1
SUPPORTING INFORMATION
2
3
Insights on the regulation of the phenylacetate degradation pathway from Escherichia coli
4
Cristina Fernández1, Eduardo Díaz, and José Luis García*
5
6
7
Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de
8
Maeztu 9, 28040 Madrid, Spain
9
1
Current address: Department of Cellular and Molecular Biology.
10
11
Experimental procedures
12
13
Bacterial strains, plasmids, and culture conditions
14
Bacterial strains and plasmids used in this study are listed in Table S1. Unless otherwise stated,
15
bacteria were grown in Luria-Bertani (LB) medium (Sambrook and Russell, 2001) at 37°C.
16
Growth in M63 minimal medium (Miller, 1972) was achieved at 30°C using the corresponding
17
necessary nutritional supplements and 20 mM glycerol as carbon source. When required, 1-5 mM
18
PA was added to the M63 minimal medium. Media were solidified with 1.5% w/v Bacto agar
19
(Pronadisa). When required, 1 mM IPTG was added to the culture medium. Where appropriate,
20
antibiotics were added at the following final concentrations: ampicillin (100 μg ml-1),
21
chloramphenicol (35 μg ml-1), kanamycin (50 μg ml-1), and rifampicin (50 μg ml-1). Bacterial
22
growth was followed by turbidity at 600 nm with a Beckman DU-520 spectrophotometer.
23
24
DNA manipulations and sequencing
1
25
DNA manipulations and other molecular biology techniques were essentially as described
26
(Sambrook and Russell, 2001). Plasmid DNA was prepared with a High Pure plasmid isolation
27
kit (Roche Applied Science). DNA fragments were purified with Gene-Clean Turbo (Q-
28
BIOgene). Transformation of E. coli cells was carried out by using the RbCl method or by
29
electroporation (Gene Pulser; Bio-Rad) (Sambrook and Russell, 2001). Oligonucleotides were
30
synthesized on an Oligo-1000M nucleotide synthesizer (Beckman Instruments). All cloned
31
inserts and DNA fragments were confirmed by DNA sequencing through an ABI Prism 377
32
automated DNA sequencer (Applied Biosystems Inc.).
33
34
Expression and purification of PaaX repressor
35
The PaaX coding sequence was amplified by PCR using oligonucleotides PXE5 5´-
36
GGGAATTCTAAATGAAGGAGAAAGATAATGAGTAAACTTGTTACTTTA-3´
37
codon is indicated in bold and an engineered EcoRI site is underlined) and PY3-BamHI 5´-
38
GTCGGATCCACAAACTCTCTTC-3´ (an engineered BamHI site is underlined) and plasmid
39
pAAD (Table 1) as a template. The amplified DNA fragment was digested with EcoRI/BamHI
40
and then cloned into EcoRI/BamHI double-digested pUC18 plasmid (Table S1) under control of
41
the Plac promoter. The resulting recombinant plasmid, pUCX2, was transformed into E. coli
42
JM109. SDS-PAGE analyses of crude lysates from E. coli JM109 cells harboring plasmid
43
pUCX2 (Table S1) showed the presence of an intense band corresponding to the PaaX protein,
44
thus indicating that the paaX gene was overexpressed in this recombinant plasmid. Crude extracts
45
containing the PaaX protein were prepared from E. coli JM109 (pUCX2) strain. Cells were
46
grown at 37ºC in ampicillin-containing LB medium and harvested at an optical density of 600 nm
47
of 1. Cell cultures were then centrifuged (3000 x g, 10 min at 20°C), and cells were washed and
48
resuspended in 0.01 volumes of 20 mM Tris-HCl buffer, pH 7.5, containing 10% glycerol, 2 mM
(start
2
49
β-mercaptoethanol, 10 mM EDTA and 50 mM KCl, prior to disruption by passage through a
50
French press (Aminco Corp.) operated at a pressure of 20000 pounds/square inch. Crude extracts
51
were clarified by removal of cell debris by centrifugation at 26000 x g for 30 min at 4 °C. The
52
clear supernatant fluid was decanted and used as crude cell extract. Protein concentration was
53
determined by the method of Bradford (1976) using bovine serum albumin as standard. To purify
54
PaaX, crude lysate was mixed with polyethyleneimine (PEI) to a final concentration of 0.05%,
55
incubated on ice for 30 min and centrifuged (14000 x g, 15 min). The supernatant was dialyzed
56
for several hours at 4 ºC in TRRG buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM –
57
mercaptoethanol, 10 mM EDTA and 50 mM KCl) and centrifuged at 14000 x g, 15 min. The
58
dialyzed supernatant was processed by ammonium sulphate (AS) precipitation at 40%. Proteins
59
were recovered from the 40% AS pellet and this pellet was resuspended in buffer TRRG and
60
dialyzed at 4 ºC in 20 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM –mercaptoethanol, and 25
61
mM KCl buffer. After dialysis the protein solution becomes cloudy. The precipitate was
62
recovered by centrifugation at 14000 x g, 15 min. The pellet was resuspeded in 1-2 ml TRRG
63
buffer and loaded onto a Sephadex G-100 column pre-equilibrated with buffer TRRG. Fractions
64
containing protein, as determined by SDS-PAGE, were pooled and stored with 40% (vol/vol)
65
glycerol at –70ºC.
