN is decreased when
356
utilizing glucose as the sole carbon source (Fig. 2A). Glucose is known to affect gene
357
expression through cAMP-CRP (32, 33). The cAMP-CRP complex binds to the
358
consensus CRP binding motif in the promoter region and affects the affinity of RNA
359
polymerase for the promoter DNA. The addition of glucose (Glc) causes the
360
dephosphorylation of glucose specific phospho- enzyme II A (P-EIIAGlc) (33). The
361
dephosphorylation process deactivates adenylate cyclase and hence lowers the
362
intracellular concentration of cAMP and the cAMP-CRP complex.
363
To address the mechanism of glucose inhibition of heparosan biosynthesis, we
364
have deleted the gene crp, cya, and constructed a region 3 promoter-lacZ fusion
365
plasmid. The promoter-lacZ fusion plasmid derives from a low-copy-number plasmid,
366
pFZY1 and carries a region 3 promoter. As shown by the β-galactosidase activity
367
assay, the addition of 10 mM cAMP offsets the decreased β-galactosidase activity
368
caused by glucose inhibition (Fig. 2B). Furthermore, the deletion of crp and cya have
369
blocked the expression of β-galactosidase from the region 3 promoter (Fig. 5). The
370
in-frame deletion of crp and cya are complemented by Red-mediated homologous
371
recombination (Fig. 3 and Fig. 4). The β-galactodidase activity of the complementary 17
372
strains is restored to that of the WT strain (Fig. 5). These results clearly demonstrate
373
that the expression from region 3 promoter is positively regulated by the cAMP-CRP
374
complex.
375
In general, cAMP-CRP binds to a palindromic sequence in which two
376
conserved motifs, TGTGA, and TCACA, are separated by a 6 bp spacer (40, 41). We
377
have performed gel shift assay to determine whether cAMP-CRP directly stimulates
378
the transcription from region 3 promoter upon binding. We have compared the
379
cAMP-CRP
380
(TGTGAtataaaTCACA)
381
(cGatctataaaTtcgc). As shown in Fig. 6C, the cAMP-CRP complex directly binds to
382
the wild type CRP binding motif in a dose-dependent manner, and the base
383
substitutions in the mutant CRP binding motif abolish its binding ability. Meanwhile,
384
the mutant CRP binding motif results in a failed transcription from region 3 promoter
385
as shown by the dramatically decreased β-galactosidase activity (Fig. 6D).
binding
capability with
of that
the of
wild the
type
CRP
binding
motif
mutant
CRP
binding
motif
386
Heparosan production is further analyzed by the PAGE electrophoresis and 1H
387
NMR. The biosynthesis of heparosan is significantly inhibited by the in-frame
388
deletion of crp gene (Fig. 7A). These results further consolidate our conclusion that
389
the binding of cAMP-CRP to the consensus CRP site is essential for the transcription
390
from region 3 promoter. Shake flask expriments are further performed to evaluate the
391
yield of heparosan in sugar-defined MM cultures. EcN and E. coli BL21 are
392
inoculated into MM and shaked at 250 rpm, 37 oC to an optical density of 1.0 at 600
393
nm. EcN heparosan in the supernatant is purified with a DEAE-Sepharose column and 18
394
analyzed with 1H NMR. The addition of glucose results in a lower yield of heparosan
395
(5 mg/L versus 10-13 mg/L) than that of fructose and mannose (Fig. 8C). Since the
396
uptake of glucose lowers the intracellular concentration of cAMP, the decreased
397
production of heparosan is reasonable. In the stationary phase, the depletion of
398
glucose will result in the increase in the intracellular cAMP concentration and
399
influence the heparosan production.
400
In summary, we have shown that the binding of cAMP-CRP to the consensus
401
CRP binding site is essential for the expression of region 3 operon. The deletion of
402
crp, cya, and the mutation of the consensus crp binding site inhibit the expression
403
from region 3 promoter and prevent the biosynthesis of heparosan. Glucose has an
404
adverse impact on intracellular cAMP concentration and the production of heparosan.
405
We have aligned the region 3 promoter of group 2 strains including EcN, E. coli K5,
406
E. coli UTI89 (O18:K1:H7), and E. coli K4 (O5:K4:H4) (39). EcN is a probiotic
407
bacterium without any known toxins while the other three bacteria are pathogenic
408
strains. These strains have a highly conserved region 3 promoter with a >95%
409
sequence identity while that of EcN and K5 is over 99%. Both the CRP binding motif
410
and JUMPstart sequence locate in the region 3 promoter of these four strains. These
411
results indicate the group 2 bacteria might use common mechanisms to escape the
412
host elimination and enhance host colonization.
413 414 415 19
416
ACKNOWLEDGMENTS
417
This study was funded by a Hefei University of Technology startup fund
418
(407-037064). We have no conflict of interest to declare.
419 420
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421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454
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Liu H, Zhang Z, Linhardt RJ. 2009. Lessons learned from the contamination of heparin. Nat Prod Rep 26:313-321.
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Finke A, Jann B, Jann K. 1990. CMP-KDO-synthetase activity in Escherichia coli
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Finke A, Bronner D, Nikolaev AV, Jann B, Jann K. 1991. Biosynthesis of the Escherichia
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Rowe S, Hodson N, Griffiths G, Roberts IS. 2000. Regulation of the Escherichia coli K5 capsule gene cluster: evidence for the roles of H-NS, BipA, and integration host factor in regulation of group 2 capsule gene clusters in pathogenic E. coli. J Bacteriol 182:2741-2745.
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Simpson DA, Hammarton TC, Roberts IS. 1996. Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster. J Bacteriol 178:6466-6474.
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Corbett D, Bennett HJ, Askar H, Green J, Roberts IS. 2007. SlyA and H-NS regulate transcription of the Escherichia coli K5 capsule gene cluster, and expression of slyA in Escherichia coli is temperature-dependent, positively autoregulated, and independent of H-NS. J Biol Chem 282:33326-33335.
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Doyle RJ. 2002. Glycomicrobiology. Kluwer Academic Publishers, New York.
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Stevens MP, Clarke BR, Roberts IS. 1997. Regulation of the Escherichia coli K5 capsule
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Hobbs M, Reeves PR. 1994. The JUMPstart sequence: a 39 bp element common to several
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Xue P, Corbett D, Goldrick M, Naylor C, Roberts IS. 2009. Regulation of Expression of
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Ebright RH, Ebright YW, Gunasekera A. 1989. Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site. Nucleic Acids Res 17:10295-10305.
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Vann WF, Schmidt MA, Jann B, Jann K. 1981. The Structure of the Capsular Polysaccharide (K5 Antigenn) of Urinary-Tract-Infective Escherichia coli 010:K5:H4. Eur J Biochem 116:359-364.
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Zhang C, Liu L, Teng L, Chen J, Liu J, Li J, Du G, Chen J. 2012. Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor. Metab Eng 14:521-527.
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Hafez M, Hayes K, Goldrick M, Warhurst G, Grencis R, Roberts IS. 2009. The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction. Infect Immun 77:2995-3003.
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Kolb A, Busby S, Buc H, Garges S, Adhya S. 1993. Transcriptional regulation by cAMP
41.
