AEM Accepted Manuscript Posted Online 18 September 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01883-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Metabolic pathway involved in 2-methyl-6-ethylaniline
2
degradation by Sphingobium sp. strain MEA3-1 and the
3
cloning of a novel flavin-dependent monooxygenase system
4
meaBA
5 6
Weiliang Dong,a† Qiongzhen Chen,a† Ying Hou,b Shuhuan Li,a Kai Zhuang,a Fei Huang,a Jie
7
Zhou,a Zhoukun Li,a Jue Wang,a Lei Fu,a Zhengguang Zhang,d Yan Huang,a Fei Wang,c
8
Zhongli Cuia*
9 10
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture,
11
College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, P.R. Chinaa;
12
College of Food and Bioengineering, Henan University of Science and Technology, Luoyang,
13
471003, P.R. Chinab;
14
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang,
15
330045, P.R. Chinac;
16
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, P.R. Chinad
17 18 19 20
*Author for correspondence: Zhongli Cui
21
E-mail address:
[email protected]; Tel. (+86) 25 84396753; Fax. (+86) 25 84396753.
22
†
These authors have contributed equally to this work.
23
ABSTRACT
24
2-methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the
25
chloroacetanilide herbicides acetochlor and metochlor. Sphingobium sp. strain
26
MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol
27
compounds as sole carbon and energy sources for growth. The mutant strain
28
MEA3-1Mut, which only converts MEA to 2-methyl-6-ethyl-hydroquinone (MEHQ)
29
and 2-methyl-6-ethyl-benzoquinone (MEBQ), was isolated. MEA may be oxidized by
30
the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA),
31
which can be hydrolytically deaminated to MEBQ or MEHQ spontaneously. The
32
MEA microbial metabolic pathway was reconstituted based on the substrate spectra
33
and the identification of the intermediate metabolites both in the wild-type and mutant
34
strains. Plasmidomic sequencing indicated that both strains harbored 7 plasmids with
35
the sizes ranging from 287,745-bp to 6,108-bp. Among the 7 plasmids, 6 were
36
identical and pMEA02’ in strain MEA3-1Mut lost a 37,000-bp fragment compared
37
with pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and
38
protein mass fingerprints (PMF) showed that MEA3-1Mut lost a two-component
39
flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded in the lost
40
fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with
41
oxygenase components of some TC-FDMs, whileas MeaB showed no sequence
42
identity with the reductase components of those TC-FDMs. Complementation with
43
meaBA in MEA3-1Mut and heterologous expression in Psedomonas putida KT2440
44
resulted in the production of an active MEHQ monooxygenase.
45
INTRODUCTION
46
Chloroacetanilide herbicides are widely used throughout the world (1) for the control
47
of most annual grasses and certain broadleaf weeds (2). The majority of commonly
48
used chloroacetanilide herbicides, such as alachlor, acetochlor, butachlor and
49
metolachlor, are N-alkoxyalkyl-N-chloroacetyl-substituted aniline derivatives (3). The
50
long-term application of these herbicides has caused negative impacts in both the
51
aquatic environment and agricultural ecosystems (4, 5). Acetochlor has been
52
classified as a B-2 carcinogen and a probable human carcinogen by the United States
53
Environmental Protection Agency (EPA) (4) and several chloroacetanilide herbicides
54
have been proven to cause tumors in rats (6). Thus, there is great concern about the
55
behavior and fate of chloroacetamide herbicides and their degradation metabolites in
56
the environment.
57
Although chloroacetamide herbicides may be degraded through chemical and
58
physical processes, microbial metabolism is the main mechanism responsible for the
59
herbicides degradation in natural soils (7, 8). A variety of bacterial strains that are
60
able to degrade butachlor, alachlor, acetochlor, and metolachlor have been
61
characterized (9, 10). In the degradation pathway, these herbicides are N-dealkylated
62
to 2-chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) (for alachlor and butachlor) or
63
2-chloro-N-(2-methyl-6-ethylphenyl) acetamide (CMEPA) (for acetochlor and
64
metolachlor),
65
2-methyl-6-ethylaniline (MEA), respectively (2, 10). Oxygenase systems are involved
66
in bacterial degradative N-dealkylation. A novel three-component Rieske non-heme
which
are
then
converted
to
2,6-diethylaniline
(DEA)
or
67
iron oxygenase system was determined to catalyze the N-dealkylation of
68
chloroacetamide herbicides in Sphingomonas strains DC-6 and DC-2 (3). A
69
cytochrome
70
N-deethoxymethylation of acetochlor by Rhodococcus sp. strain T3-1 (11). Two genes,
71
cmeH and damH, were cloned from Sphingobium quisquiliarum strain DC-2 and
72
Delftia sp. strain T3-6, respectively, and shown to encode amidases that catalyzes the
73
amide bond cleavage of CDEPA or CMEPA to DEA or MEA (10, 12).
P450
system
EthBAD,
was
reported
to
involve
in
the
74
Alkyl substituents of the aniline derivatives of MEA are located on both sides of
75
the amine group. This special chemical structure makes MEA more difficult to be
76
degraded in natural soils (13). Under both batch and continuous operations, an
77
oxidation pathway for 2,6-dimethylaniline (DMA) via hydroxyl radicals (OH•) was
78
proposed to use Fenton’s reactions in which 2,6-dimethyl-phenol (DMP),
79
2,6-dimethyl-hydroquinone (DMHQ), 2,6-dimethyl-p-benzoquinone (DMBQ), and
80
3-hydroxy-2,6-dimethyl-p-benzoquinone (3-OH-DMBQ) were detected as aromatic
81
byproducts
82
4-hydroxy-2,6-dimethylaniline (4-OH-DMA) in cultured mammalian cells by a P450
83
monooxygenase system (14). Sphingobium baderi strain DE-13 is capable of
84
degrading MEA to the intermediate 4-OH-MEA, which is further transformed to
85
2-methyl-6-ethyl-benzoquinone imine (MEBQI) (10). Zhang et al proposed a DEA
86
dealkylation process during the degradation of butachlor by Paracoccus sp. strain
87
FLY-8 (9). However, the molecular basis and further metabolic pathways for DEA,
88
MEA, DMA and DMP in microorganisms remain unclear.
(13).
Chao
reported
that
DMA
was
transformed
to
89
The biochemical pathway of acetochlor degradation by a three-bacterial
90
consortium was proposed (acetochlor to CMEPA by Rhodococcus sp. strain T3-1,
91
CMEPA to MEA by Delftia sp. strain T3-6 and MEA by Sphingobium sp. strain
92
MEA3-1) based on the identified degradation intermediates (2). Recently,
93
acetochlor and CMEPA metabolisms have been described in Rhodococcus sp. strain
94
T3-1 and Delftia sp. strain T3-6 (11, 12). In this study, we clarified the partial
95
metabolic pathway responsible for MEA degradation and cloned a novel
96
flavin-dependent monooxygenase system MeaBA involve in MEA degradation.