66
67
Expression and purification of the PaaY protein
68
The paaY gene was expressed from the PT5 promoter in the high copy number pQE32 plasmid
69
(Table S1). Plasmid pQEYES, expressing the native protein, was constructed by cloning into the
70
double-digested pQE32 plasmid an EcoRI/SacI PCR-amplified fragment containing the paaY
71
gene, by using the oligonucleotides Y5-Eco TTTGAATTCTGAACAGGAGGCGAT/EcoRI and
3
72
Y3-Sac AACGAGCTCAGCGCCGCATC/SacI. The protein was overproduced in the E. coli
73
M15 strain harboring the plasmid pREP4 (Table S1), which produces the LacI repressor to
74
strictly control gene expression from pQE32 derivatives in the presence of isopropyl-1-thio-β-D-
75
galactopyranoside (IPTG). E. coli M15(pREP4, pQEYES) cells were grown at 37ºC in
76
ampicillin- and kanamycin-containing LB medium until the cultures reached an absorbance at
77
600 nm of 1. Overexpression of PaaY was then induced for 4 h by the addition of 0.5 mM IPTG.
78
Cells were harvested, resuspended in 40 ml of buffer (25 mM Tris-HCl buffer, pH 8), and
79
disrupted by passage through a French press (Aminco Corp.) operated at a pressure of 20000
80
p.s.i.. The cell debris was removed by centrifugation at 30000 x g for 30 min at 4 ºC. The clear
81
supernatant fluid was decanted and used as crude cell extract. The PaaY protein was recovered by
82
precipitation with ammonium sulfate between 40-60%. The resulting pellet was dissolved in 25
83
mM Tris-HCl, pH 8 containing 0.5 M ammonium sulfate and loaded into a Phenyl-Sepharose
84
column equilibrated with the same buffer. The PaaY protein was eluted by using a 500-0 mM
85
gradient of ammonium sulfate. The fractions containing the PaaY protein were pooled and
86
concentrated to a final volume of 2 ml. This protein sample was loaded into a Sephadex G-100
87
column equilibrated with 25 mM Tris-HCl, pH 8 containing 10% glycerol. The PaaY protein was
88
eluted using the same buffer at 0.4 ml/min. Finally, the pooled fractions containing the PaaY
89
protein were loaded into a DEAE-cellulose column equilibrated with 25 mM Tris-HCl pH 8
90
containing 10% glycerol. The elution of PaaY was performed using 25 mM Tris-HCl, pH 8,
91
containing 10% glycerol and 100 mM NaCl. The purified PaaY protein was conserved at -70ºC
92
in 25 mM Tris-HCl, pH 8 containing 10-40% glycerol. Protein purity and molecular mass were
93
confirmed by SDS-PAGE. PaaY concentration was determined spectrophotometrically by using
94
the molar extinction coefficient at 280 nm (10,810 M-1 cm-1) calculated on the basis of its amino
95
acid sequence.
4
96
97
Construction of plasmids pAAD::Tn1000-XY and pAFK7
98
Plasmid pAAD::Tn1000-6 (Table S1) was digested with BamHI. The resulting 17.9 kb DNA
99
fragment was ligated to generate plasmid pAAD::Tn1000-XY, which expresses a paa cluster
100
harboring a deletion of the paaXY genes. Plasmid pAFK3 (Table S1) was digested with NsiI and
101
BamHI, and the resulting 7.2 kb fragment was incubated with T4 DNA polymerase to generate
102
blunt ends and then ligated to generate plasmid pAFK7 (Table S1).
103
104
Construction of E. coli strains harboring a chromosomal insertion of the Px::lacZ translational
105
fusion
106
By means of RP4-mediated mobilization, plasmid pAFPX-T, which contain mini-Tn5Km2
107
hybrid transposon expressing the Px::lacZ fusion (Table S1), was transferred from E. coli S17–
108
1λpir into rifampicin-resistant E. coli recipient strains, i.e. AF15, AFMC, S90CRif, DPB101Rif
109
as described previously (de Lorenzo and Timmis, 1994). Exconjugants containing the lacZ
110
translational fusions stably inserted into the chromosome were selected for the transposon
111
marker, kanamycin, on rifampicin-containing LB medium. The resulting strains, AF141X,
112
AFMCPX, S90CPX, DPB101PX and their relevant genotype are indicated in Table S1.
113
114
Synthesis of DNA fragments covering the Px, Pa and Pz promoter regions
115
The target DNA (200 bp) used as PX probe was generated by PCR using plasmid pAAD as
116
template,
117
AGATGTGCCACTGACCGGAAC-3´). To prepare the PX2 fragment (230 bp) that contains the
118
Px promoter, plasmid pUCPX (Table S1) was digested with HincII/EcoRI restriction enzymes
and
primers
PX5
(5´-TCGGGTGTTTGATCTGCGC-3´)
and
PX3
(5´-
5
119
and purified with Gene-Clean Turbo (Q-BIOgene). To construct plasmid pUCPX, plasmid
120
pAFPX (Ferrández et al., 1998) was digested with KpnI and BamHI. The DNA fragment
121
containing the Px promoter was cloned into KpnI/BamHI double-digested pUC18 plasmid under
122
the control of the Plac promoter. The PA-PZ probe was amplified by PCR using 10 ng of plasmid
123
pAAD
124
GGGGTGAATCAAACGGCTACG-3’) and PA5-1 (5’-CAATCTCGGAATGCGCATG-3’). The
125
PA and PZ DNA fragments were prepared as described previously (Ferrández et al., 2000).
