bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes
2 the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes
3 faecalis JQ135
4
5 Jiguo Qiu,a Bin Liu,a Lingling Zhao,a Yanting Zhang,a Dan Cheng,b Xin Yan, a
∗ 6 Jiandong Jiang,a Qing Hong,a and Jian He a
7
8 a Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of
9 Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China
10 b Laboratory Centre of Life Science, College of Life Sciences, Nanjing Agricultural University,
11 Nanjing, 210095, China
12
13 ∗Address correspondence to Jian He
14 Email: [email protected]; Tel: (86)-25-84396685, Fax: (86)-25-84396314
15
16 Runing title: 5-Hydroxypicolinic Acid 2-Monooxygenase HpaM
17
18 Key words: Alcaligenes faecalis JQ135, 5-hydroxypicolinic acid, biodegradation,
19 5-hydroxypicolinic acid 2-monooxygenase, 2-decarboxylative hydroxylation
20
21
1 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
22 Abstract
23 5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be
24 microbially degraded. However, the physiological, biochemical, and genetic
25 foundation of the microbial catabolism of 5HPA remains unknown. In this study, a
26 gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis
27 JQ135) was cloned and HpaM was identified as a novel monocomponent
28 FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with
29 the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of
30 Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli
31 BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography.
32 HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating
33 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the
34 presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values
35 of HpaM toward 5HPA and NADH were 45.4 μΜ and 37.8 μΜ, respectively. Results
36 of gene deletion and complementation showed that hpaM was essential for 5HPA
37 degradation in Alcaligenes faecalis JQ135.
38
39
2 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
40 Importance
41 Pyridine derivatives are ubiquitous in nature and important chemical materials
42 that are currently widely used in agriculture, pharmaceutical, and chemical industries.
43 Thus, the microbial degradation and transformation mechanisms of pyridine
44 derivatives received considerable attention. Decarboxylative hydroxylation was an
45 important degradation process in pyridine derivatives, and previously reported
46 decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study,
47 we cloned the gene cluster hpa, which is responsible for 5HPA degradation in
48 Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent
49 monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed
50 decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic
51 acid. These findings deepen our understanding of the molecular mechanism of
52 microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for
53 applications to transform useful pyridine derivatives.
54
55
3 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
56 Introduction
57 Pyridine derivatives are common natural products, as well as important artificial
58 compounds that are widely used in agriculture, pharmaceutical, and chemical
59 industries as solvents, dyes, pharmaceuticals, herbicides, and pesticides (1-3).
60 However, the increasing use of pyridine derivatives causes large amounts entering the
61 environment, thus leading to severe environmental problems (4, 5). Therefore, the
62 biodegradation or detoxication of pyridine derivatives, and their transformation to
63 useful products are of significant interest.
64 Pyridine derivatives could either be degraded or transformed by a variety of
65 bacteria, and the degradation processes are typically initialed via hydroxylation (6, 7).
66 Nicotinic acid (NA, 3-pyridinecarboxylic acid) and nicotine often served as models
67 for explore the catabolic mechanisms of pyridine derivatives (8-11). NA was initially
68 hydroxylated at the C2 of the pyridine ring via NA monooxygenase (NicAB), thus
69 producing 6-hydroxynicotinic acid (6HNA) (8). 6HNA was further decarboxylatively
70 hydroxylated at the C3 of the pyridine ring via 6HNA monooxygenase (NicC)
71 yielding 2,5-dihydroxypyridine (2,5-DHP), which was then subjected to ring-cleavage.
72 Nicotine could be degraded via both the pyridine and pyrrolidine pathways, and both
73 pathways include two hydroxylation steps. In the pyrrolidine pathway, the
74 intermediate 3-succinoylpyridine (SP) was hydroxylated at the C2 of the pyridine ring
75 via SP monoxygenase (Spm), thus generating 6-hydroxy-3-succinoylpyridine (HSP),
76 which was further 3-decarboxylatively hydroxylated to 2,5-DHP via HSP
77 monoxygenase (HspB). In the pyridine pathway, nicotine was hydroxylated at the C6
78 of the pyridine ring via nicotine hydroxylase (NDH) to 6-hydroxynicotine, while the
79 downstream intermediate 2,6-dihydroxypyridine was 3-hydroxylated to
80 2,3,6-trihydroxypyridine via 2,6-dihydroxypyridine 3-monoxygenase (DHPH) (10,
4 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
81 12). NicAB and Spm are multicomponent molybdenum-containing monooxygenases,
82 while the NicC and HspB are monocomponent flavin-dependent monooxygenases,
83 catalyzing the 3-decarboxylatively hydroxylation. However, gene coding of
84 monooxygenase catalyzing the 2-decarboxylatively hydroxylation of pyridine
85 derivatives has not been reported to date.