97 98 99 100 101 102 103 104 105 106 107 108 109 110
111
MATERIALS AND METHODS
112
Chemicals and Media. MEA was purchased from Qingdao Vochem Co., Ltd.
113
(Qingdao, China), 4-OH-DMA and DMHQ (structural analogues of 4-OH-MEA and
114
MEHQ)
115
alkyl-substituted aniline or phenol compounds were purchased from Sinopharm
116
Chemical Reagent Co., Ltd. (Beijing, China). All molecular reagents were purchased
117
from TaKaRa Co., Ltd. (Dalian, China). All chemicals used in this study were of
118
analytical grade or higher purity. Minimal salts medium (MSM), Lysogeny Broth (LB)
119
medium and 1/3 LB medium were used for the strains cultured in this study (15).
were
purchased
from
Sigma-Aldrich
(Shanghai,
China).
Other
120 121
Bacterial strains, plasmids, and culture conditions. The strains, plasmids and
122
primers used in this study are listed in Table 1. Escherichia coli strains were routinely
123
grown aerobically at 37°C and 180 r/min in LB medium. Strains MEA3-1 and
124
MEA3-1Mut were grown aerobically at 30°C and 180 r/min in 1/3 LB medium (16)
125
unless otherwise indicated. Pseudomonas putida strain KT2440 was grown
126
aerobically at 30°C and 180 r/min in LB medium.
127 128
Isolation of spontaneous mutant strains. Sphingobium sp. strain MEA3-1 (China
129
Center for Type Culture Collection [CCTCC] no. M 2012527) was cultured on 1/3 LB
130
plates without selection, and isolated colonies were transferred to fresh 1/3 LB plates.
131
After continuous transfers, colonies with different morphotypes were observed and
132
purified. The ability of the resulting isolates to degrade MEA was determined as
133
described below. The mutant strain that lost the ability to mineralize MEA was
134
designated MEA3-1Mut. The ERIC-PCR pattern (17) and 16S rRNA gene sequence
135
of strain MEA3-1Mut were determined and compared with the wild type strain
136
MEA3-1.
137 138
Degradation experiment. The wild-type and mutant strains were cultured in 1/3 LB
139
medium, harvested by centrifugation (Allegra X-22R Centrifuge, F0630 rotor,
140
Beckman Coulter, USA) at 2,180 ×g for 10 min at 4°C , washed twice with fresh
141
MSM, and then resuspended in MSM to an approximate optical density at 600 nm
142
(OD600) of 2.0 as the inoculum. The 1.0-ml inocula of wild-type and mutant strains
143
were inoculated into a 100-ml Erlenmeyer flask containing 50 ml MSM supplemented
144
with 100 mg/liter different aromatic compounds as the sole carbon source at 180
145
r/min and 30°C, respectively. These aromatic compounds included phenol and
146
the aniline derivatives DEA, DMA, aniline, 2,3-dimethyl-aniline (2,3-DMA),
147
2,4-dimethyl-aniline (2,4-DMA), DMHQ, 4-OH-DMA, 2-methyl-aniline (2-MA),
148
toluene, O-xylene, phenol, catechol, hydroxyquinol, 2-methyl-phenol (2-MP),
149
2,6-dimethyl-phenol
150
3,4-dimethyl-phenol (3,4-DMP). The degradation of these aromatic compounds was
151
measured using HPLC. All treatments were performed in triplicate, and control
152
experiments without inoculation or without substrates were performed under the same
153
conditions.
154
(2,6-DMP),
3,5-dimethyl-phenol
(3,5-DMP)
and
155
Identification of MEA degradation metabolites. The 2.0-ml inocula of strains
156
MEA3-1 and MEA3-1Mut as described above were inoculated into a 250-mL
157
Erlenmeyer flask containing 100 ml MSM supplemented with 100 mg/liter MEA,
158
respectively. When the concentration of MEA decreased to approximately 50 mg/liter,
159
the supernatant was collected by centrifugation at 12,580 ×g for 10 min at 4°C, and
160
lyophilized. Then, the residual was dissolved in 1 ml of methanol, which was filtered
161
through 0.22-μm Millipore membrane (Sangon Biotech., Shanghai, China) for
162
HPLC-MS/MS analysis.,A separation column (internal diameter, 4.6 mm; length, 250
163
mm) filled with Kromasil 100-5-C18 was used for HPLC analysis. The mobile phase
164
was methanol:water (80:20, vol/vol) and the flow rate was 0.8 ml/min. The detection
165
wavelength was 240 nm, and the injection volume was 20 μl. MS analysis were
166
performed in ESI mode with an Agilent G6410B Triple Quad Mass Spectrometer. In
167
the MS analysis, the metabolites were separated and ionized by electrospray with a
168
positive polarity. Characteristic fragment ions were identified by second-order MS
169
and compared to those generated with authentic or structural analogue standards.
170 171
Plasmid profiling. Plasmids from strains MEA3-1 and MEA3-1Mut were isolated
172
using a modified alkaline extraction method (18). After 12 hours of incubation
173
(logarithmic phase), approximate 5×109 cells were harvested by centrifugation at
174
12,580 ×g for 10 min at 4°C, and resuspended in 1.0 ml of buffer I (1 M NaCl and 50
175
mM glucose in TE buffer, pH 8.0) after washing with 5 ml TE buffer (10 mM
176
Tris-HCl, 1 mM EDTA, pH 8.0). The cells were incubated with 20 μg/ml RNase and
177
100 μg/ml lysozyme at 37°C for 30 min until the cells began to lyse. Next, 2.0 ml
178
freshly prepared buffer II (0.2 M NaOH, 10 g/liter SDS) was added and mixed gently.
179
The samples were placed on ice for 15 min. Then, 1.5 ml ice-cold buffer III (5 M
180
potassium acetate, 11.5% acetic acid, vol/vol) was added and mixed gently until white
181
precipitate formed. The supernatant was collected by centrifugation at 12,580 ×g for
182
15 min at 4°C. Other procedures and manipulations (phenol/chloroform extraction
183
and isopropanol precipitation) were operated as described by O’Sullivan (18).
184
The isolated plasmids were visualized using pulsed-field gel electrophoresis
185
(PFGE, Bio-Rad) (19). PFGE was performed in TBE buffer (20 mM Tris-HCl, 20
186
mM boric acid, 0.5 mM EDTA, pH 8.0) at 12.5°C at 6 V/cm with linearly increasing
187
pulse times from 10 to 20 s for 24 h. HindШ-digested lambda phage DNA was used
188
as a molecular size standard.
189 190
Plasmidomic sequencing, assembly, and annotation. The plasmids from strains
191
MEA3-1 and MEA3-1Mut were isolated as described above, and the linear DNA in
192
the plasmidomes was digested using ATP-dependent DNase. The 16S-F/16S-R primer
193
pair, which targets the 16S rRNA gene, was used to detect residual chromosomal
194
DNA.