Pyles EA, Lee JC. 1998. Escherichia coli cAMP Receptor Protein-DNA Complexes. 2.
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Connell N, Han Z, Moreno F, Kolter R. 1987. An E. coli promoter induced by the cessation
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530 531 532 533 534 22
535 536
TABLE 1
Strains and plasmids used in this study
Strain or plasmid
Characteristics
Source
E.coli strains BL21
Expression host
Takara
Nissle 1917
Wild type
DSMZ
K5
Wild type
ATCC
ZK126
W3110 Δlac tna-2
(42)
YHH1301
EcN ΔlacZ::Kan
This work
YHH1302
EcN ΔlacZ
This work
YHH1303
EcN ΔlacZ Δcrp::Cm
This work
YHH1304
ZK126 Δcrp::Cm
This work
YHH1305
ZK126 Δcya::Cm
This work
YHH1306
ZK126 Δcrp(comp)
This work
YHH1307
ZK126 Δcya(comp)
This work
Plasmid pFZY1
galK′-lacZYA transcriptional fusion vector, Ap
(28)
pFZY1- KpsMP
pFZY1 carrying the region 3 promoter with a CRP binding
This work
pFZY1- KpsMPm
pFZY1 carrying the region 3 promoter with the mutated CRP
motif, Ap This work
binding motif, Ap pET28a-crp
pET28a(+) derivative for CRP expression, Kan
537 538 539 540 541 542 543 544 545 546 23
This work
547 548 549 550
551 552 553
TABLE 2
Oligonucleotide primers used in this study
Name
Sequencea,b
0007
ATGAGCGGATAACAATTTCACACAGGATACAGCTATGACGTGTAGGCTGGAGCTGCTTC
0008
TACGCGAAATACGGGCAGACATAGCCTGCCCGGTTATTAATGGGAATTAGCCATGGTCC
0009
GGAATTCCATATGGTGCTTGGCAAACCGCAAAC
0010
TCCGCTCGAGTTAACGAGTGCCGTAAAGCA
0011
ACGGGGTACCGTATTGCCATTTCCTTAACCCCA
0012
ATCGGAATTCTTGATGATGTGATCCTAATCTCTTC
0013
ATGGTGCTTGGCAAACCGCAAACAGACCCGACTCTCGAACTGTCAAACATGAGAATTAA
0014
TTAACGAGTGCCGTAAACGACGATGGTTTTACCGTGTGCGTGTAGGCTGGAGCTGCTTC
0017
AGGAGACACAAAGCGAAAGC
0018
CGTTTATGAGGCGTATCAAGG
0060
TTTAAAcGatcTATAAATtcgcAATATGGCTGTAAAGAGGGGGC
0061
TTACAGCCATATTgcgaATTTATAgatCgTTTAAATGGTGTTATTTAAGTCGCA
0056
TTGTACCTCTATATTGAGACTCTGAAACAGAGACTGGATATGGGAATTAGCCATGGTCC
0072
TCACGAAAAATATTGCTGTAATAGCGGCGTATCGTGATCGTGTAGGCTGGAGCTGCTTC
0058
ATAAACGGTGCTACACTTGTATGTA
0059
GCAAAATCATTATCAACCGC
0080
ATAACACCATTTAAATGTGATATAAATCACAAATATGGCTGT
0081
CTTTACAGCCATATTTGTGATTTATATCACATTTAAATGGTG
0082
ATAACACCATTTAAAcGatcTATAAATtcgcAATATGGCTGT
0083
CTTTACAGCCATATTgcgaATTTATAgatCgTTTAAATGGTG
0084
TCTTCATCTCCGGTTCTGCTGGCGGAGGTGGATCTGGCGGAGGTGGATCGG
0004
CCGATCCACCTCCGCCAGATCCACCTCCGCCAGCAGAACCGGAGATGAAGA
a
The underline sequences anneal to the template plasmids while the remaining sequences correspond to the ends
of the deleted genes. b
The boxed-in sequences represent the CRP binding motif while the base substitutions are shown in lowercases.
554 555 556 557 558 559 24
560 561
FIGURE LEGENDS
562 563
FIG 1 The structure of heparosan and the transcriptional organization of the
564
kps locus. (A) The structure of a native chain of heparosan. Heparosan is comprised of
565
a [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc)
566
(1→)]n repeating disaccharide unit. The average value of n is about 70. (B) The
567
transcriptional organization of the kps locus. Both the biosynthesis and export of
568
heparosan are carried out by the kps-locus-coded proteins. The transcription start
569
points are indicated by broken arrows. The horizontal arrows show the primary
570
transcripts from region 1 and region 3. The region 2 promoters are weak and
571
insufficient for the synthesis of detectable heparosan. (C) DNA sequence of the region
572
3 promoter. The broken arrow marks the transcriptional start site (23). The stop codon
573
of upstream gspM gene, -10 sequence, Shine-Dalgarno (SD) sequence, and kpsM start
574
codon are denoted by thick underlining. The JUMPstart sequence is indicated by
575
double underline, and the ops sequence (shaded box), which is essential for the action
576
of RfaH, is contained within JUMPstart (23). The H-NS binding regions are
577
underlined, and the SlyA binding region is indicated by shaded box (25). The putative
578
CRP binding motif is shown in an unshaded box.
579 580 581
FIG 2
Glucose has a negative effect on heparosan expression. (A) PAGE
analysis of the capsular polysaccharides extracted from EcN grown on MM plates 25
582
with different carbon sources. Lane 1, glucose; Lane 2, fructose; Lane 3, mannose. (B)
583
Effects of glucose, cAMP, and CRP on the transcription from region 3 promoter. Both
584
E.coli ZK126 (wild type) and the isogenic crp mutant are carrying the promoter
585
fusion plasmid pFZY1-KpsMP. The strains are grown in LB, LB plus 0.8% glucose,
586
or LB plus 0.8% glucose and 10mM cAMP. At different time points during cell
587
growth, aliquots are collected for the measurement of OD600 (squares and triangles)
588
and β-galactosidase activity (bars).
589
FIG 3
Complement of the in-frame deletion of crp. (A) The growth curve of
590
ZK126 (WT), crp mutant, and the complemented strain Δcrp(comp). (B) The
591
doubling time of ZK126 (WT), crp mutant and Δcrp(comp). (C) PCR verification of
592
the disruption and complement of crp gene. Lane 1, the DNA ladder; lane 2, ZK126
593
(WT); lane 3, Δcrp::Cm; lane 4, Δcrp(comp).
594 595
FIG 4
Complement of the in-frame deletion of cya. (A) The growth curve of
596
ZK126(WT), cya mutant and Δcya(comp). (B) The doubling time of ZK126(WT), cya
597
mutant and Δcya(comp). (C) PCR verification of the disruption and complement of
598
the cya gene. Lane 1, the DNA ladder; lane 2, ZK126 (WT); lane 3, Δcya::Cm; lane 4,
599
Δcya(comp).
600 601
FIG 5 The complement crp (A) and cya (B) recover the expression from
602
region 3 promoter. The cells carrying pFZY1-KpsMP are grown in LB medium at 37 26
603
o
604
determination of growth curve (squares, triangles, and circles) and β-galacotsidase
605
activity (bars).