86 5-Hydroxypicolinic acid (5HPA) is a isomer of 6HNA and a natural pyridine
87 derivative produced by bacteria (such as Nocardia sp.) or plants (such as Gynura
88 divaricata) (13, 14). The degradation of 5HPA has only been reported in Pusillimonas
89 sp. 5HP (15). A 5-hydroxypicolinate 2-monooxygenase (catalyzing the
90 2-decarboxylative hydroxylation of 5-HPA to 2,5-DHP) was partially purified from
91 strain 5HP. However, both the amino acid sequence of the 5-hydroxypicolinate
92 2-monooxygenase, and the genetic foundation of 5HPA degradation remain unknown.
93 In this study, the gene cluster hpa involved in 5HPA degradation was cloned, and
94 a 5-hydroxypicolinic acid 2-monooxygenase HpaM was identified from Alcaligenes
95 faecalis JQ135 (Fig. 1A, B). HpaM is FAD and NADH-dependent, and catalyzes the
96 2-decarboxylative hydroxylation of the pyridine-ring in 5HPA to produce 2,5-DHP.
97
98 Results
99 Degradation of 5HPA by strain A. faecalis JQ135.
100 A. faecalis JQ135 was formerly identified as a picolinic acid (PA)-degrading
101 bacterium (16). 5HPA is a 5-hydroxylated derivate of PA that was tested for
102 degradation and utilization by the strain in a carbon and nitrogen-absent MSM. The
103 results showed that the strain A. faecalis JQ135 could completely degrade 1 mM 5HPA
104 within 36 h, and correspondingly, the OD600 of the culture increased from 0.2 to 0.5
105 ± 0.1 (Fig. 2). These results indicated that strain A. faecalis JQ135 could degrade and
5 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
106 utilize 5HPA as sole source of carbon and nitrogen for growth. In addition, attempts to
107 detect metabolic intermediates of 5HPA within the culture failed. This might because
108 little or no intermediates were excreted from strain JQ135 cells during degradation of
109 5HPA.
110
111 Transposon mutagenesis and cloning of a gene cluster involved in 5HPA
112 degradation
113 To clone the genes involved in 5HPA degradation, a transposon mutagenesis
114 library of A. faecalis JQ135 was constructed. One mutant (Z10) could not grow in
115 MSM agar containing 1 mM 5HPA and was screened from approximately 5000
116 mutants. When inoculated into liquid MSM, containing 1 mM 5HPA, mutant Z10
117 could not degrade 5HPA (Fig. 2). Furthermore, the genome of A. faecalis JQ135 was
118 determined by the PacBio system. The complete genome of the strain contained one
119 circular chromosome (4,078,346 bp) and no plasmid could be found. A total of 3,723
120 ORFs were predicted. The insertion position of the transposon, determined via the
121 DNA walking method (17), was located in gene AFA_18575 (genome position
122 4,070,825). AFA_18575 is 1,218 bp in length with a G+C content of 56.08%. The
123 deduced protein was searched against the NCBI database
124 (http://blast.ncbi.nlm.nih.gov/), using the BLASTP program (Table 1). The results
125 showed that the proteins that were related the most were flavin-containing
126 monooxygenases, such as 6-hydroxynicotinate 3-monooxygenase (NicC, sequence ID:
127 Q88FY2) from Pseudomonas putida KT2440 (identity of 31%) (8), and salicylate
128 1-monooxygenase (SalM or NahG, sequence ID: P23262) from P. p u t i d a KF715
129 (identity of 28%) (18) (Fig. 3, S1). NicC and SalM catalyzed the decarboxylatively
130 hydroxylation of 6HNA and salicylate, respectively. Downstream of AFA_18575, a
6 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
131 gene (AFA_18580) with unknown function in the DUF2236 family and a TetR-type
132 regulator gene (AFA_18585) followed (Fig. 1B; Table 1). Upstream of AFA_18575,
133 eight genes were found (AFA_18535 to AFA_18570). AFA_18535, AFA_18540, and
134 AFA_18545 were predicted to be branched-chain amino acid ABC transporter
135 substrate-binding proteins, while AFA_18550 and AFA_18555 were branched-chain
136 amino acid ABC transporter ATP-binding proteins, indicating that these five genes
137 encoded an ABC-type transporter. The next three genes AFA_18560, AFA_18565, and
138 AFA_18570, showed 57%, 53%, and 41% identities, respectively, with NicD
139 (N-formylmaleamaic acid deformylase), NicX (2,5-DHP dioxygenase), and NicF
140 (maleamate amodohydrolase) from P. p ut ida KT2440 at amino acid levels (Fig. 3).