(Table
S1)
as
template,
and
the
oligonucleotides
PZ-5
(5’-
126
127
DNase I footprinting assays
128
The PX2 fragment was singly 3'-end-labeled by filling in the overhanging EcoRI-digested end
129
with [-32P]dATP (6000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of E. coli
130
DNA polymerase I as described (Sambrook and Russell, 2001). The labeled fragment (PX2
131
probe) was purified by using Gene-Clean Turbo (Q-BIOgene). For DNase I footprinting assays,
132
the reaction mixture contained 2 nM DNA probe, 500 µg/ml bovine serum albumin, and purified
133
protein in 15 µl of 20 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM β-mercaptoethanol, 50 mM
134
KCl buffer. This mixture was incubated for 20 min at 30 °C, after which 3 µl (0.05 units) of
135
DNase I (Amersham Biosciences) (prepared in 10 mM CaCl2, 10 mM MgCl2, 125 mM KCl, and
136
10 mM Tris-HCl pH 7.5) was added, and the reaction further incubated at 37 °C for 20-25 s. The
137
reaction was terminated by the addition of 180 µl of a solution containing 0.4 M sodium acetate,
138
2.5 mM EDTA, 50 µg/ml calf thymus DNA, and 0.3 µl/ml glycogen. After phenol/chloroform
139
extraction, DNA fragments were precipitated with absolute ethanol, washed with 70% ethanol,
140
dried, and directly resuspended in 5 µl of 90% (v/v) formamide-loading gel buffer (10 mM Tris-
141
HCl, pH 8.0, 20 mM EDTA, pH 8.0, 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene
6
142
cyanol). Samples were denatured at 95 °C for 2 min and analyzed on a 6% denaturing
143
polyacrylamide-urea gel. Protected bands were identified by comparison with the migration of the
144
same fragment treated for the A + G sequencing reaction (Maxam and Gilbert, 1980). The gels
145
were dried onto Whatman 3MM paper and exposed to Hyperfilm MP.
146
147
Gel retardation assays
148
The DNA fragments used as probes were labelled at their 5’-end with phage T4 polynucleotide
149
kinase (Amersham Pharmacia Biotech) and [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences).
150
The fragment was purified on a glass fiber column (High Pure PCR purification kit, Roche
151
Applied Science). The reaction mixtures contained 20 mM Tris-HCl, pH 7.5, 10% glycerol, 2
152
mM β-mercaptoethanol, 50 mM KCl, 0.5 nM DNA probe, 500 g/ml bovine serum albumin, 50
153
g/ml salmon sperm (competitor) DNA, and the indicated amount of crude extract or purified
154
PaaX protein in a 9-µl final volume. After incubation of the retardation mixtures for 20 min at
155
30°C, mixtures were fractionated by electrophoresis in 5% polyacrylamide gels buffered with
156
0.5x TBE (45 mM Tris borate, 1 mM EDTA). The gels were dried onto Whatman 3MM paper
157
and exposed to Hyperfilm MP (Amersham Pharmacia Biotech). In reactions containing RNAP
158
(Epicenter), the DNA probe was preincubated at 37ºC for 30 min. When CRP was present, gel
159
retardation assays were performed in the presence of 0.2 mM of cAMP.
160
161
RT-PCR analyses
162
For reverse transcription-PCR (RT-PCR) experiments, cultures of E. coli W cells grown
163
aerobically in PA were collected at an optical density at 600 nm of 1. Total RNA was isolated
164
with a High Pure RNA isolation kit (Roche). Any contamination by DNA was eliminated by the
7
165
use of a DNase treatment and removal kit (Ambion). One microgram of purified total RNA was
166
used to prepare cDNA by the use of 3 U of avian myeloblastosis virus reverse transcriptase
167
(Promega). PCRs were carried out with 2.5 U of AmpliTaq DNA polymerase (Roche). Control
168
reactions in which reverse transcriptase was omitted from the reaction mixture ensured that DNA
169
products resulted from the amplification of cDNA rather than from DNA contamination. The
170
primers used for these experiments were IX3 (5’-ACGCAGTGCTGCACGAGAAAGC-3’), IX5
171
(5’-GAAGGCTGGCTGGATGTTTCCC-3’), IX25 (5’-AAACCTCGGTCGGTGAACTGCC-3’),
172
IY3 (5’-TTCCATGCCAGCTCCTGCTCAC-3’), IY5 (5’-AGGCAAGGGCGTTTACGTTGG-
173
3’), and IY25 (5’-GCGGCGGATAATGCAACCATG-3’) (Figure S2).
174
175
Western blotting
176
To confirm that paaX and paaY genes constitute a single operon, we analyzed the production of
177
the PaaY protein in different E. coli W14 recombinant cell extracts by Western blot using anti-
178
PaaY antibodies (Figure S2). E. coli cell extract proteins were separated in 12.5 % SDS-PAGE
179
and electrotransferred to a Nitrocellulose Membrane, 0.45 m (BIO-RAD) as described
180
previously (Sambrook and Russell, 2001). Additional protein binding sites were blocked by
181
incubating the membrane in PBS-Tween buffer (10 mM Na phosphate, pH 7.4 containing 140
182
mM NaCl and 0.05% (w/v) Tween 80) with the addition of 3% dry milk at 4ºC for 12 h. Then,
183
the membrane was washed in PBS-Tween and incubated at room temperature 4 h with a 1:6000
184
dilution of anti-PaaY antiserum in buffer PBS. After that, the membrane was washed in PBS-
185
Tween several times and incubated with the secondary antibody (Jackson Immunoresearch). The
186
antibody-antigen complex was detected with hydrogen peroxide and 4-chloro-1-naftol (Sigma) in
187
PBS buffer.