195
shotgun-sequenced (20) with a Roche 454 Genome Sequencer FLX Titanium platform
196
(Han-Yu Biological Technology Co. Ltd.) (21). The depth of sequencing and the
197
fold-coverage of the plasmid draft were 20× and 99%, respectively. Sequencing reads
198
were
The
plasmidomes
assembled
using
of
the
strains
SOAP
MEA3-1
de
novo
and
MEA3-1Mut
software
(version
were
1.05;
199
http://soap.genomics.org.cn/soapdenovo.html).
200
conducted
201
http://ccb.jhu.edu/software/glimmer/index.shtml).
202
combined with sequences from the KEGG, COG, Swiss-Prot and non-redundant
203
protein databases, was used to accomplish functional annotation with an E-value
204
cutoff of 1E-5.
using
De
novo
Glimmer
gene
prediction
(version The
BLAST
was 3.0;
program
(22),
205
The gaps predicted by MUMmer and BLAST were closed using ContigScape
206
(23), which interactively displayed the relationships between plasmidome contigs,
207
thereby allowing a faster and more precise determination of linkages and greatly
208
improving the efficiency of gap closure (23). Gap closure was verified by PCR and
209
SEFA-PCR (24) amplification; all gap closure primers are shown in Table S1.
210 211
Protein extraction and 2-DE analysis. Crude enzyme extracts of strains MEA3-1
212
and MEA3-1Mut were prepared using ultrasonic disruption (Sonicator 201 M, Kubota,
213
Japan) for 10 min at 4°C in a lysis buffer (7 M urea, 2 M thiourea, 4% wt/vol CHAPS,
214
0.2% wt/vol Bio-lytes, pH 3-10, 65 mM DTT) and centrifuged at 12,580 ×g for 20
215
min at 4°C to remove cell debris. Protein concentration was determined using a
216
modified Bradford assay (25) with ovalbumin as the standard.
217
The proteins in the supernatant were analyzed using 2-DE. For each replicate,
218
100 μg of total protein extract was loaded onto immobilized pH gradient dry strips (17
219
cm, pH 4-7 linear gradient, Bio-Rad, USA) during the rehydration step (13 h),
220
followed by focusing for a total of 60,000 Vh using a Protean IEF Cell (Bio-Rad,
221
USA). After isoelectric focusing, the gel strips were equilibrated in 5 mL equilibration
222
buffer (0.375 M Tris-HCl at pH 8.8, 6 M urea, 20% vol/vol glycerol, 2% wt/vol SDS,
223
and 2% wt/vol DTT) for 15 min and then re-equilibrated again for 15 min in the same
224
buffer, except DTT was replaced with iodoacetamide (2.5% wt/vol) (26). SDS-PAGE
225
in the second dimension was performed in 12% SDS-polyacrylamide gels (25 cm×20
226
cm) and sealed with low-melting agarose (0.5% wt/vol). Electrophoresis was
227
performed using a Protean Plus Dodeca cell apparatus (Bio-Rad, USA) at 50 V for the
228
first 30 min, followed by 150 V for 8 h. The protein spots were visualized using mass
229
spectrometry-compatible silver staining (27).
230 231
Protein mass fingerprints (PMF). The gels were scanned using a UMAX Powerlook
232
III scanner (UMAX Technologies, USA) at 300-dpi resolution, and images of the gels
233
were analyzed with PDQuest (version 8.0, Bio-Rad). Selected protein spots were
234
manually excised from the gels for MALDI-TOF MS analysis (Bo-Yuan Biological
235
Technology Co. Ltd.). The resulting peptide fragments were analyzed by searching
236
against the plasmidomic databases of strains MEA3-1 and MEA3-1Mut as described
237
below.
238 239
Cloning of meaBA. The lost genes in Sphingobium sp. strain MEA3-1 that may be
240
involved in MEA degradation were analyzed in detail by searching against the NCBI
241
and PDB databases. Possible monooxygenase genes involved in MEA degradation,
242
designated as meaBA, were further studied by sequence analysis and functional
243
verification as described below.
244
For phylogenetic analysis, the amino acid sequences of MeaA and MeaB were
245
first aligned using ClustalX (version 2.1) (28) and then imported into MEGA (version
246
5.0) (29) to construct a phylogenetic tree via the neighbor-joining method. Distances
247
were calculated using the Kimura two-parameter distance model. Confidence values
248
for the branches of the phylogenetic tree were determined using bootstrap analysis
249
based on 1,000 resamplings.
250 251
Functional complementation of MEA3-1Mut and KT2440 with MeaBA. Genomic
252
DNA was extracted and purified from strain MEA3-1 via high-salt precipitation (11).
253
Three PCR fragments (1,224-bp, 2,069-bp and 6,247-bp) containing the 60-bp
254
potential promoter region of meaB were amplified using the primers pairs
255
MeaAp-F/MeaA-R，MeaBp-F/MeaA-R and MeaBp-F/Orf4-R (Table 1), designated
256
meaA, meaBA and meaBA-orf1-4, respectively. The three fragments were digested
257
with SacI and XhoI and inserted into the corresponding sites of the broad-host-range
258
plasmid pBBR1MCS-2 (30), to generate pBBR-meaA, pBBR-meaBA and
259
pBBR-meaBA-orf1-4, respectively. The inserted fragments in the three plasmids were
260
verified by sequencing. The resulting three plasmids were transformed into E. coli
261
DH5α. Then, three plasmids were introduced into strains MEA3-1Mut and KT2440
262
(47) via tri-parental mating (31) using pRK600 (56) as the helper plasmid. The
263
abilities of the strains harboring different plasmids to degrade MEA and MEHQ were
264
determined using a whole cell biotransformation assay as described by Liu et al. (32).
265
Samples were taken at regular intervals, the concentrations of the substrates were
266
determined rapidly using an ultraviolet-visible spectrophotometer, and the bacterial
267
growth was monitored by measuring the colony-forming units (cfu/ml).
268 269
Effect of metyrapone on the degradation of MEA. Metyrapone is a specific
270
inhibitor of cytochrome P450 monooxygenase systems (33). Therefore, 200 μl MSM
271
containing 0.5 mM MEA and different concentrations of metyrapone (100 mg/liter,
272
200 mg/liter, or 300 mg/liter) were added into 96-well plates inoculated with strain
273
MEA3-1. The concentration of MEA was measured by color development with
274
4-aminoantipyrene and potassium hexacyanoferrate (12). Reaction mixtures without
275
inoculation and metyrapone were used as the negative and positive controls,
276
respectively.