141 Interestingly, NicD, NicX, and NicF, in combination with NicC, formed a complete
142 metabolic pathway for the transformation of 6HNA into maleic acid (8). Considering
143 the structural similarity between 5HPA and 6HNA, we presumed that these contiguous
144 genes were organized in a single cluster as well as involved in the degradation of 5HPA
145 in A. faecalis JQ135. For this study, this gene cluster was designated as hpa
146 (hydroxypicolinic acid).
147 To confirm the function of hpaM (AFA_18575), a fragment containing
148 AFA_18575 was amplified from the genome of A. faecalis JQ135 and ligated into the
149 broad-host-range vector pBBR1MCS-5, thus generating pBBR-hpaM. The mutant
150 Z10, which was introduced with pBBR-hpaM (Z10-pBBR-hpaM) regained the ability
151 to degrade 5HPA (Fig. 2), suggesting that HpaM enabled the conversion of 5HPA.
152
153 Gene knockout of hpaM and the phenotype analysis
154 To further determine the physiological function of hpaM, both the in-frame
155 deletion mutant JQ135ΔhpaM and complemented strain JQ135ΔhpaM-pBBR-hpaM
7 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
156 were constructed. The strain JQ135ΔhpaM had lost the ability to degrade and utilize
157 5HPA, while the complemented strain JQ135ΔhpaM-pBBR-hpaM recovered the
158 ability to degrade and grow on 5HPA. These results clearly demonstrate the gene
159 hpaM to be essential for the catabolism of 5HPA in A. faecalis JQ135.
160
161 Transcription levels of genes in cluster hpa in response to 5HPA
162 Real-time PCR (RT-PCR) was conducted to determine the transcription of the
163 hpa cluster and to investigate whether these genes could be induced by 5HPA. The
164 results showed that the five putative transporter-coding genes (AFA_18535 to
165 AFA_18555), hpaD, hpaX, hpaF, and hpaM comprised a transcriptional operon, and
166 were transcribed in cells grown on 5HPA containing medium, but not on glycerol (Fig.
167 1C), indicating that the cluster hpa was induced by 5HPA. These results further
168 indicate that cluster hpa was involved in the degradation of 5HPA in A. faecalis
169 JQ135.
170
171 Heterogenous expression and purification of HpaM and function determination
172 hpaM was cloned into pET29a(+) and expressed in E. coli BL21(DE3). The
173 N-terminal 6×His-tag HpaM was purified via nickel affinity chromatography. The
174 purified enzyme migrated as a single band with the size of about 45 kDa (obtained via
175 SDS-PAGE analysis), which was in agreement with its theoretical value (Fig. S2).
176 HpaM was predicted to contain FAD binding domains (such as GXGXXG) (Fig.
177 S1). However, the purified HpaM remained colorless, suggesting that no flavin was
178 associated after purification. The purified HpaM showed enzymatic activity only after
179 addition of external FAD (but not FMN). NADH (but not NADPH) could be used as
180 electron donor. These results indicated that HpaM was FAD- and NADH-dependent.
8 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
181 The rapid degradation of 5HPA was monitored via spectrophotometric changes (250
182 nm to 400 nm). First, the absorption spectra of authentic 5HPA (maximum UV
183 absorption (λmax) at 278 and 320 nm), 2,5-DHP (λmax at 322 nm), and NADH (λmax at
184 272 and 340 nm) in 50 mM PBS (pH7.0) were determined (Fig. 4B). Then, the
185 reaction was started with addition of 5HPA, and the consumptions of 5HPA (at 278 nm)
186 and NADH (between 340 to 400 nm) were observed (Fig. 4C). HPLC results showed
187 that 5HPA (with a retention time of 5.52 min) decreased and a product (with a
188 retention time of 5.22 min) accumulated (Fig. S3). The retention time of the
189 compound was equal to that of the authentic 2,5-DHP standard, and the LC/MS
190 analysis indicated that the product showed a molecular ion at m/z 112.1 [M+H]+,
191 which was identical to that of 2,5-DHP in previous reports (12, 19). Thus, based on the
192 above analysis, the product could be identified as 2,5-DHP.