C with shaking at 250 rpm. At different time intervals, the aliquots are taken for the
606 607
FIG 6 The cAMP-CRP complex binds to a CRP binding motif and stimulates
608
the transcription from region 3 promoter. (A) The DNA fragment of region 3
609
promoter carries a CRP binding motif located 487 base pairs 5’ of the kpsM gene. (B)
610
The DNA fragment carries a mutant CRP binding motif with base substitutions shown
611
in lowercase. The conserved base pairs of CRP binding motif is randomly mutated.
612
(C) Gel shift assays of the DNA fragments containing the WT/mutant CRP binding
613
motif. Variant amounts of the purified CRP protein (0-80 nM) are utilized to bind to
614
the digoxigenin-labeled DNA fragments with the present of 100 μM cAMP. The
615
arrowhead denotes the cAMP-CRP-DNA complex. (D) The β-galactosidase activity
616
assays of the region 3 promoter carrying a WT/mutant CRP binding motif. The lined
617
squares and triangles represent the growth curves; The bars indicate the
618
β-galactosidase activity.
619 620
FIG 7
Heparosan is not detectable in the isogenic crp mutant of EcN. (A)
621
PAGE analysis of the heparosan extracted from EcN (lane 1) and its isogenic crp
622
mutant (lane 2). The bacteria are grown on the glucose-defined MM plates at 37 oC
623
for 24 h. Then the cells are harvested from plates and heparosan is extracted. (B) The
27
624
1
625
on the glucose-defined MM plates.
H NMR analysis of EcN heparosan. The heparosan is extracted from EcN cultured
626 627
FIG 8 Analysis of heparosan purified from the shake flask cultures. EcN and
628
E. coli BL21 are inoculated into MM and shaked at 250 rpm, 37 oC to an optical
629
density of 1.0 at 600 nm. EcN heparosan in the supernatant is precipitated with
630
ethanol and purified with a DEAE-Sepharose column (1.6 X 60 cm) as described in
631
Materials and Methods. The supernatant of E. coli BL21 culture is purified in the
632
same way as that of EcN and used as a negative control. (A) The 1H NMR spectra of
633
the purified EcN heparosan from shake flask cultures is compared with that of E. coli
634
BL21. (B) Standard curve for the quantification of heparosan. E. coli K5 is grown in
635
LB medium at 250 rpm, 37 oC for 20 h. Heparosan in the supernatant is purified with
636
a DEAE-Sepharose column and used as standards. (C) The yield of purified EcN
637
heparosan in MM supplemented with different carbon sources. The 1H NMR analysis
638
is performed with sodium terephthalate as an internal standard for the heparosan
639
quantification.
28
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6
Fig 7
Fig 8
1
cAMP-CRP regulates the heparosan production in Escherichia coli Nissle 1917
2 3
Huihui Yan1,2, Feifei Bao1,2, Liping Zhao3, Yanying Yu1,2, Jiaqin Tang1,2, Xianxuan Zhou1,2,*
4 5 6
1. Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus,
7
Xuancheng 242000, China
8
2. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009,
9
China
10
3. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027，
11
China
12 13
* Address correspondence to Xianxuan Zhou. E-mail: [email protected]
14 15 16 17 18 19 20 21 22 1
23
ABSTRACT
24
Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis
25
of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is
26
a significant concern for the cost-effective synthesis of bioengineered heparin. The
27
carbon source is known as the pivotal factor affecting heparosan production. However,
28
the mechanism by which carbon sources control the biosynthesis of heparosan is
29
unclear. In this study, we found that the biosynthesis of heparosan was influenced by
30
different carbon sources. Glucose inhibits the biosynthesis of heparosan while the
31
addition of either fructose or mannose increases the yield of heparosan. Further study
32
demonstrated that the cAMP-CRP complex binds to the upstream of region 3
33
promoter, and stimulates the transcription of the gene cluster for heparosan
34
biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its binding
35
capability of CRP and eliminated the stimulative effect on transcription. The 1H NMR
36
analysis was further performed to determine the Escherichia coli Nissle 1917 (EcN)
37
heparosan structure and quantify the extracellular heparosan production. Our results
38
add to the understanding of the regulation of heparosan biosynthesis and may
39
contribute to the study of other exopolysaccharide producing strains.
40 41 42 43 44 2
45
INTRODUCTION
46
Heparosan is the starting carbon backbone for the chemoenzymatic synthesis of
47
heparin. Heparin is a widely used clinical anticoagulant drug with a worldwide
48
production exceeding 100 tons/year (1). Pharmaceutical heparin is currently produced
49
from the porcine intestinal mucosa through a long supply chain that posts a potential
50
risk of contaminants and adulteration (2). The worldwide outbreak of heparin
51
contamination crisis in 2008 underscores the vulnerability of heparin supply chain.
52
The in vitro chemoenzymatic synthesis of bioengineered heparin-like polysaccharide
53
has shown promise as an alternative approach to producing heparin from non-animal
54
source (3-5). The chemoenzymatic synthesis of heparin-like polysaccharide starts
55
from heparosan, comprised of a [(→4) β-D-glucuronic acid (GlcA) (1→4)
56
N-acetyl-α-D-glucosamine (GlcNAc) (1→)]n repeating disaccharide unit (Fig. 1A).
57
The
58
N-deacetylase/N-sulfotransferase,
59
6-O-sulfotransferase and 3-O-sulfotransferase to produce the fully elaborated heparin.
60
As the starting carbon backbone for the cost-effective synthesis of
61
bioengineered heparin, the availability of heparosan is a big concern (6-9). Heparosan
62
is extracted from the capsular polysaccharide (CPS) of E. coli K5, Pasteurella
63
multicida, and EcN (8, 10, 11). The proteins encoded by the kps locus govern the
64
biosynthesis and export of heparosan (Fig 1B). The kps locus comprises a
65
serotype-specific region 2 (kfiABCD) flanked by two conserved regions (region 1 and
66
3) (12, 13). Region 1 includes the genes of the kpsFEDUCS cluster, and region 3
backbone
is
then
further
modified
by
C5-epimerase,
3
the
enzymes
including
2-O-sulfotransferase,
67
includes kpsMT. The gene products encoded by kfiABCD are responsible for the
68
biosynthesis of heparosan (14). Translocation across the cytoplasmic membrane is
69
mediated by the products of kpsC, kpsS, and kpsMT while translocation across the
70
periplasm and outer membrane involves the KpsD and KpsE proteins (15). The kpsU
71
gene encodes for a functional CMP-Kdo synthetase, and the specific activity levels of
72
CMP-Kdo synthetase elevate at capsule-permissive temperatures (16, 17). KpsF
73
catalyzes the conversion of the pentose pathway intermediate Ru5P (D-ribulose
74
5-phosphate) into A5P (D-arabinose 5-phosphate), the precursor of Kdo (18).
75
A complex regulatory network controls the expression of the kps locus (Fig. 1C).
76
The kps locus is temperature-regulated, being expressed at 37 oC but not at 20 oC (19).