188
8
189
Protein determination
190
Proteins were analyzed by SDS-PAGE as described previously (Sambrook and Russell, 2001).
191
The protein concentration in cell extracts was determined by the method of Bradford (Bradford,
192
1976) by using bovine serum albumin as the standard. PaaY concentration was determined by
193
measuring the absorbance at 280 nm using a theoretical monomer extinction coefficient of 10,810
194
cm-1 M-1 and a compute monomer molecular mass of 21,439 Da. The concentration of purified
195
PaaX was determined spectrophotometrically by using the molar extinction coefficient at 280 nm
196
(48,700 M-1 cm-1) calculated on the basis of its amino acid sequence. The N-terminal amino acid
197
sequence of PaaY from E. coli W was determined by Edman degradation with a 477 automated
198
protein sequencer (Applied Biosystems Inc.). The phenylthiohydantoin derivatives were
199
identified with an on-line Applied Biosystems liquid chromatograph. PaaY was loaded in a
200
12.5% SDS-PAGE, and was electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad)
201
as described previously (Speicher, 1994)
202
203
β-Galactosidase assays
204
β-Galactosidase activities were measured with permeabilized cells when cultures reached mid
205
exponential phase as described (Miller, 1972). At least three independent assays were performed
206
in each case. E. coli AF1411 (pAAD) and E. coli AF1411 (pAAD::Tn1000-84) cells were
207
cultured at 30C in minimal medium with 20 mM glycerol and chloramphenicol (35 µg ml−1) to
208
reach an optical density at 600 nm of 1.5. Cells were harvested by centrifugation, washed twice
209
with two volumes of 0.9% NaCl, and suspended in M63 minimal medium. For 0.5 ml of cells we
210
added 4.5 ml of M63 minimal medium and 25 µl of 1M PA. Cells were incubated at 37C and
211
samples were taken at different times to determine the -galactosidase activity.
9
212
213
PaaY enzymatic assays
214
The method used to determine the carbonic anhydrase activity was based on the observation that
215
carbonic anhydrases show esterase activity in vitro. Esterase activity was measured
216
spectrophotometrically using p-nitrophenylacetate as substrate. The hydrolysis of p-
217
nitrophenylacetate was determined at 25ºC, using a modification of the described method
218
(Armstrong et al., 1966). The reaction mixture (0.5 ml) contained 0.316 ml of 50 mM Tris-HCl,
219
pH 7.6, 0.167 ml of freshly prepared 3 mM p-nitrophenylacetate (prepared by dissolving 13.6 mg
220
of p-nitrophenylacetate in 1 ml of acetone which was further diluted with 24 ml of water) and
221
stored in dark. The reaction was started by adding 10 μl of enzyme solution (1.2 mg/ml). The
222
change in absorbance at 348 nm was measured over 5 min. Commercial bovine carbonic
223
anhydrase (Sigma) was used as control (1-2 U/ml).
224
Thioesterase activity was determined at 30ºC, in a final volume of 0.5 ml containing 25
225
mM Tris-HCl pH 8, glycerol 25%, 1 mM DTNB (5,5′-dithio-bis(2-nitrobenzoic acid)) as a
226
colorimetric developing agent, 0.005 mM CoCl2 and the corresponding acyl-CoA derivative at a
227
final concentration of 0.2 mM. The reaction was started by adding 5 l of enzyme solution. The
228
5-thio-2-nitrobenzoate was produced by the reaction of DTNB with the CoA liberated from the
229
acyl-CoA substrate upon hydrolysis. The reaction was followed at 412 nm using a Beckman
230
DU®529 spectrophotometer.
231
PaaY kinetic parameters such as Km and Vmax were calculated by changing acyl-CoA
232
concentrations from 0 to 0.5 mM in three independent experiments (with S.D. values of <10%)
233
and directly fitting the Michaelis-Menten equation. Temperature and pH optimal were calculated
234
by assaying the thioesterase activity over a temperature range from 15ºC to 50ºC and a pH range
10
235
from 6 to 9, respectively. The influence of different metals on the PaaY thioesterase activity was
236
tested as previously described at 30ºC, pH 8, with acetoacetyl-CoA 0.2 mM.
237
238
Sedimentation equilibrium experiments
239
A Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics was
240
employed for analytical ultracentrifugation measurements by using an An50Ti rotor. We used
241
standard (12-mm optical path) double-sector centerpieces of charcoal-filled Epon. The
242
equilibrium temperature was 4°C. PaaY was equilibrated in 25 mM Tris-HCl buffer (pH 8.0).
243
Short-column sedimentation equilibrium experiments (70 µl of protein) with loading
244
concentrations ranging from 5.9 to 47.0 M were done at two successive speeds (10000 and
245
13000 rpm) by taking absorbance scans at the appropriate wavelengths at sedimentation
246
equilibrium. Samples were judged to be at equilibrium by the absence of systematic deviations in
247
successive scans taken at 2h intervals. High-speed sedimentation (40000 rpm) was conducted
248
afterwards for baseline corrections in all cases. Conservation of protein mass in solution was
249
controlled during the experiment. The equilibrium temperature was 4ºC. Average molecular
250
masses were determined by fitting a sedimentation equilibrium model for a single solute to
251
individual data sets with XLAEQ and EQASSOC (Minton, 1994). These values were converted
252
to the molecular mass using the specific partial volume, calculated with the amino acid sequence
253
and corrected for the temperature (Laue et al., 1992).