193
194 Kinetics analysis constants and biochemical properties
195 Kinetic analysis revealed Km and kcat values of HpaM for 5HPA of 45.4 ± 4.2 μΜ
-1 196 and 10.2 ± 0.3 s , respectively (Fig. S4). The apparent catalytic efficiency (kcat/Km)
-1 -1 197 was 225.5 s mM . Additionally, the apparent Km value of HpaM for NADH was 37.8
198 ± 3.4 μM (Fig. S4). HpaM showed activities within a narrow pH range (pH 6.0 to 8.0)
199 (Fig. S5). The optimal pH was found to be 7.0 in 50 mM PBS. Most of the activity
200 was lost at pH 3.0 and 9.0. The optimal temperature was 25°C. It retained
201 approximately 60% of its activity at 20°C and 35°C. HpaM remained stable for seven
202 days at 4°C in PBS (50 mM, pH 7.0). As the temperature increased, the enzyme was
203 unstable. It lost 60% of the activities after incubation in 40°C for 1 h. When 1 mM of
204 various metal ions were added into the reaction mixture, the Ag+, Cu2+, Hg2+, Ni2+, or
205 Zn2+ completely inhibited the activities, while only a slight inhibition was found for
9 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
206 Ca2+, Co2+, Fe3+, Li+, or Mg2+, and slight activation was found for Mn2+.
207 The following structural analogs of 5HPA were tested as substrates in a standard
208 activity test: PA, NA, 5-aminopicolinic acid, 5-methylpicolinic acid, 5-chloropicolinic
209 acid, 5-bromoopicolinic acid, 3-hydroxypicolinic acid, 6-hydroxypicolinic acid,
210 2,3-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 2,6-pyridinedicarboxylic
211 acid, 2-hydroxynicotinic acid, 4-hydroxynicotinic acid, 5-hydroxynicotinic acid,
212 6-hydroxynicotinic acid, and 3-hydroxyisonicotinic. HpaM showed no activities
213 toward any of these compounds, indicating that the hydroxylase activity was 5HPA
214 specific.
215
216 Discussion
217 In this study, the 5HPA-degradation-deficient mutant JQ135m was screened from
218 the Tn5-transposon mutant library of A. faecalis JQ135. DNA walking results and
219 bioinformatics analysis revealed that the transposon was inserted into the
220 monoxygenase gene hpaM. HpaM was shown to be responsible for the initial
221 2-decarboxylative hydroxylation of 5HPA based on the following indications, 1)
222 deletion of hpaM resulted in a deficient 5HPA degradation in A. faecalis JQ135ΔhpaM,
223 while introduction of hpaM into JQ135ΔhpaM regained the ability to degrade 5HPA;
224 2) the transcription of the hpaM was evidently induced by 5HPA; and 3) purified
225 HpaM could transform 5HPA to 2,5-DPH in vitro.
226 Hydroxylation increased the hydrophilicity and polarity of substrates, thus this is
227 typically the initial and key degradation step of aromatic compounds. So far, a number
228 of flavin-dependent monooxygenases have been described that are responsible for the
229 hydroxylation of aromatic compounds. Most of them catalyzed the direct
230 hydroxylation of unsubstituted carbon atoms within the aromatic rings, and there were
10 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
231 also a few flavin-dependent monooxygenases that catalyzed the decarboxylative
232 hydroxylation of carboxyl-substituted carbon atoms in the aromatic ring (Fig. 5C). So
233 far, only two of them (NicC and HspB) catalyzed the 3-decarboxylative hydroxylation
234 of the pyridine-ring (Fig. 5B) (8, 20, 21). To the best of our knowledge, HpaM was
235 the first identified flavin-dependent monooxygenase that catalyzed the
236 2-decarboxylative hydroxylation of pyridine or pyridine derivatives. Our report
237 provides a novel insight into the decarboxylative 2-hydroxylation of pyridine
238 derivatives.
239 HpaM showed low similarities (identities of only 31-28%) to several
240 flavin-dependent monooxygenases. Flavin-dependent monooxygenase, which contains
241 the vitamin B2 derivatives FAD and FMN as redox-active prosthetic group, is the
242 largest family of flavoenzymes. To date, more than 230 flavin-dependent
243 monooxygenases have been registered in class 1.14.13.-
244 (http://enzyme.expasy.org/EC/1.14.13.-). These catalyze a variety of reactions such as,
245 hydroxylation, Baeyer–Villiger oxidation, sulfoxidation, epoxidation, and
246 halogenation, while playing an important role in the catabolism of natural and
247 xenobiotic compounds (22). They are divided into six groups (A to F), and groups A
248 and B are monocomponent systems, while groups C–F are two-component systems.
249 Group A flavin monooxygenases rely on NAD(P)H as external electron donor and
250 contain a glutathione reductase (GR-2) type Rossmann fold (GXGXXG) for FAD
251 binding (22, 23). Group B flavin monooxygenases are similar to group A except that
252 they contain two Rossmann folds. Sequences alignment showed that HpaM contained
253 several conserved motifs, such as GXGXXG, DGX5R, and GDAX10GX6DX3L (Fig.
254 S1) (23, 24). On the phylogenetic tree, which was constructed based on related
255 monocomponent flavin-dependent monooxygenases, HpaM clearly located within
11 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
256 Group A, forming a subclade with 6-hydroxynicotinate 3-monooxygenase NicC (Fig.
257 5A). The evidence indicates that HpaM is a novel group of A flavin monooxygenases.