77
The IHF protein is required for maximum transcription from the region 1 promoter at
78
37 oC and binds to a single site located 130 bp 3’ to the transcription start point (19,
79
20). Three additional regulators including SlyA, BipA, and H-NS play a crucial role
80
in the temperature regulation of region 1 promoter (19, 21). The region 2 promoters
81
are weak and generate low levels of expression, which in the absence of
82
RfaH-mediated readthrough transcription from the region 3 promoter are insufficient
83
for the synthesis of detectable heparosan (22). The region 3 promoter is located 741
84
base pairs 5’ of the kpsM gene (23). The transcription of the region 3 promoter
85
proceeds through region 2 with the aid of the transcription antitermination factor
86
RfaH (23). The RfaH function is dependent on a short sequence present in the region
87
3
mRNA
known
as
the
JUMPStart
4
element
(just
upstream
of
many
88
polysaccharide-associated gene starts) (24). Besides the region 1 promoter, the region
89
3 promoter is also temperature-regulated via both SlyA and H-NS (25).
90
As a nonpathogenic probiotic strain without known toxins, EcN can be used as a
91
safe source of heparosan preparation. EcN carries all the genes for heparosan
92
biosynthesis and export. Although the above-mentioned studies have not been
93
performed on the kps locus of EcN, it is possible to investigate the heparosan
94
production in EcN based on the knowledge of the studied strains. Recent studies have
95
indicated that carbon sources have differential impacts on heparosan production. The
96
glycerol-defined medium allows a 3-fold increase of heparosan production than that
97
of LB medium (26). Another report has demonstrated that the glucose-defined
98
medium compares favorably to the glycerol-defined medium in heparosan yield (27).
99
However, the mechanism by which carbon sources control the biosynthesis of
100
heparosan is unclear.
101
In this study, we have found that glucose, fructose, and mannose have
102
discriminative impacts on the biosynthesis of heparosan. Further studies have
103
demonstrated that the expression of the region 3 promoter is regulated by the complex
104
of cyclic AMP (cAMP) and cAMP receptor protein (CRP). Gel shift assay shows that
105
the cAMP-CRP complex binds to a CRP binding motif in region 3 promoter. The
106
deletion of crp, the deletion of cya, and the base substitutions of the CRP binding site
107
dramatically decrease the expression from the region 3 promoter. Furthermore, the
108
yield of heparosan is apparently lower when glucose is used as the sole carbon source
109
than that of fructose and mannose. 5
110
MATERIALS AND METHODS
111
Bacterial strains, plasmids and culture conditions. Strains and plasmids used in
112
this study were listed in Table 1. E. coli strains were grown in Luria-Bertani (LB)
113
medium or on LB plates containing 1.5% agar. Polymerase chain reactions (PCR)
114
were performed on the Arktik Thermal Cycler (Thermo Fisher Scientific Inc.,
115
Waltham, MA, USA). The minimal medium (MM) was containing 2% carbon source
116
(glucose, fructose or mannose), 0.24 g/L MgSO4, 0.01 g/L CaCl2, 6 g/L Na2HPO4, 3
117
g/L KH2PO4, 0.5 g/L NaCl, and 1 g/L NH4Cl. The MM was used to culture EcN for
118
heparosan preparation. 1H NMR was performed on an Agilent VNMRS 600MHz
119
NMR Spectrometer (Agilent Technologies, Inc., USA). Unless otherwise stated,
120
glucose and cAMP were utilized at 0.8% and 10 mM, respectively. Ampicillin (Ap),
121
kanamycin (Kan), and chloramphenicol (Cm) were added to 50 μg/ml, 50 μg/ml, and
122
25 μg/ml when necessary. The chemicals were provided by Sangon Co. Ltd.,
123
Shanghai, China.
124
Plasmid construction. The low-copy-number vector pFZY1 was used to
125
construct the promoter fusion plasmids in this study (28). The region 3 promoter was
126
amplified by the primers 0011/0012 and inserted into pAH125 at the Kpn I- EcoR I
127
site to create pAH125-KpsMP. Then pAH125-KpsMP was cleaved with BamH I. The
128
purified DNA fragment carrying the region 3 promoter was cloned into the plasmid
129
pFZY1 to create pFZY1-kpsMP. Site-directed mutagenesis was used to create
130
pFZY1-KpsMPm that carried a mutated CRP binding site. Briefly, the primer sets
131
0060 and 0061 were used to introduce the mutant base pairs with pFZY1-KpsMP as 6
132
the template. Then the PCR products were digested with the enzyme Dpn I, purified,
133
and transformed into competent cells. The crp gene was cloned by the primers 0009
134
and 0010 to create pET28a-crp. The purified PCR product was digested and inserted
135
into pET28a(+) at the Nde I - Xho I site. The constructed plasmids were sequenced to
136
verify their integrity.
137
Gene
disruption
and
complement.
The
Red-mediated
homologous
138
recombination system was used to construct the in-frame deletions (29). A kanamycin
139
resistance cassette was amplified using pKD4 as template and the primers 0007/0008
140
and used to delete the lacZ gene in EcN. The PCR products were treated by the
141
enzyme Dpn I and introduced by electroporation into EcN containing the pKD46
142
expressed Red recombinase. Transformants were selected on LB plates supplemented
143
with kanamycin. The helper plasmid pKD46 was later cured by incubation at 42 oC.
144
In orde to construct the strain YHH1302 (EcN ΔlacZ), the kanamycin resistance
145
cassette of the strain YHH1301 (EcN ΔlacZ::Kan) was eliminated using pCP20 as
146
previously described (29). To construct the strain YHH1303 (EcN ΔlacZ Δcrp::Cm), a
147
chloramphenicol cassette was amplified from pKD3 using the primers 0013/0014.
148
Then the chloramphenicol cassette was introduced into YHH1302 (EcN ΔlacZ )
149
containing the plasmid pKD46. The strain YHH1304 (ZK126 Δcrp::Cm) and
150
YHH1305 (ZK126 Δcya::Cm) was created similarly using the chloramphenicol
151
cassette amplified from pKD3 with the primers 0013/0014 and 0056/0072,
152
respectively. To create the complementary strains, the DNA fragments carrying the
153
crp and cya gene were amplified with the primers 0017/0018 and 0058/0059. Then 7
154
the DNA products were purified and introduced into YHH1304/pKD46 and
155
YHH1305/pKD46. The complements, which grew faster than the isogenic crp/cya
156
mutants, were selected on the LB plates without any antibiotics. All the deletions and
157
complements were verified by PCR test.
158
Expression and purification of CRP. E. coli BL21(DE3) carrying the plasmid
159
pET28a-crp was grown in LB at 37 oC to an optical density of 0.6 at 600 nm, then
160
induced with 0.2 mM IPTG overnight at 22 oC. All subsequent procedures were
161
performed at 4 oC. The cells were harvested and resuspended in 30 ml of solution I
162
(20mM Tris-HCl, pH 7.6, 200mM NaCl). After the addition of 100 μM PMSF, the
163
cells were lysed by sonication. The lysate was centrifuged at 12000 rpm for 20min,
164
and the supernatant was applied to a nickel-NTA column. The column was washed
165
with 30 ml solution II (20mM Tris-HCl, pH 7.6, 200mM NaCl, 50mM imidazole).
166
CRP was eluted with a gradient of 50 to 250 mM imidazole in solution I. The
167
His-tagged CRP were dialyzed against solution I and stored at -80 oC until use. The
168
purity of CRP was analyzed by SDS-PAGE.