254
255
Plasma emission spectrometric analyses of PaaY
256
The metal content in purified PaaY was determined by inductively coupled plasma emission
257
spectroscopy (ICP) using an ICP-OES Optima 2000DV equipment (PerkinElmer).
11
258
259
3D-Modeling of PaaY
260
The three-dimensional model of PaaY from E. coli W was built using the program Loopp
261
(http://ser-loopp.tc.cornell.edu/cbsu/loopp.htm). The crystal structures of Fbp from Pyrococcus
262
horikoshii (Protein Data Bank 1V3W) (Kawarabayasi et al., 1998) and Cam from
263
Methanosarcina thermophila (Protein Data Bank 1QRG) (Iverson et al., 2000) were used as
264
templates. According to this 3D-model, the PaaY structure is dominated by a coiled structural
265
domain formed by a left-handed parallel –helix (LH). Each hexapeptide repeat is used to form
266
one side of a triangular coil with a canonical 18-residue length. The overall appearance of the
267
LH structural domain is that of an equilateral prism, with each flat surface representing a single
268
untwisted parallel –sheet (Figure S10B).
269
270
271
272
References
273
properties of human erythrocyte carbonic anhydrases. J Biol Chem 241: 5137-5149.
Armstrong, J.M., Myers, D.V., Verpoorte, J.A., and Edsall, J.T. (1966) Purification and
274
275
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities
276
of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
277
278
de Lorenzo, V., and Timmis, K.N. (1994) Analysis and construction of stable phenotypes in
279
gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:
280
386-405.
281
282
Ferrández, A., García, J.L., and Díaz, E. (2000) Transcriptional regulation of the divergent paa
283
catabolic operons for phenylacetic acid degradation in Escherichia coli. J Biol Chem 275: 12214-
284
12222.
285
12
286
Ferrández, A., Miñambres, B., García, B., Olivera, E.R., Luengo, J.M., García, J.L., and Díaz, E.
287
(1998) Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic
288
hybrid pathway. J Biol Chem 273: 25974-25986.
289
290
Iverson, T.M., Alber, B.E., Kisker, C., Ferry, J.G., and Rees, D.C. (2000) A closer look at the
291
active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the
292
carbonic anhydrase from Methanosarcina thermophila. Biochemistry 39: 9222-9231.
293
294
Kawarabayasi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y., Yamamoto, S. et al.
295
(1998) Complete sequence and gene organization of the genome of a hyper-thermophilic
296
archaebacterium, Pyrococcus horikoshii OT3 (supplement). DNA Res 5: 147-155.
297
298
Laue, T.M., Shah, B.D., Ridgeway, T.M., and Pelletier, S.L. (1992) In Analytical
299
Ultracentrifugation in biochemistry and polymer science. Harding, S., Rowe, A., and Horton, J.
300
(ed). Cambridge, UK, pp. 90-125.
301
302
Maxam, A.M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical
303
cleavages. Methods Enzymol 65: 499-560.
304
305
Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor, NY.
306
307
Minton, A.P. (1994) Conservation of signal: a new algorithm for the elimination of the reference
308
concentration as an independently variable parameter in the analysis of sedimentation
309
equilibrium. In Modern analytical ultracentrifugation. In Schuster, T.y.L., T. (eds.). (ed).
310
Birkhauser Boston, Inc., Cambridge, MA, pp. 81-93.
311
312
Sambrook, J., and Russell, D.W. (2001) Molecular cloning: A laboratory Manual. NY: Cold
313
Spring Harbor, NY.
314
315
Silhavy, T.J., Berman, M.L., and Enquist, L.W. (1984) Experiments with gene fusions. Cold
316
Spring Harbor, NY.
317
13
318
Speicher, D.W. (1994) Methods and strategies for the sequence analysis of proteins on PVDF
319
Membranes. Methods 6: 262-273.
320
321
Teufel, R., Friedrich, T., and Fuchs, G. (2012) An oxygenase that forms and deoxygenates toxic
322
epoxide. Nature 483: 359-362.
323
324
325
14
326
327
Table S1. Bacterial strains and plasmids used in this study.