277 278
Simulation of 4-OH-MEA hydrolytic deamination. 4-OH-DMA, a structural
279
analogue of 4-OH-MEA, was used to simulate the spontaneous hydrolytic
280
deamination process. Briefly, 200 μl of pure water containing 0.5 mM 4-OH-DMA
281
were added into 96-well plates and Nessler's reagent was added into at an intervals to
282
determine the release of ammonia (34).
283 284
Nucleotide sequence accession numbers. The GenBank accession number of the
285
12,052-bp DNA fragment containing the meaBA gene cluster and orf1-9 is KP752077.
286
The sequence of the plasmidome from Sphingobium sp. strain MEA3-1 has been
287
deposited with GenBank under accession numbers CM003352-CM003358.
288 289
RESULTS
290
Isolation of MEA-degradation-deficient mutants and metabolites identification.
291
The MEA degradation phenotype of Sphingobium sp. strain MEA3-1 was quite
292
unstable. Large and small colonies appeared when freshly streaked from -80°C frozen
293
samples or after continuous transfers on 1/3 LB medium without selective pressure.
294
The 16S rRNA gene sequence of the mutant strain exhibited 100% similarity with that
295
of strain MEA3-1. The ERIC-PCR (17, 35) patterns of both strains exhibited identical
296
fragment distributions (Fig. S1). These results indicated that the larger-size colony
297
was indeed an MEA-degradation-deficient mutant of strain MEA3-1 and was
298
designated Sphingobium sp. strain MEA3-1Mut.
299
Strain MEA3-1 was able to completely degrade MEA as detected by HPLC
300
analysis, and utilize MEA as the sole carbon source for growth (2). However, the
301
whole cell transformation experiments indicated that strain MEA3-1Mut could only
302
transform MEA into an unidentified metabolite (Fig. 1). The MS/MS results indicated
303
that the MEA substrate, with a tR (retention time) of 5.68 min, has a prominent
304
protonated molecular ion at m/z 136 and fragment ions of m/z 120, 108, and 91 (Fig.
305
1A). Product A, with a tR of 5.08 min, has a molecular ion at m/z 152 and fragment
306
ions of m/z 135, 123, and 107, which correspond to a hydroxylated form of MEA (Fig.
307
1C). Product A was proposed to be 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA).
308
Product B, with a tR of 4.61 min, has a molecular ion at m/z 153, and fragment ions of
309
m/z 136, 121, and 108, which correspond to the hydrolytic deamination of product A
310
(Fig. 1B). Product B was proposed to be 2-methyl-6-ethyl-hydroquinone (MEHQ).
311
The possible spontaneous oxidation products of 4-OH-MEA and MEHQ,
312
2-methyl-6-ethyl-benzoquinone imine
313
2-methyl-6-ethyl-benzoquinone (MEBQ) at m/z 151, respectively, were also detected
314
using HPLC-MS (data not shown). The MS/MS fragment results were analyzed based
315
on the compound structures (Fig. 1).
(MEBQI) at
m/z
150
and
316 317
Identification
318
Sphingobium sp. strain MEA3-1 can utilize MEA as a sole carbon and energy source,
319
but cannot utilize aniline for growth (36), indicating that strain MEA3-1 employs
320
different pathways to degrade alkyl-substituted aniline compounds. To clarify the
321
MEA degradation pathway in this strain, the degradation intermediates were analyzed.
322
The same degradation products, 4-OH-MEA and MEHQ, were detected using
323
HPLC-MS, in the culture medium extracts of strain MEA3-1 during the
324
biodegradation of MEA (data not show). Another metabolite, product C (with a tR of
325
2.70 min), exhibited a molecular ion at m/z 169, and fragment ions of m/z 152, 134
326
and 115.7, which correspond to the hydroxylation form of MEHQ. Product C was
327
proposed to be 3-hydroxy-2-methyl-6-ethyl-hydroquinone (3-OH-MEHQ, Fig. 1D),
328
and
329
3-hydroxy-2-methyl-6-ethyl-benzoquinone (3-OH-MEBQ), with a molecular ion of
330
m/z 167 and the same MS/MS fragment ions was also detected.
the
of
MEA degradation
possible
metabolites
spontaneous
from
strain
oxidation
MEA3-1.
product,
331
In addition to 4-OH-MEA, MEHQ was also detected in the MEA metabolites.
332
We speculate that MEBQI, the 4-OH-MEA oxidation product, can spontaneously
333
hydrolytically deaminate to MEBQ or MEHQ. 4-OH-DMA, a structural analogue of
334
4-OH-MEA, was used to simulate the spontaneous hydrolytic deamination process
335
using Nessler's reagent colorimetry. As shown in Fig. S2, 4-OH-DMA was rapidly
336
spontaneously deaminated by hydrolysis in pure water. Considering their similar
337
structures, the similar reaction may also occur with 4-OH-MEA.
338 339
The substrate degradation spectra of the wild-type and mutant strains. Using
340
HPLC analysis, the degradation of alkyl-substituted aniline or phenol compounds by
341
wild-type and mutant strains were monitored. The degradation results are shown in
342
Table 2. Wild-type strain MEA3-1 degraded most alkyl-substituted aniline or phenol
343
compounds except for toluene, xylene, aniline, 2,3-DMA, 2,4-DMA and 3,4-DMP.
344
These results indicated that strain MEA3-1 employs different pathways to degrade
345
alkyl-substituted aniline compounds, and this metabolic pathway is similar to that of
346
alkyl-substituted phenol compounds. However, the mutant strain MEA3-1Mut was
347
only able to transform alkyl-substituted aniline or phenol compounds, it is worth
348
noting the mutant strain could not degrade 4-OH-DMA or DMHQ, which are
349
structural analogues of 4-OH-MEA and MEHQ, respectively. These results indicate
350
that the degradation of MEA by strain MEA3-1 is subject to para-hydroxylation of
351
the ring. Given the MEA degradation metabolites of strains MEA3-1 and
352
MEA3-1Mut, a possible pathway for MEA degradation by Sphingobium sp. MEA3-1
353
is illustrated in Fig. 2, and MEA3-1Mut has likely lost a key gene or gene cluster for
354
MEBQ degradation.
355 356
Plasmidomic sequencing. Considering the unstable degradative phenotype, we
357
deduced that the genes encoding the MEA-degrading enzymes were located on
358
plasmids. The plasmid profiles of MEA3-1 and MEA3-1Mut were analyzed using
359
PFGE. Seven plasmids, designated pMEA01, pMEA02 (pMEA02’ for strain
360
MEA3-1Mut), pMEA03, pMEA04, pMEA05, pMEA06, and pMEA07, were detected
361
(Fig. 3). By comparing the plasmid profiles of strains MEA3-1 and MEA3-1Mut, six
362
of the seven plasmids were found to shift identically in the PFGE of both strains.
363
Compared with its counterpart pMEA02 in strain MEA3-1, a smaller plasmid
364
pMEA02’, was observed in the mutant (Fig. 3). We deduced that pMEA02’ is a
365
derivative of pMEA02 with some portions deleted.