169
β-Galactosidase Assay. E. coli ZK126 was used as the wild-type (WT) strain.
170
ZK126 and its derivatives were used in the β-Galactosidase activity assays. Overnight
171
cultures of E. coli were diluted 1:100 into fresh LB medium. The cultures were
172
incubated at 37 oC with shaking at 250 rpm. At different time points during cell
173
growth, aliquots were removed for the determination of OD600 and β-Galactosidase
174
activity as previously described (30). The β-Galactosidase activity was expressed in
8
175
Miller units. All assays were performed in triplicate. The error bars in the graphs
176
indicated the standard deviations.
177
Gel shift assay. The double-stranded region 3 promoter fragments containing
178
the WT/mutated CRP binding motif were produced by boiling and slowly cooling the
179
synthetic DNA oligonucleotides 0080/0081 and 0082/0083 (Table 2). The DIG gel
180
shift kit (Roche Ltd., Mannheim, Germany) was used for DNA labeling and signal
181
detection. A DNA fragment without the CRP binding motif was used as the
182
competitive probe (primers 0004/0084). The labeled DNA fragments (1.6 nM) were
183
incubated with various amounts of purified CRP at 37 oC for 10 min in the
184
CRP-binding buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1 mM EDTA; 1 mM
185
DTT ; 50 μg/ml BSA; 100μM cAMP; and 160 nM of the competitive DNA probe).
186
The formed DNA-protein complexes were separated by 8% PAGE in 1 × TBE buffer
187
containing 100 μM cAMP.
188
Preparation of heparosan from liquid cultures. The overnight cultures of
189
EcN and E. coli K5 were expanded into fresh medium, shaked at 250 rpm, 37 oC for
190
variable times. The supernatant of bacterial cultures was recovered by centrifugation,
191
filtered
192
vacuum evaporator. Heparosan in the supernatant was precipitated with 3 volumes of
193
ethanol at -20 oC overnight and pelleted by centrifugation at 12000 rpm for 15 min at
194
4 oC. After washing with 75% ethanol, the heparosan was resuspended in deionized
195
water. The phenol/chloroform extraction was performed to remove proteins. Then, the
196
heparosan sample was dialyzed using the Spectra/Por dialysis membrane (MWCO 10
through
a
0.45-µm
membrane,
9
and
concentrated
by
a
rotary
197
kDa) against Buffer A (20 mM NaAcO, 50 mM NaCl, pH4) and applied to
198
DEAE-Sepharose column (1.6 X 50 cm) purification. The sample was loaded into the
199
column at 1 ml/min. After loading of heparosan, the column was washed with 10
200
column volumes of Buffer A at 3 ml/min. Then heparosan was eluted at 3 ml/min
201
with Buffer B (20 mM NaAcO, 1 M NaCl, pH4). Fractions containing heparosan were
202
pooled, dialyzed against water, and freeze-dried. The purified heparosan was stored at
203
-80 oC for later use.
204
Preparation of heparosan from bacteria grown on the MM plates. The
205
extracellular capsular polysaccharide from EcN was purified using the method
206
described previously with modifications (31). The overnight cultures of EcN were
207
diluted with fresh medium, and the 100 μl of aliquots were plated onto the MM plates
208
supplemented with the specific carbon source. The plates were incubated at 37 oC for
209
24 h for the bacteria to biosynthesize and translocate heparosan. Then, cells were
210
harvested and resuspended in the PBS buffer. To restrain the cell breakage and release
211
of large amounts of chemical compounds, we gently extracted heparosan by shaking
212
the cell resuspension solution overnight at 100 rpm, 4 oC. Then, the cell suspension
213
was centrifuged at 12000rpm for 10min at 4 oC. Heparosan in the supernatant was
214
precipitated with 3 volumes of ethanol at -20 oC overnight and pelleted by
215
centrifugation at 12000 rpm for 15 min at 4 oC. After washing with 75% ethanol, the
216
heparosan was dried and resuspended in deionized water. DNA in the heparosan
217
suspension was degraded by the addition of 20 units/ml Dpn I. Then proteinase K was
218
added at a final concentration of 400 μg/ml and the suspension was incubated at 37 oC 10
219
overnight. Following the phenol/chloroform extractions, the CPS preparation was
220
dialyzed using the Spectra/Por dialysis membrane (MWCO 10 kDa) against deionized
221
water. Then the sample was freeze-dried and stored at -80 oC for later use.
222
PAGE analysis of the capsular polysaccharide. The isocratic large-slab
223
PAGE was cast as previously described (6). A total of 45 μl sample plus 5 μl loading
224
buffer were loaded into each well. The gel electrophoresis was performed at 120V for
225
5h. Following electrophoresis, the gel was gently shaken at room temperature in the
226
washing buffer (10% acetic acid and 25% ethanol) for 30min. After staining with a
227
solution of Alcian blue (0.5%) in acetic acid (2%) for 30min, the gel was destained in
228
the washing buffer until the gel background was transparent.
229
1
H NMR analysis. The
1
H NMR analysis was performed as described
230
previously with sodium terephthalate as an internal standard for the heparosan
231
quantification (7). The CPS preparations were lyophilized and dissolved in 0.5 ml
232
D2O, and this step was repeated two times. Then the lyophilized CPS was redissolved
233
in 0.5 ml of D2O containing 71 μg of sodium terephthalate before being transferred to
234
a 5-mm NMR tube. 1H NMR was performed on an Agilent VNMRS 600MHz NMR
235
Spectrometer (Agilent Technologies, Inc., USA), and acquisition of the spectra was
236
carried out using VnmrJ Rev. 3.2A software. The 1H NMR spectra were processed in
237
MestReNova software. The integration of the peaks was performed using the
238
“integration” function, with the peak area being selected manually.
239 240 11
241
RESULTS
242
The biosynthesis of heparosan is affected by catabolite repression. To evaluate the
243
impact of carbon sources on heparosan production, we have extracted heparosan from
244
EcN grown on the MM plates. The medium has been supplemented with 2% of
245
glucose, fructose or mannose as the sole carbon source. The overnight bacterial lawns
246
have been scraped off, and heparosan is extracted according to the method described
247
in Materials and Methods. The amount of heparosan is checked by PAGE
248
electrophoresis. As shown in Fig. 2A, the yields of heparosan from bacteria using
249
fructose and mannose versus glucose as the carbon source are obviously higher.
250
Glucose is well known to affect gene expression through the cAMP-CRP
251
complex (32, 33). Thus, we set out to check the sequence of the kps locus and find a
252
potential CRP binding site located in the region 3 promoter (Fig. 1C). To study
253
whether cAMP-CRP is involved in the regulation of heparosan biosynthesis, we have
254
constructed the Δcrp::Cm strain and a region 3 promoter-lacZ fusion plasmid. The
255
deletion of crp gene has dramatically dropped the expression from region 3 promoter
256
(Fig. 2B). On the other hand, the addition of 0.8% glucose to LB medium
257
significantly inhibits the expression from region 3 promoter, and the corresponding
258
β-galactosidase activity is halved. However, the addition of 0.8% glucose plus 10 mM
259
cAMP offsets the decreased β-galactosidase activity. These results suggest that the
260
expression of the region 3 promoter is downregulated by the addition of glucose and
261
stimulated by both cAMP and CRP.