Relevant genotype and characteristic(s)
Strains and plasmids
E. coli strains
E.coli K12
DH5α
Reference / source
F- endA1 hsdR17 supE44 thi-1 recA1 gyrA(NalR) relA1 Δ(argF-lac) U169 deoR
Φ80dlacΔ(lacZ)M15
(Sambrook and Russell, 2001)
S17-1λpir
Tpr Smr recA thi hsdRM+ RP4::2-Tc::Mu::Km Tn7 λpir phage lysogen
(de Lorenzo and Timmis, 1994)
MC4100
F- araD139 Δ(argF-lac) U169 rpsL150(Smr) relA1 flbB5301 deoC1 ptsF25 rbsR
(Silhavy et al., 1984)
JM109
recA1, supE44, endA1, hsdR17, gyrA96, relA1, thi, (lac-proAB)
AFMC
MC4100 Rifr, F- araD139 Δ(argF-lac) U169 rpsL150(Smr) relA1 flbB5301 deoC1
ptsF25 rbsR
(Ferrández et al., 2000)
Δ(lac pro) rpsL Smr Rifr
(Ferrández et al., 2000)
DPB101Rif
S90C him D451::mini-Tc Rifr
(Ferrández et al., 2000)
AFMCPX
AFMC with chromosomal insertion of mini-Tn5Km2 Px::lacZ; Rifr, Kmr
This work
S90CRif with chromosomal insertion of mini-Tn5Km2 Px::lacZ; Rifr, Kmr
This work
DPB101Rif with chromosomal insertion of mini-Tn5Km2 Px::lacZ; Rifr, Kmr
This work
S90CRif
S90CPX
DPB101PX
(Sambrook and Russell, 2001)
E. coli W
W
W14
AF15
W ATCC11105
(Ferrández et al., 1998)
(Ferrández et al., 1998)
W (paa)
r
W14Rif (lac), Rif
(Ferrández et al., 2000)
AF141X
AF141 with a chromosomal insertion of a Px::lacZ translational fusion; Rifr, Kmr
AF15PZ
AF15 with a chromosomal insertion of mini-Tn5Km2 Pz-lacZ; Rifr, Kmr
(Ferrández et al., 2000)
AF1411
W14 Rif lacZ-, with a chromosomal insertion of mini-Tn5Km2 Pa-lacZ; Rifr, Kmr
(Ferrández et al., 1998)
Plasmids
pUC19
Apr; oriColE1 high copy number cloning vector, lacZa
(Sambrook and Russell, 2001)
pUC18
Apr; oriColE1 high copy number cloning vector, lacZa
(Sambrook and Russell, 2001)
pQE32
Apr , oriColE1, T5 promoter, lac operator, N-terminal 6-His tag
Qiagen
pREP4
Kmr , plasmid that expresses the lacI repressor
Qiagen
pCK01
Cmr, oripSC101, low copy number cloning vector
This work
(Ferrández et al., 1998)
15
pAFPX-T
Apr, Kmr; pUTmini-Tn5 Km2 containing the Px::lacZ fusion
(Ferrández et al., 1998)
This work
pUCX2
Apr, pUC18 derivative overexpressing the paaX gene under Plac
pAFX
Apr, pUC18 derivative carrying the paaX gene under Plac
(Ferrández et al., 1998)
pAFX2
Cmr, pCK01 derivative carrying the paaX gene under Plac
(Ferrández et al., 1998)
pAFY
Apr , pUC18 derivative carrying the paaY gene under Plac
(Ferrández et al., 1998)
pQEYES
Apr , pQE32 derivative carrying the paaY gene under Plac
This work
pAFK5
Apr, pUC18 derivative carrying the paaK gene under Plac
(Ferrández et al., 1998)
pAFK3
Cmr, pCK01 derivative carrying the paaK, paaX and paaY genes under Plac
(Ferrández et al., 1998)
pAFK7
Cmr , pAFK3 derivative carrying the paaK and paaX genes under Plac
pAAD
Cmr, pCK01 containing a 15.5-kb DNA fragment carrying the paa cluster
pUCPX
Apr, pUC18 derivative carrying the Px promoter
This work
pAAD::Tn1000-6
Cmr, pAAD derivative, Tn1000 insertion in paaX
(Ferrández et al., 1998)
pAAD::Tn1000-84
Cmr, pAAD derivative, Tn1000 insertion in paaY
(Ferrández et al., 1998)
pAAD::Tn1000-ΔXY
Cmr , pAAD derivative with paaX and paaY deletion
This work
(Ferrández et al., 1998)
This work
328
329
16
330
331
Table S2. PaaY thioesterase activity. % activity represents the percentage of activity considering
332
100% that obtained with acetoacetyl-CoA. * ND, not detectable.
333
Km (M)
Vmax
(molmin-1)
146.6
0.659
136.4
0.373
23.2
42.7
0.131
Lauroyl-CoA
56.0
23.2
0.171
Miristoyl-CoA
46.9
20.0
0.139
Palmitoyl-CoA
9.7
CoA derivatives
% Activity
Acetyl-CoA
ND
Butiryl-CoA
ND
Isobutyryl-CoA
ND
Acetoacetyl-CoA
100
Succinyl-CoA
ND
Hexanoyl-CoA
9.7
Octanoyl-CoA
4.7
Phenylacetyl-CoA
28.6
Benzoyl-CoA
ND
Decanoyl-CoA
334
335
17
336
FIGURE LEGENDS
337
338
Figure S1. Aerobic phenylacetate degradation pathway. Reactions and intermediates are
339
according to Teufel et al. (2012). The Paa enzymes involved in the different enzymatic steps
340
(solid arrows) are shown in bold. The spontaneous formation of 2-hydroxycyclohepta-1,4,6-
341
triene-1-carboxyl-CoA, a side product of the pathway that is likely a precursor for primary and
342
secondary metabolites, e.g, antibiotics and -cycloheptyl fatty acids (grey shadow), is indicated
343
by a dashed arrow. The thioesterase activity of PaaY is also shown.