366
The plasmidomes of strains MEA3-1 and MEA3-1Mut were sequenced and
367
subjected to gap closure. The total contig numbers of strain MEA3-1 were 213 with a
368
total length of 785,046-bp. Gaps between the contigs were closed via bioinformatic
369
analysis and primer walking with PCR products. Seven complete plasmid sequences
370
(pMEA01-pMEA07) were obtained, and a circle graph of pMEA02 from strain
371
MEA3-1 was shown in Fig. S3.
372
The largest and smallest plasmids were 287,745 bp and 6,108 bp with 413 and 10
373
ORFs, respectively. The seven plasmids were not homologous to other large plasmids
374
from Sphingomonas sp., and most of the ORFs were annotated as hypothetical
375
proteins, plasmid-relevant proteins or transposases. The 134,691-bp pMEA02
376
contained 164 ORFs with six IS6100 transposase regions and many other
377
transposase-family regions. A 24,948-bp fragment (from 132,840 to 23,097 bp in
378
pMEA02) and a 12,052-bp fragment (from 43,824 to 55,875 bp in pMEA02) were
379
lost to generate the plasmid pMEA02’ in strain MEA3-1Mut (Fig. S3). Therefore,
380
MEA degradation-related genes may be located in these contigs in pMEA02 from
381
strain MEA3-1.
382 383
2-DE and PMF analyses. Sphingobium sp. strains MEA3-1 and MEA3-1Mut total
384
proteins were analyzed using 2-DE to determine the differences associated with
385
pMEA02 in the wild-type and mutant strains. As revealed by PDQuest software
386
analysis, the protein profiles produced from the 2-DE gels were highly reproducible
387
among the three independent extractions. Figure S4 shows representative gels of the
388
soluble proteins extracted from strains MEA3-1 and MEA3-1Mut. Compared with
389
MEA3-1Mut, strain MEA3-1 had three significantly distinct protein spots, which
390
were designated A, B and C.
391
PMF analysis identified several peptide fragments from the three protein spots
392
(Table S2). Genes encoding the three proteins were located at 9382-10158 bp,
393
12,713-13,816 bp and 45,926-47,104 bp in pMEA02, which were lost in MEA3-1Mut.
394
The three proteins exhibited 100%, 99% and 99% amino acid sequence similarity
395
with
396
dehydrogenase (spot B) and hypothetical protein (spot C) from Sphingomonas sp.
3-hydroxy-2-methylbutyryl-CoA
dehydrogenase
(spot
A),
acyl-CoA
397
DC-6, respectively, as determined by searching against the NCBI protein database. It
398
is worth nothing that the strain DC-6 was reported to mineralize acetochlor
399
completely (3, 10).
400 401
ORF analysis. Spots A and B are located in the lost 24,948-bp fragment and spot C is
402
located in the lost 12,052-bp fragment. Both fragments are surrounded by the same
403
transposable element, Tnp1, in pMEA02. Tnp1 exhibited high levels of amino acid
404
sequence identity (99% and 100%) with the IS6100 transposase-like protein from a
405
carbazole-degrading strain of Sphingomonas sp. XLDN2-5 and Escherichia coli,
406
respectively (39).
407
There were no relevant hydroxylase genes in the 24,948-bp fragment encoding
408
the proteins in spots A and B (data not shown), therefore, it was not analyzed in detail.
409
A detailed search for ORFs in the 12,052-bp fragment revealed that orf5, encoding the
410
hypothetical protein (HP1) in spot C, was an oxygenase gene (Table 3). Phylogenetic
411
analysis showed that orf5 encodes an amino acid sequence that shares significant
412
identity with the oxygenase components of some TC-FDMs (Fig. 4), for example,
413
HP1 exhibits 25% identity with C2-hpaH (p-hydroxyphenylacetate 3-hydroxylase)
414
from
415
(3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione
416
Rhodococcus sp. RHA1 (41), and 22% identity with NcnH (naphthocyclinone
417
hydroxylase) from Streptomyces arenae (42).
418
Acinetobacter
baumannii
(40),
24%
identity
with
monooxygenase)
HsaA from
HsaA, NcnH and C2-hpah were previously classified as enzymes in the TC-FDM
419
family, and the consensus sequences of this family were determined (43). HP1 shares
420
partial conserved residues (W86, W183, S145, W143, R264, H367 and M363)
421
identical to the consensus amino acid sequences interacting with FMN in TC-FDM
422
(Fig. S5) (43). HP1 also contained the conserved domains COG1960 and KOG0139
423
belonging to the acyl-CoA dehydrogenase (ACAD) superfamily (44). These results
424
indicate that HP1 is a novel oxygenase component of the TC-FDM family and its
425
absence in mutant MEA3-1Mut suggests that HP1 is responsible for the hydroxylation
426
of MEHQ. orf5 was designated meaA and encodes the oxygenase component MeaA
427
of the MEHQ monooxygenase.
428
A BLAST analysis indicated that another hypothetical protein (HP2, encoded by
429
orf6), located upstream of the meaA gene shared significant identity with cytochrome
430
C (30% amino acid sequence identity with 40% coverage) from Bacillus altitudinis.
431
We aligned HP2 with the reductase components of the TC-FDMs described above and
432
found that the sequence identities with some hypothetical proteins ranged from 6 to
433
14% (40-42). We deduced that HP2 was the reduction component of a TC-FDM. The
434
HP2 was assumed to be the reductase component of MeaA and designated MeaB. The
435
reductase component of the MEHQ hydroxylase (MeaB) may perform the
436
NADH-dependent reduction of free FMN, which may be subsequently transferred to
437
the larger monooxygenase component (MeaA) and used for MEHQ monooxygenation
438
(Fig. 5C).
439 440
Functional expression of MeaBA. To further confirm the function of the genes meaA,
441
meaBA, and the completely missing fragment (meaBA-orf1-4, excluding the
442
transposable element), plasmids pBBR1MSC-2, pBBR-meaA, pBBR-meaBA, and
443
pBBR-meaBA-orf1-4 were introduced into strains MEA3-1Mut, DH5α and KT2440.
444
The whole cell transformation experiments revealed that strains MEA3-1Mut and
445
KT2440 harboring pBBR-meaBA or pBBR-meaBA-orf1-4, but not pBBR-meaA,
446
acquired the ability to degrade or thansform MEHQ (Fig. 5A). The initial and final
447
cell
448
pBBR-meaBA-orf1-4 were approximately 2×106 and 7.2×106 cfu/ml, respectively
449
(Fig.
450
pBBR-meaBA-orf1-4 acquired the ability to transform MEHQ, but could not utilize
451
MEHQ as a sole carbon source for growth (data not shown). Therefore, we confirmed
452
that MeaBA were the two components of the MEHQ monooxygenase, and orf1-orf4
453
in the 12,052-bp fragment likely do not participate in the degradation of MEA.