12
262
The complementary crp and cya genes restore the expression from region 3
263
promoter. To further study the regulatory relationship between cAMP-CRP and the
264
region 3 promoter, the Δcrp::Cm, Δcya::Cm, and the corresponding complementary
265
strains are constructed. All the in-frame deletions and the complementary
266
manipulations are carried out with the help of the λ Red homologous recombination
267
system (29). The in-frame deletion of crp and cya gene results in a clearly decreased
268
growth ability (Fig. 3AB and Fig. 4AB). The crp and cya gene are amplified with
269
EcN chromosome as template and utilized to complement the in-frame deletions of
270
both the Δcrp::Cm and Δcya::Cm constructs. The complements with restored
271
doubling time exhibit as the bigger colonies on the LB plate than other ones in the
272
bacteria lawn. The complements are selected and repurified on the LB plate without
273
any antibiotics. The in-frame deletions and complementary strains are verified via
274
colony PCR analysis (Fig. 3C and 4C).
275
Complementary experiments are conducted further to verify the stimulation of
276
expression from region 3 promoter by the cAMP-CRP complex. The region 3
277
promoter-lacZ fusion plasmid is transformed into WT, Δcrp::Cm, Δcya::Cm, and the
278
complementary strains including Δcrp(comp) and Δcya(comp). The β-galactosidase
279
activity expressed in Miller Units is determined to evaluate the impacts of different
280
genotypes on the expression from region 3 promoter. As shown in Fig. 5A, the
281
expression from region 3 promoter is significantly inhibited in the Δcrp::Cm strain,
282
and that of the crp complementary strain recovers to the level of the WT strain.
283
In-frame deletion of cya will eliminate the intracellular cAMP, leading to the loss 13
284
function of the cAMP-CRP complex (33). Similar to the phenotype of the Δcrp::Cm
285
strain, the deletion of cya gene eliminates the transcription from region 3 promoter
286
(Fig. 5B). The β-galactosidase activity of the cya complementary strain restores to the
287
level of the WT strain.
288
The cAMP-CRP complex directly binds to a CRP binding motif in the
289
region 3 promoter and stimulates the expression of this operon. The cAMP-CRP
290
complex controls gene transcription via binding to the consensus CRP binding site (34,
291
35). The region 3 promoter is located 741 base pairs 5’ of the kpsM gene (23). In the
292
region 3 promoter, there is a potential CRP binding site (TGTGAtataaaTCACA)
293
located 487 base pairs 5’ of the kpsM gene (Fig. 6A). To determine whether the
294
cAMP-CRP complex directly modulates the expression of region 3 promoter via the
295
potential CRP binding site, we have performed gel shift assays. Our results reveal that
296
cAMP-CRP directly binds to the DNA fragment of region 3 promoter in a
297
dose-dependent manner. However, the DNA fragment with a mutant CRP binding site
298
(cGatctataaaTtcgc) loses the ability to form complex with cAMP-CRP (Fig. 6BC).
299
Further studies are performed to verify the essentiality of the CRP binding site
300
for the expression from the region 3 promoter. The CRP binding motif of region 3
301
promoter is base-substituted, and the β-galactosidase activity is determined. As shown
302
in Fig. 6D, the region 3 promoter carrying a mutated CRP binding site fails to express
303
LacZ and exhibits no substantial β-galactosidase activity when compared to that of
304
the WT region 3 promoter. This result is consistent with the deficiency of
305
β-galactosidase activities in the Δcrp::Cm and Δcya::Cm strains that carry a WT 14
306
region 3 promoter-lacZ fusion plasmid. These results indicate a requirement for the
307
CRP binding site in the cAMP-CRP mediated activation.
308
The substitution of selective hexose for glucose increases the yield of
309
heparosan. As mentioned above, our data has clearly demonstrated that both the
310
consensus CRP binding site and the cAMP-CRP complex are essential for the
311
expression of genes regulated by the region 3 promoter. To further confirm the impact
312
of the global regulator CRP on the biosynthesis of heparosan, the CPS is extracted
313
from the wild-type EcN and its isogenic crp mutant grown on glucose-defined MM
314
plates. Both the PAGE electrophoresis and 1H NMR are performed to analyze the
315
extracted heparosan. As shown by the PAGE electrophoresis, there is a substantial
316
amount of heparosan purified from the wild-type EcN, but the heparosan extracted
317
from the isogenic crp mutant is not detectable (Fig. 7A). Furthermore, the 1H NMR
318
spectrum for the wild type EcN heparosan is similar to the previously published
319
spectra for K5 polysaccharide (Fig. 7B) (7, 36, 37). The peak at 2.04 ppm,
320
corresponding to the methyl protons in N-acetyl groups of heparosan, is clearly shown
321
for the EcN heparosan. These results from both the PAGE electrophoresis and 1H
322
NMR analysis consolidate the conclusions that biosynthesis of heparosan is
323
stimulated by the cAMP-CRP complex.
324
To further address the impacts of different hexoses on the biosynthesis of
325
heparosan, shake flask experiments are performed. The liquid MM supplemented with
326
glucose, fructose, and mannose as the sole carbon source is used to culture the
327
wild-type EcN and E. coli BL21. The cultures are shaked at 250 rpm, 37 oC to an 15
328
optical density of 1.0 at 600 nm. The heparosan in the supernatant of bacterial cultures
329
is purified with a DEAE-Sepharose column. The supernatant of E. coli BL21 culture
330
is purified in the same way as that of EcN and used as a negative control. 1H NMR is
331
utilized to analyze the purified EcN heparosan (Fig. 8A). Heparosan purified from E.
332
coli K5 (50 μg, 100 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg) is used to develop the
333
standard curve (Fig. 8B). Sodium terephthalate is selected as a water-soluble, stable,
334
and nonreactive internal standard for the heparosan quantification as previously
335
described (7). The N-acetyl peak (2.04 ppm) area is selected and normalized to the
336
sodium terephthalate peak area. The yields of heparosan in the sugar-defined MM
337
cultures are determined (Fig. 8C). The addition of glucose results in 5 mg/L of
338
heparosan production, lower than that of fructose (13 mg/L)and mannose (10 mg/L )
339
(Fig. 8C).
340 341
DISCUSSION
342
Pharmaceutical heparin produced from porcine intestinal mucosa bears the potential
343
risk of contaminants and adulteration (2). The in vitro chemoenzymatic synthesis of
344
bioengineered heparin and oligosaccharides has shown promise as an alternative
345
approach to producing the anticoagulant drug from non-animal source (3-5). As the
346
starting material for the cost-effective synthesis of novel anticoagulant drugs, the
347
availability of heparosan is a significant concern (6-8). Recent studies show that
348
carbon sources have differential impacts on the yield of heparosan (26, 27). However,
349
the mechanism by which carbon sources control the biosynthesis of heparosan is 16
350
unclear. Furthermore, both the well-known probiotic (EcN) and urinary tract pathogen
351
(E. coli K5) share their extracellular CPS composed of heparosan as the molecular
352
camouflage for host colonization (38, 39). Understanding the regulation mechanism
353
of heparosan biosynthesis will also contribute to the study on the resistance of the host
354
to pathogens.