344
345
Figure S2. The paaX and paaY genes constitute an operon. (A) RT-PCR analysis of the
346
expression of paaX and paaY genes in PA-grown E. coli W cells was performed as indicated in
347
Experimental procedures. The schematic representation of the paaX-paaY region, the Px
348
promoter, and the localization of the primers used for PCR amplification are shown at the
349
bottom. Lane M, HaeIII-digested ΦX174 DNA ladder (in bp). Lane 1, amplification of a paaX
350
internal fragment (primers IX5 and IX3). Lane 3, amplification of a paaY internal fragment
351
(primers IY5 and IY3). Lane 5, amplification of a paaXY intergenic fragment (primers IX25 and
352
IY25). Lanes 2, 4 and 6, control reactions of paaX, paaY and paaXY expression, respectively, in
353
which reverse transcriptase was omitted from the reaction mixture. (B) Western blot analysis of
354
PaaY content in different PA-grown E. coli cells. Lane 1, cell extract of E. coli W14 (pAAD;
355
contains a wild-type paa cluster); lane 2, cell extract of E. coli W14 (pAAD::Tn1000-6; contains
356
a disrupted paaX gene); lane 3, cell extract of E. coli W14 (pAAD::Tn1000-84; contains a
357
disrupted paaY gene); lane 4, cell extract of E. coli W14 (pCK01; control plasmid); lane M,
358
molecular mass markers. Approximately 10 g of total protein and an anti-PaaY antiserum was
359
used to detect the presence of PaaY (indicated by an arrow) in each cell extract.
18
360
361
Fig S3. (A) Nucleotide sequence of the Px promoter region of E. coli W. The sequence is
362
numbered relative to the Px transcription start site (+1). The –10 extended promoter box is
363
indicated in grey. The ATG start codon of paaX and TGA stop codon of paaK are shown in
364
lowercase letters. Direction of transcription is indicated by arrows. The PX5 and PX3 primers
365
used to amplify the PX DNA fragment (200 bp) are underlined. The PaaX-mediated protection
366
from DNase I digestion is boxed in the non-coding strand, and the nucleotides matching the
367
consensus PaaX-binding sequence are shown in bold letters.
368
(B) Comparison of PaaX operator regions in different promoters. Pa, Pz and Px refer to the
369
promoters that control the transcription of the three paa operons of E. coli W. Ppac is the
370
promoter of penicillin G acylase gene from E. coli W or Kluyvera citrophila. Nucleotides
371
matching the consensus sequence are shown in uppercase bold letters. N, indicates the distance
372
(in nt) between the inverted repeats of the operator. The -35 and -10 boxes for RNAP binding to
373
the promoters are underlined, except for the boxes of Ppac promoters which are located further
374
downstream the operator region.
375
376
Figure S4. Purification of the PaaX protein. Analysis on a 12.5% SDS-PAGE of the purification
377
process of PaaX from E. coli JM109 (pUCX2) cells as detailed in Experimental procedures . (A)
378
Lane M, “Broad Range” (BioRad) molecular mass markers; lane 1, soluble fraction of the crude
379
extract of E. coli JM109 containing pUC18 as a negative control; lane 2, soluble fraction of the
380
crude extract from E. coli JM109 (pUCX2) cells; lane 3, soluble fraction after polyethylenimine
381
precipitation; lane 4, soluble fraction after dialysis; lane 5, protein fraction resuspended after
382
ammonium sulfate precipitation; lane 6, protein fraction resuspended after dialysis. (B) Lane M,
19
383
molecular mass markers; lane 1, purified PaaX protein after Sephadex G-100 chromatography.
384
The PaaX protein is indicated with an arrow.
385
386
Figure S5. PaaX competes with the RNAP for the interaction at the Px promoter. The interaction
387
of RNAP and PaaX to the Px promoter was monitored by gel retardation assays as detailed in
388
Experimental procedures. Lane 1, PX probe. Lane 2, PX probe and RNAP (150 nM). Lanes 3-5,
389
increasing concentrations of PaaX (10, 50 and 100 nM, respectively), the PX probe and RNAP
390
(150 nM) were added simultaneously. Lane 6, PaaX (100 nM), PX probe and RNAP (150 nM)
391
were incubated in the presence of 500 µM PA-CoA. Lane 7, PX probe and PaaX (100 nM). - and
392
+, indicate the absence or presence of purified proteins, respectively. The unbound PX probe and
393
the PX/RNAP and PX/PaaX complexes are indicated by arrows.
394
395
Figure S6. In vitro binding of purified PaaX, RNAP and CRP to the Pz promoter. Gel retardation
396
analyses were performed as indicated under Experimental procedures. - and +, indicate the
397
absence or presence of purified proteins and PA-CoA. The unbound PZ probe and the
398
PZ/RNAP/CRP and PZ/PaaX complexes are indicated by arrows. Lane 1, PZ probe. Lanes 2 to
399
7 contain 150 nM of RNAP. Lanes 3 to 7, contain 100 nM CRP. Lanes 4 to 6, contain 10, 50 and
400
200 nM of PaaX, respectively; in the rest of cases PaaX was added at 200 nM. Lane 7, PA-CoA
401
was added at 500 µM. Proteins were incubated simultaneously. Increasing concentrations of
402
PaaX cause a decrease in the yield of the RNAP-PZ complex and the appearance of a gel
403
retardation band corresponding to the PaaX-PZ complex. We performed the retardation assays in
404
the presence of CRP and cAMP, although we have observed that RNAP can bind to the Pz
405
promoter even in the absence of CRP. The addition of PA-CoA to the retardation assays
406
abolished the binding of PaaX and restored the formation of the RNAP-PZ complex.
20
407
408
Figure S7. In vitro binding of purified PaaX, RNAP and CRP to the Pa promoter. Gel retardation
409
analyses were performed as indicated under Experimental procedures. - and +, indicate the
410
absence or presence of purified proteins and PA-CoA. The unbound PA probe and the
411
PA/RNAP/CRP/PaaX, PA/RNAP/CRP, PA/CRP and PA/PaaX complexes are indicated by
412
arrows. Lanes 2 to 7, contain 150 nM of RNAP. Lanes 3 to 7, contain 100 nM CRP. Lanes 4 to 6,
413
contain 10, 50 and 200 nM of PaaX, respectively; in the rest of cases PaaX was added at 200 nM.