454
However, E. coli DH5α harboring either pBBR-meaBA or pBBR-meaBA-orf1-4
455
could not degrade MEHQ (Fig. 5A). This failure might be caused by codon usage,
456
protein folding or the low efficiency of the native meaBA promoter in E. coli DH5α
457
(48). To exclude the latter possibility, meaBA was placed under the control of a T7
458
promoter in the vector pET-29a(+) and introduced into E. coli BL21(DE3) (48). The
459
results of whole cell transformation assay showed that IPTG-induced E. coli BL21
460
(DE3) harboring pET29a-meaBAT7 was still unable to degrade MEHQ.
densities
5B).
In
of
strains
addition,
MEA3-1Mut
strain
KT2440
harboring
harboring
pBBR-meaBA
pBBR-meaBA
or
or
461 462
The possible enzyme involved in the upstream MEA metabolic pathway. We
463
deduced that a P450 monooxygenase system initiates the degradation of MEA via
464
aromatic ring hydroxylation at the para position. Metyrapone is a P450 inhibitor, and
465
its effect on MEA degradation by strain MEA3-1 was tested (Fig. S6). MEA
466
degradation could be visually observed because MEA reacts with 4-aminoantipyrene
467
to form a purple compound, and color gradually deepens as MEA concentration
468
increases. MEA degradation was inhibited when the metyrapone concentration
469
exceeded 200 mg/liter. These results show that a P450 monooxygenase system
470
possible initiates MEA degradation to transform MEA to 4-OH-MEA.
471 472 473
DISCUSSION MEA and
DEA are
important
degradation
intermediates
of
several
474
chloroacetanilide herbicides. Located on both sides of the amine group, the
475
alkyl-substituents of these aniline derivatives make their degradation processes
476
different from that of anilines in natural soils (3, 10). Microbial degradation is an
477
important process in the biogeochemical cycling and detoxification of organic
478
pollutants in the environment. Several strains have been reported to degrade MEA,
479
but the detailed degradation process is unclear (2, 10). It was proposed that DEA, a
480
metabolite of butachlor, was degraded through dealkylation to an aniline, oxidation to
481
a catechol, and ring-cleavage through an ortho-cleavage pathway in Paracoccus sp.
482
FLY-8 in vitro (9). In the present study, a novel flavin-dependent monooxygenase
483
system MeaBA for MEHQ hydroxylation was cloned and characterized, and the
484
possible metabolic pathway responsible for MEA degradation was proposed.
485
MEA was first transformed to the transitory intermediate 4-OH-MEA by a
486
potential P450 monooxygenase. The degradation of DMA, a structural analogue of
487
MEA, has been extensively studied in mammals (6, 14). It was reported that DMA
488
was transformed to 4-OH-DMA in cultured mammalian cells by a P450
489
monooxygenase system (14). It has also been shown that the human cytochrome P450
490
isoforms responsible for the metabolism of alkylated aniline derivatives were
491
CYP3A4 and CYP2B6 (6). Cytochrome P450 was proposed to involve in the first
492
degradation step of MEA by Sphingobium sp. MEA3-1. The cytochrome P450
493
inhibitor metapyrone (33) was found to inhibit the degradation of MEA by strain
494
MEA3-1 (Fig. S6). Therefore, we hypothesize that MEA is transformed to
495
4-OH-MEA in the presence of a P450 monooxygenase system. We found five
496
cytochrome P450 oxygenases annotated in the genome of strain MEA3-1. A blastp
497
anylysis indicates that these cytochrome P450 oxygenases may invovle in the
498
metabolism or synthesis of N-(1-pyrene)-acetamide, N-butyl-isocyanide and
499
cholesterol. However, whether they are invovled in MEA degradation still needs to be
500
clarified.
501
4-OH-MEA is an unstable intermediate that is oxidized rapidly to MEBQI
502
in animals and microbes (10, 14). In this study, using Nessler’s reagent to determine
503
the release of ammonia from 4-OH-DMA (34), we found that MEBQI was
504
spontaneously deaminated to MEHQ (Fig. S2). Levenbery reported the hydrolytic
505
deamination of the naturally occurring 2-amino-4-hydroxy-pteridines (pterins), which
506
yielded the corresponding 2,4-dihydroxy compounds (lumazines) (37). 4-OH-DMA,
507
the degradation product of sulfonated azo dyes, was not observed in the reaction
508
mixtures because it hydrolyzed to DMBQ spontaneously and rapidly (38).
509
Based on metabolite identification, MEHQ is further hydroxylated to form
510
3-OH-MEHQ (Fig. 1). Similar transformations have been reported in the degradation
511
of xylenol compounds. Cell extracts of Mycobacterium sp. strain DM1 catalyzed the
512
reduction
513
3-hydroxy-2,6-dimethyl-hydroquinone (3-OH-DMHQ) (49). Hofrichter et al. also
514
reported that the metabolism of DMHQ occurred via DMHQ, DMBQ, 3-OH-DMHQ,
515
and
516
biotransformation mechanism of DMHQ and MEHQ to 3-OH-DMHQ and
517
3-OH-MEHQ, respectively, is still unclear.
of
3-OH-DMBQ
2,6-dimethyl-3-hydroxyquinone
in
Penicillium
frequentans
Bi7/2
(DMHQ)
(50).
However,
to
the
518
In this study, using heterologous expression and complementation experiments,
519
we identified two genes, meaA and meaB, that encode enzymes responsible for
520
MEHQ hydroxylation. The oxygenase MeaA and reductase MeaB were obviously
521
different from previously reported TC-FDMs at the amino acid sequence level, such
522
as C2-hpaH (41), HsaAB (43) and NcnH (42). MeaB shared significant sequence
523
identity with cytochrome C from Bacillus altitudinis. The hydroxylation of
524
4-ethylphenol by the bacterial flavocytochrome p-cresol methylhydroxylase (PCMH)
525
from Pseudomonas putida has been proposed to involve in the initial formation of an
526
enzyme-bound p-quinone methide product intermediate (45, 46). The reductase
527
component of MEHQ hydroxylase (MeaB) performs the NADH-dependent reduction
528
of free FMN, which is subsequently transferred to the larger monooxygenase
529
component (MeaA) and used for the reaction of MEHQ monooxygenation (51).
530
Degradative plasmids are frequently isolated from Sphingobium species. Seven
531
plasmids sequences (pMEA01-pMEA07) were obtained from strain MEA3-1 (Fig. 3).
532
Several instances of the plasmid-encoded degradation of aromatic compounds have
533
been previously reported, such as pLB1 (a γ-hexachlorocyclohexane-degradative
534
plasmid)
535
carbofuran-degradative plasmid) from Sphingomonas sp. strain CF06 (53), and
536
pCAR3 (a carbazole-degradative plasmid) from Sphingomonas sp. strain KA1 (54).