258 Bioinformatic analysis and RT-PCR results indicated that hpaM and its upstream
259 eight genes (AFA_18535 to AFA_18570) constituted a 5HPA-inducible transcriptional
260 operon. AFA_18535 to AFA_18555 were predicted to encode the five subunits of an
261 ABC-type transporter, which may be responsible for the transmembrane transport of
262 5HPA. AFA_18560 (hpaD), AFA_18565 (hpaX), and AFA_18570 (hpaF) showed the
263 highest similarities with 2,5-DHP catabolic enzymes NicD, NicX, and NicF from P.
264 putida KT2440, respectively (Fig. 3 and Table 1). This suggests that the genes in
265 cluster hpa constituted a catabolic pathway of 5HPA. However, it is interesting that
266 the cluster hpa lacked a maleate isomerase gene, which is essential for the
267 transformation of maleic acid to fumaric acid, while this maleate isomerase gene
268 (nicE or iso) is present in the 2,5-DHP metabolic gene cluster in P. putida strains
269 KT2440 and S16 (8, 25). However, unpublished data showed that strain JQ135 could
270 utilize maleic acid as the sole carbon source for growth. Bioinformatics analysis
271 showed that a gene (AFA_16520), which was physically separated from the hpa
272 cluster, showed 99% identity with a previously reported maleic acid cis-trans
273 isomerase (MaiA) from A. faecalis IFO13111 and 59% identity to NicE from P. putida
274 KT2440 at the amino acid level (26). MaiA catalyzed the reversible conversion of
275 maleate to fumarate. Thus AFA_16520 was predicted to convert maleate to fumarate
276 in A. faecalis JQ135. Based on the above analysis, we proposed a 5HPA degradation
277 pathway in A. faecalis JQ135: (a) 5HPA is 2-decarboxylative hydroxylated by HpaM,
278 generating 2,5-DHP; (b) 2,5-DHP is further ring-cleaved into N-formylmaleamic acid
279 (NFM); (c) NFM is subsequently transformed to fumaric acid (a metabolite of citric
280 acid cycle) via maleamic acid and maleic acid as intermediates (Fig. 1). Besides
12 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
281 5HPA, A. faecalis JQ135 could also utilize PA, NA, and 6-hydroxypicolinic acid.
282 Maleic acid was the common intermediate metabolite of all above compounds, thus
283 AFA_16520 might be involved in the mineralization of all these compounds. The
284 separation of AFA_16520 from the hpa cluster might facilitate the flexible regulation
285 of AFA_16520 for the degradation of these substrates in strain JQ135. Moreover, the
286 homologous genes of AFA_16520 and hpa cluster were highly conserved (> 95%
287 identities at the level of amino acid sequences) in most (13 out of 16) A. faecalis
288 strains with available genome in GenBank
289 (https://www.ncbi.nlm.nih.gov/genome/genomes/13038?; Table S1), suggesting that
290 the degradation of 5HPA was a common feature of A. faecalis.
291 Additionally, 2,5-DHP (the hydroxylated product of 5HPA) could easily be
292 chemically transformed to 5-aminolevulinic acid, which is an important chemical
293 intermediate used for synthesis of plant hormones and drugs in cancer diagnosis and
294 therapy. Thus, our present study might provide a new biological approach to produce
295 2,5-DHP.
296
297 Materials and Methods
298 Chemicals and media
299 5HPA (99%) and its structural analogs were purchased from J&K Scientific Ltd.
300 (Shanghai, China). 2,5-DHP (98%) was purchased from SynChem OHG (Altenburg,
301 Germany). All other chemicals and solvents used in this experiment were
302 commercially available. Enzymes used in this study were purchased from Vazyme
303 Biotech Co., Ltd (Nanjing, China). Mineral salts medium (MSM) and Luria-Bertani
304 medium (LB) have been described in previous report (16), and to prepare
305 nitrogen-absent MSM, NH4NO3 was replaced.
13 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
306
307 Bacterial strains, vectors, and growth conditions
308 Bacterial strains and vectors used in this study are listed in Table 2. A. faecalis
309 JQ135 has previously been identified as a picolinic acid-degrading bacterium (CCTCC
310 M 2015812) (16). E. coli strains were grown at 37°C, while other strains were grown
311 at 30°C. The media were supplemented with chloramphenicol (Cm, 34 μg/mL),
312 kanamycin (Km, 50 μg/mL), gentamicin (Gm, 50 μg/mL), or streptomycin (Str, 50
313 μg/mL) as required.
314
315 Isolation of 5HPA growth-deficient mutant and determination of the transposon
316 insertion site
317 Both generation and selection of 5HPA growth-deficient mutants of A. faecalis
318 JQ135 were performed according to the selection method via picolinic acid (PA)
319 growth-deficient mutants as previously described (16). PA was replaced by 5HPA.