355
Our study shows that the yield of heparosan from EcN is decreased when
356
utilizing glucose as the sole carbon source (Fig. 2A). Glucose is known to affect gene
357
expression through cAMP-CRP (32, 33). The cAMP-CRP complex binds to the
358
consensus CRP binding motif in the promoter region and affects the affinity of RNA
359
polymerase for the promoter DNA. The addition of glucose (Glc) causes the
360
dephosphorylation of glucose specific phospho- enzyme II A (P-EIIAGlc) (33). The
361
dephosphorylation process deactivates adenylate cyclase and hence lowers the
362
intracellular concentration of cAMP and the cAMP-CRP complex.
363
To address the mechanism of glucose inhibition of heparosan biosynthesis, we
364
have deleted the gene crp, cya, and constructed a region 3 promoter-lacZ fusion
365
plasmid. The promoter-lacZ fusion plasmid derives from a low-copy-number plasmid,
366
pFZY1 and carries a region 3 promoter. As shown by the β-galactosidase activity
367
assay, the addition of 10 mM cAMP offsets the decreased β-galactosidase activity
368
caused by glucose inhibition (Fig. 2B). Furthermore, the deletion of crp and cya have
369
blocked the expression of β-galactosidase from the region 3 promoter (Fig. 5). The
370
in-frame deletion of crp and cya are complemented by Red-mediated homologous
371
recombination (Fig. 3 and Fig. 4). The β-galactodidase activity of the complementary 17
372
strains is restored to that of the WT strain (Fig. 5). These results clearly demonstrate
373
that the expression from region 3 promoter is positively regulated by the cAMP-CRP
374
complex.
375
In general, cAMP-CRP binds to a palindromic sequence in which two
376
conserved motifs, TGTGA, and TCACA, are separated by a 6 bp spacer (40, 41). We
377
have performed gel shift assay to determine whether cAMP-CRP directly stimulates
378
the transcription from region 3 promoter upon binding. We have compared the
379
cAMP-CRP
380
(TGTGAtataaaTCACA)
381
(cGatctataaaTtcgc). As shown in Fig. 6C, the cAMP-CRP complex directly binds to
382
the wild type CRP binding motif in a dose-dependent manner, and the base
383
substitutions in the mutant CRP binding motif abolish its binding ability. Meanwhile,
384
the mutant CRP binding motif results in a failed transcription from region 3 promoter
385
as shown by the dramatically decreased β-galactosidase activity (Fig. 6D).
binding
capability with
of that
the of
wild the
type
CRP
binding
motif
mutant
CRP
binding
motif
386
Heparosan production is further analyzed by the PAGE electrophoresis and 1H
387
NMR. The biosynthesis of heparosan is significantly inhibited by the in-frame
388
deletion of crp gene (Fig. 7A). These results further consolidate our conclusion that
389
the binding of cAMP-CRP to the consensus CRP site is essential for the transcription
390
from region 3 promoter. Shake flask expriments are further performed to evaluate the
391
yield of heparosan in sugar-defined MM cultures. EcN and E. coli BL21 are
392
inoculated into MM and shaked at 250 rpm, 37 oC to an optical density of 1.0 at 600
393
nm. EcN heparosan in the supernatant is purified with a DEAE-Sepharose column and 18
394
analyzed with 1H NMR. The addition of glucose results in a lower yield of heparosan
395
(5 mg/L versus 10-13 mg/L) than that of fructose and mannose (Fig. 8C). Since the
396
uptake of glucose lowers the intracellular concentration of cAMP, the decreased
397
production of heparosan is reasonable. In the stationary phase, the depletion of
398
glucose will result in the increase in the intracellular cAMP concentration and
399
influence the heparosan production.
400
In summary, we have shown that the binding of cAMP-CRP to the consensus
401
CRP binding site is essential for the expression of region 3 operon. The deletion of
402
crp, cya, and the mutation of the consensus crp binding site inhibit the expression
403
from region 3 promoter and prevent the biosynthesis of heparosan. Glucose has an
404
adverse impact on intracellular cAMP concentration and the production of heparosan.
405
We have aligned the region 3 promoter of group 2 strains including EcN, E. coli K5,
406
E. coli UTI89 (O18:K1:H7), and E. coli K4 (O5:K4:H4) (39). EcN is a probiotic
407
bacterium without any known toxins while the other three bacteria are pathogenic
408
strains. These strains have a highly conserved region 3 promoter with a >95%
409
sequence identity while that of EcN and K5 is over 99%. Both the CRP binding motif
410
and JUMPstart sequence locate in the region 3 promoter of these four strains. These
411
results indicate the group 2 bacteria might use common mechanisms to escape the
412
host elimination and enhance host colonization.
413 414 415 19
416
ACKNOWLEDGMENTS
417
This study was funded by a Hefei University of Technology startup fund
418
(407-037064). We have no conflict of interest to declare.
419 420
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530 531 532 533 534 22
535 536
TABLE 1
Strains and plasmids used in this study
Strain or plasmid
Characteristics
Source
E.coli strains BL21
Expression host
Takara
Nissle 1917
Wild type
DSMZ
K5
Wild type
ATCC
ZK126
W3110 Δlac tna-2
(42)
YHH1301
EcN ΔlacZ::Kan
This work
YHH1302
EcN ΔlacZ
This work
YHH1303
EcN ΔlacZ Δcrp::Cm
This work
YHH1304
ZK126 Δcrp::Cm
This work
YHH1305
ZK126 Δcya::Cm
This work
YHH1306
ZK126 Δcrp(comp)
This work
YHH1307
ZK126 Δcya(comp)
This work
Plasmid pFZY1
galK′-lacZYA transcriptional fusion vector, Ap
(28)
pFZY1- KpsMP
pFZY1 carrying the region 3 promoter with a CRP binding
This work
pFZY1- KpsMPm
pFZY1 carrying the region 3 promoter with the mutated CRP
motif, Ap This work
binding motif, Ap pET28a-crp
pET28a(+) derivative for CRP expression, Kan
537 538 539 540 541 542 543 544 545 546 23
This work
547 548 549 550
551 552 553
TABLE 2
Oligonucleotide primers used in this study
Name
Sequencea,b
0007
ATGAGCGGATAACAATTTCACACAGGATACAGCTATGACGTGTAGGCTGGAGCTGCTTC
0008
TACGCGAAATACGGGCAGACATAGCCTGCCCGGTTATTAATGGGAATTAGCCATGGTCC
0009
GGAATTCCATATGGTGCTTGGCAAACCGCAAAC
0010
TCCGCTCGAGTTAACGAGTGCCGTAAAGCA
0011
ACGGGGTACCGTATTGCCATTTCCTTAACCCCA
0012
ATCGGAATTCTTGATGATGTGATCCTAATCTCTTC
0013
ATGGTGCTTGGCAAACCGCAAACAGACCCGACTCTCGAACTGTCAAACATGAGAATTAA
0014
TTAACGAGTGCCGTAAACGACGATGGTTTTACCGTGTGCGTGTAGGCTGGAGCTGCTTC
0017
AGGAGACACAAAGCGAAAGC
0018
CGTTTATGAGGCGTATCAAGG
0060
TTTAAAcGatcTATAAATtcgcAATATGGCTGTAAAGAGGGGGC
0061
TTACAGCCATATTgcgaATTTATAgatCgTTTAAATGGTGTTATTTAAGTCGCA
0056
TTGTACCTCTATATTGAGACTCTGAAACAGAGACTGGATATGGGAATTAGCCATGGTCC
0072
TCACGAAAAATATTGCTGTAATAGCGGCGTATCGTGATCGTGTAGGCTGGAGCTGCTTC
0058
ATAAACGGTGCTACACTTGTATGTA
0059
GCAAAATCATTATCAACCGC
0080
ATAACACCATTTAAATGTGATATAAATCACAAATATGGCTGT
0081
CTTTACAGCCATATTTGTGATTTATATCACATTTAAATGGTG
0082
ATAACACCATTTAAAcGatcTATAAATtcgcAATATGGCTGT
0083
CTTTACAGCCATATTgcgaATTTATAgatCgTTTAAATGGTG
0084
TCTTCATCTCCGGTTCTGCTGGCGGAGGTGGATCTGGCGGAGGTGGATCGG
0004
CCGATCCACCTCCGCCAGATCCACCTCCGCCAGCAGAACCGGAGATGAAGA
a
The underline sequences anneal to the template plasmids while the remaining sequences correspond to the ends
of the deleted genes. b
The boxed-in sequences represent the CRP binding motif while the base substitutions are shown in lowercases.