414
Lane 7, PA-CoA was added at 500 µM. Lane 9, PA probe and 200 nM CRP. Binding of RNAP to
415
the Pa promoter depends on the presence of cAMP-CRP. RNAP is able to bind to the Pa
416
promoter even in the presence of increasing concentrations of PaaX, and a low mobility complex
417
due to the interaction of the PA probe with RNAP, PaaX and cAMP-CRP is observed.
418
419
Figure S8. The paaY mutation causes a change in cell morphology. Cultures of E. coli W14
420
harboring plasmid pAAD (paa wild type) or pAAD::Tn1000-84 (paaY mutant) grown in PA-
421
containing minimal medium at an A600 of 0.7 were observed by phase contrast microcopy.
422
423
Figure S9. Gel retardation analysis of PaaX binding to the Pa-Pz promoter region in the absence
424
(A) or presence (B) of the PaaY protein. Protein extracts of E. coli W14 (pAFX), that
425
overexpresses paaX, and E. coli W14 (pQEYES), that overexpresses paaY (Table S1), were used.
426
The PA-PZ probe used contains the divergent Pa and Pz promoters. The positions of the free
427
probe and the PA-PZ/PaaX complexes I and II are shown with arrows. (A) 0.08 µg of crude
428
extract from E. coli W14 (pAFX) was used. Increasing concentrations of PA-CoA, 10, 25, 50,
429
250, 500 and 1000 µM, were added in lanes 3 to 8, respectively. (B) 0.08 µg of crude extract
430
from E. coli W14 (pAFX) and 0.03 µg of crude extract from E. coli W14 (pQEYES) were used.
21
431
Increasing concentrations of PA-CoA, 10, 25, 50, 250, 500 and 1000 µM, were added in lanes 3
432
to 8, respectively.
433
434
Figure S10. Structure of PaaY. (A) 3D structural model of the PaaY trimer, viewed parallel to
435
the molecular threefold axis, emphasizing the triangular LH domain. The positions of the
436
putative metal atoms are indicated by small pink spheres. (B) Ribbon diagram of the PaaY
437
monomer viewed perpendicular to the molecular threefold axis. (C) Structure-based sequence
438
alignment of the LH domain of PaaY. Residues corresponding to seven complete or partial coils
439
(C1 to C7) are aligned. Each complete coil is composed of three flat -strands (PB1, PB2 and
440
PB3) separated by three turns (T1, T2 and T3). The most highly conserved residues are shaded in
441
blue [LIV], yellow [GAED] and purple [STAV]. The three conserved histidines (H65, H82 and
442
H87) are marked in red.
443
444
Figure S11. Purification process of the PaaY protein. SDS-PAGE (12.5%) analysis of the
445
fractions obtained in the purification process as described in Experimental procedures. Lane
446
M, “Broad Range” (BioRad) molecular mass markers; lane 1, 10 μg of protein extract of E.
447
coli M15 (pREP4, pQE32); lane 2, 15 μg of protein extract of E. coli M15 (pREP4, pQEYES)
448
after centrifugation at 14000 rpm for 15 min; lane 3, 12 g of extract after precipitation with
449
60% ammonium sulfate; lane 4, 6 μg of the protein fraction recovered from the Phenyl-
450
Sepharose column; lane 5, 1.5 μg of protein recovered from the Sephadex G-100 column; lane
451
6, 1 μg of protein recovered from the DEAE cellulose column. The PaaY protein is indicated
452
with an arrow.
453
22
454
Figure S12. Study of the oligomerization state of the PaaY protein in solution by analytical
455
ultracentrifugation experiments. The symbols (●) represent the experimental data obtained at
456
13000 rpm, 20 ºC, of a PaaY protein concentration of 47 M in 25 mM Tris-HCl buffer pH 8,
457
100 mM NaCl. Data did not change significantly with protein concentration over the range
458
examined (6-47 μM). The solid line represents the best fit of the experimental data to the
459
sedimentation equilibrium gradient of a single protein trimer species (Mw = 63,740). The upper
460
plot shows the residuals expressed as the difference between the experimental and the fitted
461
data.
462
23
463
Figure S1
464
465
466
24
467
Figure S2
468
469
25
470
Figure S3
471
472
473
26
474
475
Figure S4
476
477
27
478
Figure S5
479
480
481
482
28
483
Figure S6
484
485
486
29
487
Figure S7
488
30
489
Figure S8
490
491
492
493
31
494
495
496
Figure S9
497
498
32
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
Figure S10
A.
B.
C.
n
n+1
n+2 n+3 n+4
n+5
Consensus: [LIV] – [GAED] – X – X – [STAV] – X
PB1
10
28
49
71
89
106
122
TPV
DVI
RIV
DTV
CII
GAV
KAE
T1
PB2
VPEESF
LGKGVY
VKDGAN
VGEDGH
RRNAL
IGENSI
MPANYL
T2
PB3
VHPTAV
VGPNAS
IQDNCV
IGHSAI
VGHNAV
VGASAF
IVGS
L
L
M
L
V
V
T3
IG
RG
HG
HG
MD
27
45
66
88
105
121
136
C1
C2
C3
C4
C5
C6
C7
33
524
525
526
Figure S11
527
528
34
529
Figure S12
530
35