320 The insertion site of the transposon was determined via SEFA-PCR as previously
321 described (16, 17).
322
323 Deletion of hpaM and complementation
324 Standard DNA manipulation was performed as previously described (27). All
325 primers used in this study are listed in Table S2. The deletion of the hpaM gene in A.
326 faecalis JQ135 was generated via a two-step homogenetic recombination method
327 using the suicide vector pJQ200SK. The two primer pairs, koUF/koUR and
328 koDF/koDR, were used to amplify the homologous recombination-directing
329 sequences. Then, both PCR fragments were cloned into SacI/PstI-digested pJQ200SK
330 using the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd,
14 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
331 Nanjing, China). The resulting vector pJQ-ΔhpaM was then introduced into A. faecalis
332 JQ135 cells. The single-crossover mutant was screened on LB plates containing Str
333 and Gm. The double-crossover mutant (JQ135ΔhpaM) was selected on LB plates
334 mixed with Str and 10% (wt/vol) sucrose. The vector pBBR-hpaM was constructed
335 for gene complementation. The hpaM gene was amplified via primers hpaM-F and
336 hpaM-R, and then fused with the XhoI/HindIII-digested pBBR1-MCS5, thus
337 generating pBBR-hpaM. The pBBR-hpaM vector was transferred into the mutant
338 JQ135ΔhpaM via triparental mating to generate the complemented strain
339 JQ135ΔhpaM-pBBR-hpaM.
340
341 RNA extraction and RT-PCR
342 JQ135 Cells were cultured in glycerol and harvested at the mid-exponential phase,
343 washed twice with MSM, and resuspended in MSM. The cell suspension (with an
344 OD600 of 0.6) was transferred into a 50 mL flask, containing 20 mL MSM
345 supplemented with 1 mM of either glycerol or 5HPA. After incubation at 30°C and 180
346 rpm for 6 h, cells were harvested. Total RNA was isolated using an RNA isolation kit
347 (TaKaRa). Reverse transcription (RT)-PCR was conducted with a PrimeScript RT
348 reagent kit (TaKaRa). All RT-PCR primers are listed in Table S2. All samples were
349 run in triplicate.
350
351 Cloning, overexpression, and purification of HpaM
352 The hpaM gene (ignoring the stop codon) was PCR amplified with primers
353 exphpaMF and exphpaMR and fused into the NdeI/XhoI digested pET29a(+), thus
354 producing pET-hpaM. The C-terminal 6×His-tagged HpaM was overexpressed in E.
355 coli BL21(DE3) that carried pET-hpaM. The cells were grown in LB at 37°C to an
15 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
356 OD600 of 0.5 and then induced for 16 h via addition of 0.1 mM
357 isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C. HpaM was purified via
358 Ni2+-nitrilotriacetic acid agarose chromatography (Novagen) and eluted at 100 mM
359 imidazole. HpaM was dialyzed against PBS (50 mM, pH 7.0) at 4°C for 24 h and
360 analyzed via 12.5% SDS-PAGE.
361
362 Enzyme assays
363 The standard reaction mixture contained 50 mM PBS (pH 7.0), 0.2 mM FAD, 0.5
364 mM NADH, 0.2 mM 5HPA, and 1 μg purified HpaM. The reference cuvette
365 contained all of these compounds except for 5HPA. The assay was initiated via
366 addition of substrate 5HPA. For the activity assay, HpaM activity was
367 spectrophotometrically analyzed by measuring NADH oxidation at 370 nm (ε = 2,470
-1 -1 368 M cm ) (28) instead of at its λmax of 340 nm to avoid interference of the absorptions
369 of 5HPA and 2,5-DHP (Fig. 4B). One unit of HpaM activity was defined as the
370 amount of enzyme required for the oxidation of 1 μmol of NADH per min at 25°C.
371 Rapid observation of HpaM activities was monitored via spectrophotometric changes
372 from 250 nm to 450 nm at room temperature, using a UV2450 spectrophotometer
373 (Shimazu) in 1 cm path length quartz cuvettes. For determination of kinetics constants,
374 substrates were appropriately diluted into seven concentrations around the Km (3.6, 7.2,
375 18, 36, 72, 108, 144, and 360 μM for 5HPA; 5, 10, 25, 50, 75, 100, and 500 μM for
376 NADH). The kinetics values were calculated via nonlinear regression fitting to the
377 Michaelis-Menten equation. The effects of different pH (3.0 to 10.0), temperature (10
378 to 60°C), and metal ions on HpaM activities were determined as previously reported
379 (29).