537
Until this study, Stolz reported that, at most, five plasmids hav previously been
538
isolated from Sphingomonads, such as two plasmids from S. aromaticivorans strain
539
F199, S. wittichii strain RW1 and Novosphingobium pentaaromativorans strain US6-1;
540
three plasmids from S. japonicum strain UT26 and Novosphingobium sp. strain PP1Y;
541
four plasmids from S. xenophagum strain BN6 and S. fuligines strain ATCC27551;
542
and five plasmids from Sphingomonas sp. strain MM-1 (55). The meaBA genes are
543
located on plasmid pMEA02 and are surrounded by a transposable element from the
544
IS6100 family (Fig. 5A). This location might be the reason that MEA degradation
545
ability was prone to loss.
from
Sphingobium
japonicum
strain
UT26
(52),
pCF01-05
(a
546
The horizontal transfer of genes plays a key role in the evolution of catabolic
547
pathways, thereby facilitating bacterial adaptation to pollutant-contaminated sites (57).
548
Notably, many monooxygenase genes are associated with mobile genetic elements
549
such as IS6100, an insertion sequence classified in the IS6 family. Wang et al.
550
reported that a novel 3-phenoxybenzoate 1,2-dioxygenase gene in Sphingobium
551
wenxiniae strain JZ-1 was located between two IS6100 transposase genes, tnp1 and
552
tnp2 (58). Two identical nonylphenol monooxygenase genes were also surrounded by
553
an IS21-type insertion sequence (IS) and IS6100 in Sphingomonas sp. strain NP5 (59).
554
Dogra revealed that most of the lin genes in S. paucimobilis strains B90A, Sp+ and
555
UT26 are associated with IS6100 (60). Further research suggests that the IS6100
556
elements have a very broad host range, and their presence on plasmids (even in strains
557
in which their locations have not been ascertained) cannot be ruled out (60).
558 559
A downstream ring cleavage pathway and an initializing P450 monooxygenase still need to be identified in future research.
560 561
ACKNOWLEDGMENTS
562
This work was financially supported by the Natural Science Foundation of China
563
(Nos. 31400098, 31270095, and 31560031), the National Basic Research Program
564
(No.2015CB1505002), the Natural Science Foundation of Jiangsu Province (No.
565
BK2012029), the National Science and Technology Support Program (Nos.
566
2012BAD14B02 and 2014ZX08011-003), the 863 Project (No. 2013AA102804) and
567
the Graduate Culture and Innovation Project of Jiangsu Province (No. KYLX_0514).
568 569 570 571 572 573 574
575
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36. Zhang, T., J. Zhang, S.J. Liu, and Z.P. Liu.2008. A novel and complete gene
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700
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improving the sensitivity of progressive multiple sequence alignment through
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environmental stresses. Environmental microbiology. 17:76–90.
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49. Ewers, J., M.A. Rubio, H.J. Knackmuss, and D. Freier-Schröder. 1989.
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50. Hofrichter, M., F. Bublitz, and W. Fritsche. 1995. Cometabolic degradation of
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involved in γ-hexachlorocyclohexane degradation. Appl. Environ. Microbiol.
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2007. The Sphingomonas plasmid pCAR3 is involved in complete mineralization
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of carbazole. J. Bacteriol. 189:2007–2020.
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the chlorothalonil hydrolytic dehalogenase gene. Appl. Environ. Microbiol.
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Sphingobium wenxiniae JZ-1. Appl. Environ. Microbiol. 80:3811–3818.
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Negoro. 2012. Two identical nonylphenol monooxygenase genes linked to
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767
NP5. Microbiology 158:1796–1807.
768
60. Dogra, C., V. Raina, R. Pal, M. Suar, S. Lal, K.H. Gartemann and R. Lal.
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2004. Organization of lin genes and IS6100 among different strains of
770
hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for
771
horizontal gene transfer. J. Bacteriol. 186:2225–2235.
772
773
FIGURE LEGENDS
774
Fig. 1 HPLC and mass spectra detection of the intermediate metabolites (A) HPLC
775
and positive-ion mass spectra of the substrate MEA; (B) positive-ion mass spectra of
776
the metabolite product(MEHQ) by strain MEA3-1Mut; (C) positive-ion mass spectra
777
of the metabolite product (4-OH-MEA) by strain MEA3-1Mut; (D) positive-ion mass
778
spectra of the metabolite product (3-OH-MEHQ) by strain MEA3-1.
779
Fig. 2 The partial metabolic pathway of MEA mineralization by Sphingobium sp.
780
strain MEA3-1.
781
Fig. 3 Plasmid profiles of strains MEA3-1 and MEA3-1Mut using PFGE. M: Hind III
782
digest of lambda phage DNA.
783
Fig. 4 Neighbor-joining tree constructed using the alignment of MeaA with the amino
784
acid sequences of the oxidation components of some characterized FDMs. The
785
branches corresponding to partitions reproduced in less than 50% of the bootstrap
786
replicates are collapsed. The names of the strains and proteins are displayed in the
787
phylogenetic tree. The amidases and their accession numbers are as follows: MeaA
788
(KP752077),C2-hpah (AY566612), HsaA (Q0S811), NcnH (AAG44124), TcpA
789
(AAM55214), HpaB (YP003001901), PheA1 (ABS30825), and CndA (KJ461679).
790
CndA belongs to the oxygenase component of a Rieske non-heme iron
791
monooxygenase and was used to provide a frame of reference for the phylogeny.