554 555 556 557 558 559 24
560 561
FIGURE LEGENDS
562 563
FIG 1 The structure of heparosan and the transcriptional organization of the
564
kps locus. (A) The structure of a native chain of heparosan. Heparosan is comprised of
565
a [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc)
566
(1→)]n repeating disaccharide unit. The average value of n is about 70. (B) The
567
transcriptional organization of the kps locus. Both the biosynthesis and export of
568
heparosan are carried out by the kps-locus-coded proteins. The transcription start
569
points are indicated by broken arrows. The horizontal arrows show the primary
570
transcripts from region 1 and region 3. The region 2 promoters are weak and
571
insufficient for the synthesis of detectable heparosan. (C) DNA sequence of the region
572
3 promoter. The broken arrow marks the transcriptional start site (23). The stop codon
573
of upstream gspM gene, -10 sequence, Shine-Dalgarno (SD) sequence, and kpsM start
574
codon are denoted by thick underlining. The JUMPstart sequence is indicated by
575
double underline, and the ops sequence (shaded box), which is essential for the action
576
of RfaH, is contained within JUMPstart (23). The H-NS binding regions are
577
underlined, and the SlyA binding region is indicated by shaded box (25). The putative
578
CRP binding motif is shown in an unshaded box.
579 580 581
FIG 2
Glucose has a negative effect on heparosan expression. (A) PAGE
analysis of the capsular polysaccharides extracted from EcN grown on MM plates 25
582
with different carbon sources. Lane 1, glucose; Lane 2, fructose; Lane 3, mannose. (B)
583
Effects of glucose, cAMP, and CRP on the transcription from region 3 promoter. Both
584
E.coli ZK126 (wild type) and the isogenic crp mutant are carrying the promoter
585
fusion plasmid pFZY1-KpsMP. The strains are grown in LB, LB plus 0.8% glucose,
586
or LB plus 0.8% glucose and 10mM cAMP. At different time points during cell
587
growth, aliquots are collected for the measurement of OD600 (squares and triangles)
588
and β-galactosidase activity (bars).
589
FIG 3
Complement of the in-frame deletion of crp. (A) The growth curve of
590
ZK126 (WT), crp mutant, and the complemented strain Δcrp(comp). (B) The
591
doubling time of ZK126 (WT), crp mutant and Δcrp(comp). (C) PCR verification of
592
the disruption and complement of crp gene. Lane 1, the DNA ladder; lane 2, ZK126
593
(WT); lane 3, Δcrp::Cm; lane 4, Δcrp(comp).
594 595
FIG 4
Complement of the in-frame deletion of cya. (A) The growth curve of
596
ZK126(WT), cya mutant and Δcya(comp). (B) The doubling time of ZK126(WT), cya
597
mutant and Δcya(comp). (C) PCR verification of the disruption and complement of
598
the cya gene. Lane 1, the DNA ladder; lane 2, ZK126 (WT); lane 3, Δcya::Cm; lane 4,
599
Δcya(comp).
600 601
FIG 5 The complement crp (A) and cya (B) recover the expression from
602
region 3 promoter. The cells carrying pFZY1-KpsMP are grown in LB medium at 37 26
603
o
604
determination of growth curve (squares, triangles, and circles) and β-galacotsidase
605
activity (bars).
C with shaking at 250 rpm. At different time intervals, the aliquots are taken for the
606 607
FIG 6 The cAMP-CRP complex binds to a CRP binding motif and stimulates
608
the transcription from region 3 promoter. (A) The DNA fragment of region 3
609
promoter carries a CRP binding motif located 487 base pairs 5’ of the kpsM gene. (B)
610
The DNA fragment carries a mutant CRP binding motif with base substitutions shown
611
in lowercase. The conserved base pairs of CRP binding motif is randomly mutated.
612
(C) Gel shift assays of the DNA fragments containing the WT/mutant CRP binding
613
motif. Variant amounts of the purified CRP protein (0-80 nM) are utilized to bind to
614
the digoxigenin-labeled DNA fragments with the present of 100 μM cAMP. The
615
arrowhead denotes the cAMP-CRP-DNA complex. (D) The β-galactosidase activity
616
assays of the region 3 promoter carrying a WT/mutant CRP binding motif. The lined
617
squares and triangles represent the growth curves; The bars indicate the
618
β-galactosidase activity.
619 620
FIG 7
Heparosan is not detectable in the isogenic crp mutant of EcN. (A)
621
PAGE analysis of the heparosan extracted from EcN (lane 1) and its isogenic crp
622
mutant (lane 2). The bacteria are grown on the glucose-defined MM plates at 37 oC
623
for 24 h. Then the cells are harvested from plates and heparosan is extracted. (B) The
27
624
1
625
on the glucose-defined MM plates.
H NMR analysis of EcN heparosan. The heparosan is extracted from EcN cultured
626 627
FIG 8 Analysis of heparosan purified from the shake flask cultures. EcN and
628
E. coli BL21 are inoculated into MM and shaked at 250 rpm, 37 oC to an optical
629
density of 1.0 at 600 nm. EcN heparosan in the supernatant is precipitated with
630
ethanol and purified with a DEAE-Sepharose column (1.6 X 60 cm) as described in
631
Materials and Methods. The supernatant of E. coli BL21 culture is purified in the
632
same way as that of EcN and used as a negative control. (A) The 1H NMR spectra of
633
the purified EcN heparosan from shake flask cultures is compared with that of E. coli
634
BL21. (B) Standard curve for the quantification of heparosan. E. coli K5 is grown in
635
LB medium at 250 rpm, 37 oC for 20 h. Heparosan in the supernatant is purified with
636
a DEAE-Sepharose column and used as standards. (C) The yield of purified EcN
637
heparosan in MM supplemented with different carbon sources. The 1H NMR analysis
638
is performed with sodium terephthalate as an internal standard for the heparosan
639
quantification.
28
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6
Fig 7
Fig 8