380
16 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
381 Analytical methods
382 Determinations of 5HPA and 2,5-DHP were performed via HPLC analysis on an
383 Shimadzu AD20 system equipped with Phecda C18 reversed phase column (250 mm
384 × 4.60 mm, 5 μm) with array detection at both 280 nm and 310 nm. The mobile phase
385 consisted of methanol : water : formic acid (12.5:87.5:0.2, v/v/v) at a flow rate of 0.6
386 mL/min, 30°C. All assays in this study were independently performed three times, and
387 the means and standard errors of measurements were calculated. LC/MS analysis was
388 performed in a Thermo (America) DECA-60000 XLCQ Deca XP Plus instrument as
389 previously described (19).
390
391 Nucleotide sequence accession numbers
392 The hpa cluster sequence and the complete genome sequence of A. faecalis
393 JQ135 have been deposited in the GenBank database under accession numbers
394 KY230187 and CP021641, respectively.
395
396 Acknowledgments
397 This work was supported by the National Natural Science Foundation of China
398 (No. 31500082), China Postdoctoral Science Foundation (No. 2016M601826), the
399 Postdoctoral Foundation of Jiangsu Province (No. 1601035A), and Natural Science
400 Foundation of Jiangsu Province (No. BK20141366).
401
402
17 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
403 References
404 1. Guan A-Y, Liu C-L, Sun X-F, Xie Y, Wang M-A. 2016. Discovery of
405 pyridine-based agrochemicals by using intermediate derivatization methods.
406 Bioorg. Med. Chem. 24:342-353.
407 2. Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson
408 T, Collins D, De Lisle G, Jacobs Jr WR. 1994. inhA, a gene encoding a target
409 for isoniazid and ethionamide in Mycobacterium tuberculosis. Science
410 263:227-229.
411 3. Hamaker JW, Johnston H, Martin RT, Redemann CT. 1963. A picolinic
412 acid derivative: a plant growth regulator. Science 141:363-363.
413 4. Tekle-Röttering A, Reisz E, Jewell KS, Lutze HV, Ternes TA, Schmidt W,
414 Schmidt TC. 2016. Ozonation of pyridine and other N-heterocyclic aromatic
415 compounds: Kinetics, stoichiometry, identification of products and elucidation
416 of pathways. Water Res. 102:582-593.
417 5. Brinkmann M, Maletz S, Krauss M, Bluhm K, Schiwy S, Kuckelkorn J,
418 Tiehm A, Brack W, Hollert H. 2014. Heterocyclic aromatic hydrocarbons
419 show estrogenic activity upon metabolization in a recombinant transactivation
420 assay. Environ. Sci. Technol. 48:5892-5901.
421 6. Fetzner S. 1998. Bacterial degradation of pyridine, indole, quinoline, and
422 their derivatives under different redox conditions. Appl. Microbiol. Biotechnol.
423 49:237-250.
424 7. Kaiser J-P, Feng Y, Bollag J-M. 1996. Microbial metabolism of pyridine,
425 quinoline, acridine, and their derivatives under aerobic and anaerobic
426 conditions. Microbiol. Rev. 60:483-498.
427 8. Jiménez JI, Canales A, Jiménezbarbero J, Ginalski K, Rychlewski L,
18 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
428 García JL, Díaz E. 2008. Deciphering the genetic determinants for aerobic
429 nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440.
430 Proc. Nat. Acad. Sci. USA 105:11329-11334.
431 9. Li H, Xie K, Yu W, Hu L, Huang H, Xie H, Wang S. 2016. Nicotine
432 Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase
433 Involved in the Hybrid Nicotine-degrading Pathway in Agrobacterium
434 tumefaciens S33. Appl. Environ. Microbiol.:doi: 10.1128/AEM.03909-03915.
435 10. Yu H, Tang H, Li Y, Xu P. 2015. Molybdenum-Containing Nicotine
436 Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the
437 Pyridine and Pyrrolidine Pathways. Appl. Environ. Microbiol. 81:8330-8338.
438 11. Qiu J, Wei Y, Ma Y, Wen R, Wen Y, Liu W. 2014. A novel
439 (S)-6-hydroxynicotine oxidase gene from Shinella sp. strain HZN7. Appl.
440 Environ. Microbiol. 80:5552-5560.
441 12. Yu H, Tang H, Zhu X, Li Y, Xu P. 2015. Molecular mechanism of nicotine
442 degradation by a newly isolated strain Ochrobactrum sp. strain SJY1. Appl.
443 Environ. Microbiol. 81:272-281.
444 13. Makar'eva T, Kalinovskii A, Stonik V, Vakhrusheva E. 1989. Identification
445 of 5-hydroxypicolinic acid among the products biosynthesized by Nocardia sp.