792
Fi g. 5 Physical map of the 12,052-bp putative transposable element containing
793
meaBA in Sphingobium sp. strain MEA3-1. (A) The arrows indicate the size, location,
794
and direction of transcription of the ORFs. Complementation of the meaBA-disrupted
795
mutants with different regions is illustrated below the physical map; (B) Degradation
796
curve of MEHQ and the growth curve of MEA3-1Mut(pBBR-meaBA-orf1-4,
797
pBBR-meaBA and pBBR-meaA); (C) The probable scheme of the catalytic cycle of
798
MEHQ hydroxylase MeaA
799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816
817 818
Table 1 Table 1. Strains, plasmids and primers used in this study Strains or plasmids
Source or
Characteristic(s)
reference
Strains Sphingobium sp.MEA3-1
Wild-type MEA degrader; pMEA01-pMEA07;
(2)
r
Sm Sphingobium sp.MEA3-1Mut
Derivative
of
MEA3-1;
deletion
within
This study
pMEA02 Pseudomonas putida KT2440
A model strain for aromatic catabolism; Cmr,
(47)
r
Amp Escherichia coli DH5α E. coli HB101(pRK600)
Host strain for cloning vectors
TaKaRa
r
(56)
Conjugation helper strain; Cm
Plasmids pBBR1MCS-2 pBBR-meaA pBBR-meaBA pBBR-meaBA-orf1-4 pET29a-meaBAT7 pMD19-T Primers
Broad-host-range cloning vector; Kmr pBBR1MCS-5 derivative carrying meaA; Km
This study r
pBBR1MCS-5 derivative carrying meaBA; Km
This study
pBBR1MCS-5 derivative carrying 7,479-bp
This study
r
pET29a(+)derivative carrying meaBA; Km T-A cloning vectors, Amp
r
Sequence (5’ to 3’)
MeaBp-F
CTCGAGGATCGGCCATCCTATCGCTG
MeaA-R
GAGCTCTCATCGCGCCTCCGTCAGCGC
MeaAp-F
(30) r
This study TaKaRa Target gene
meaBA
CTCGAGGATCGGCCATCCTATCGCTGAACAGC
meaA
TTCGGCGGTATCTAGAGGAGGTACTTTGAATG
(fusion promoter
GAAGAGGCGCATAACAA Orf4-R
GAGCTCCTAACAATCCACCCGGACCA
16S-F
GCGTAGGATTAGCTAGTTGGT
sequence) meaBA-orf1-4
partial 16S rRNA 16S-R
819 820 821
AGCTAGTTATCATCGTTTACG
sequence
822
Table 2 Table 2. The substrate spectra of strains MEA3-1 and MEA3-1Mut a
823
Substrates
MEA3-1
MEA3-1Mut
2-methyl-6-ethyl-aniline (MEA) 2,6-diethyl-aniline (DEA) 2,6-dimethyl-aniline (DMA) Aniline 2,3-dimethyl-aniline (2,3-DMA) 2,4-dimethyl-aniline (2,4-DMA) 2-methyl-aniline (2-MA) 4-OH-DMA DMHQ Toluene O-xylene Phenol Catechol Hydroxyquinol Hydroquinone 2-methyl-phenol (2-MP) 2,6-dimethyl-phenol (2,6-DMP) 3,5-dimethyl-phenol (3,5-DMP) 3,4- dimethyl-phenol (3,4-DMP)
+b + + + + + + + + + + + + -
* * * * + + + + * * * -
824
a
825
and substrates were not degraded; *, negative for strain growth and substrates were
+, positive for strain growth and substrates were degraded; -, negative for strain growth
826
transformed to other compounds.
827
b
828
spectrophotometer. The mineralization or co-metabolic transformation of these aromatic
829
compounds was measured using HPLC, all treatments were performed in triplicate, and
830
control experiments without inoculation and without substrate were performed under the
831
same conditions.
832 833 834 835
The biomass was measured at 600 nm (OD600) using a Shimadzu ultraviolet-visible
836
Gene
Table 3
837
Table 3. Deduced function of ORFs within the missing 12,052-bp fragment
838
sequence.
Product size
Position
Homologous protein
Source
(amino acids)
GenBank
% identity
accession no.
(amino acid) 100
tnp1
264
1-795
Transposase IS6100
Escherichia coli
YP_003108355.1
orf7
138
835-1251
Aminoglycosidephosphotransferase
S. chlorophenolicum L-1
YP_004554070.1
68
orf6(meaB)
168
1251-1757
Hypothetical protein
S. chlorophenolicum
WP_013847811
31
orf5(meaA)
392
2088-3266
Pigment production hydroxylase
Rhodococcus opacus
WP_012691790
39
orf1
480
3479-4921
Isopropylmalate isomerase large subunit
S.bacterium B12
WP_022690399
81
orf2
199
4918-5517
Isopropylmalate isomerase small subunit
S. chlorophenolicum
WP_013849123
61
orf3
441
5727-7052
Homogentisate 1,2-dioxygenase
S. sp. YL-JM2C
WP_037520129
52
orf4
123
7073-7444
Hypothetical protein Veis_4652
Verminephrobacter eiseniae
YP_999367.1
58
orf8
284
7479-6625
Integrase catalytic subunit
S. wittichii RW1
YP_001260297.1
88
orf9
267
10253-11056
Putative transposase
Magnetospirillum sp. SO-1
WP_008622716.1
84
tnp1
264
12052-11258
Transposase IS6100
E. coli
YP_003108355.1
100
EF01-2
839 840 841 842 843 844 845 846 847
848
Fig. 1
849
Fig. 1 HPLC and mass spectra of the intermediate metabolites. (A) HPLC and
850
positive-ion mass spectra of the substrate MEA; (B) positive-ion mass spectra of the
851
metabolite product (MEHQ) of strain MEA3-1Mut; (C) positive-ion mass spectra of
852
the metabolite product (4-OH-MEA) of strain MEA3-1Mut; (D) positive-ion mass
853
spectra of the metabolite product (3-OH-MEHQ) of strain MEA3-1.
854 855 856
857
Fig. 2
858
Fig. 2 The proposed metabolic pathway of MEA mineralization by Sphingobium sp.
859
strain MEA3-1.
860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
886
887 888 889 890 891 892 893 894 895 896 897 898 899
Fig. 3
Fig. 3 Plasmid profiles of strains MEA3-1 and MEA3-1Mut using PFGE. M: HindIII-digested lambda phage DNA
900
Fig. 4 Sphingobium sp. MEA3-1 MeaA Acinetobacter baumannii C2-hpah Rhodococcus sp. RHA1 HsaA
61 96 97
Streptomyces arenae DSM40737 NcnH Sphingomonas sp. DC-6 CndA Ralstonia eutropha JMP134 TcpA 100
901
100
Escherichia coli BL21HpaB R. erythropolis UPV-1 PheA1
0.2
902
Fig. 4 Neighbor-joining tree constructed using the alignment of MeaA with the amino
903
acid sequences of the oxidation components of some characterized FDMs. The
904
branches corresponding to partitions reproduced in less than 50% of the bootstrap
905
replicates are collapsed. The names of the strains and proteins are displayed in the
906
phylogenetic tree. The amidases and their accession numbers are as follows: MeaA
907
(KP752077),C2-hpah (AY566612), HsaA (Q0S811), NcnH (AAG44124), TcpA
908
(AAM55214), HpaB (YP003001901), PheA1 (ABS30825), and CndA (KJ461679).
909
CndA belongs to the oxygenase component of a Rieske non-heme iron
910
monooxygenase and was used to provide a frame of reference for the phylogeny.
911 912 913 914 915 916
917
Fig. 5
918
Fig. 5 Physical map of the 12,052-bp putative transposable element containing
919
meaBA in Sphingobium sp. strain MEA3-1. (A) The arrows indicate the size, location,
920
and direction of ORF transcription. The complementation of the meaBA-disrupted
921
mutant with differing regions is illustrated below the physical map. (B) The MEHQ
922
degradation curve and the growth curve of MEA3-1Mut (pBBR-meaBA-orf1-4,
923
pBBR-meaBA and pBBR-meaA). (C) The probable scheme of the catalytic cycle of
924
MEHQ hydroxylase MeaA
925 926