446 Chem. Nat. Compd. 25:125-126.
447 14. Chen L, Song Z, Wang J, Song H, Zhang G, Wang J. 2010. Studies on the
448 chemical constituents from aerial parts of Gynura divaricata. J. Chinese Med.
449 Mater. (Zhong yao cai) 33:373-376.
450 15. Karvelis L, Gasparavičiūtė R, Klimavičius A, Jančienė R, Stankevičiūtė J,
451 Meškys R. 2014. Pusillimonas sp. 5HP degrading 5-hydroxypicolinic acid.
452 Biodegradation 25:11-19.
19 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
453 16. Qiu J, Zhang J, Zhang Y, Wang Y, Tong L, Hong Q, He J. 2017.
454 Biodegradation of picolinic acid by a newly isolated bacterium Alcaligenes
455 faecalis strain JQ135. Cur. Microbiol. 74:508-514.
456 17. Wang SM, He J, Cui ZL, Li SP. 2007. Self-formed adaptor PCR: a simple
457 and efficient method for chromosome walking. Appl. Environ. Microbiol.
458 73:5048-5051.
459 18. Lee J, Oh J, Min KR, Kim Y. 1996. Nucleotide sequence of salicylate
460 hydroxylase gene and its 5′-flanking region of Pseudomonas putida KF715.
461 Biochem. Biophys. Res. Commun. 218:544-548.
462 19. Ma Y, Wei Y, Qiu J, Wen R, Hong J, Liu W. 2013. Isolation, transposon
463 mutagenesis, and characterization of the novel nicotine-degrading strain
464 Shinella sp. HZN7. Appl. Microbiol. Biotechnol. 98:2625-2636.
465 20. Yu H, Hausinger RP, Tang H-Z, Xu P. 2014. Mechanism of the
466 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from
467 Pseudomonas putida S16. J. Biol. Chem. 289:29158-29170.
468 21. Hicks KA, Yuen ME, Zhen WF, Gerwig TJ, Story RW, Kopp MC, Snider
469 MJ. 2016. Structural and biochemical characterization of 6-hydroxynicotinic
470 acid 3-monooxygenase, a novel decarboxylative hydroxylase involved in
471 aerobic nicotinate degradation. Biochemistry 55:3432-3446.
472 22. Huijbers MM, Montersino S, Westphal AH, Tischler D, van Berkel WJ.
473 2014. Flavin dependent monooxygenases. Arch. Biochem. Biophys. 544:2-17.
474 23. Van Berkel W, Kamerbeek N, Fraaije M. 2006. Flavoprotein
475 monooxygenases, a diverse class of oxidative biocatalysts. J. Biotechnol.
476 124:670-689.
477 24. Chao H-J, Chen Y-F, Fang T, Xu Y, Huang WE, Zhou N-Y. 2016. HipH
20 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
478 catalyzes the hydroxylation of 4-hydroxyisophthalate to protocatechuate in 2,
479 4-xylenol catabolism by Pseudomonas putida NCIMB 9866. Appl. Environ.
480 Microbiol. 82:724-731.
481 25. Tang H, Yao Y, Wang L, Yu H, Ren Y, Wu G, Xu P. 2012. Genomic analysis
482 of Pseudomonas putida: genes in a genome island are crucial for nicotine
483 degradation. Sci. Rep. 2:377.
484 26. Hatakeyama K, Asai Y, Uchida Y, Kobayashi M, Terasawa M, Yukawa H.
485 1997. Gene cloning and characterization of maleate cis-trans isomerase from
486 Alcaligenes faecalis. Biochem. Biophys. Res. Commun. 239:74-79.
487 27. Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual, 3rd
488 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
489 28. Luanloet T, Sucharitakul J, Chaiyen P. 2015. Selectivity of substrate
490 binding and ionization of 2‐methyl‐3‐hydroxypyridine‐5‐carboxylic acid
491 oxygenase. The FEBS J. 282:3107-3125.
492 29. Yun H, Liang B, Qiu J, Zhang L, Zhao Y, Jiang J, Wang A. 2017.
493 Functional characterization of a novel amidase involved in biotransformation
494 of triclocarban and its dehalogenated congeners in Ochrobactrum sp. TCC-2.
495 Environ. Sci. Technol. 51:291-300.
496
497
498
21 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
499 Figure Legends
500 Fig. 1 Catabolic mechanism of 5HPA in A. faecalis JQ135. (A) The proposed
501 5HPA degradation pathway in A. faecalis JQ135. HpaM, 5-hydroxypicolinic acid
502 2-monooxygenase; HpaX, 2,5-Dihydroxypyridine 5,6-dioxygenase; HpaD,
503 N-formylmaleamic acid deformylase; HpaF, maleamic acid amidohydrolase; MaiA,
504 maleic acid cis-trans isomerase; and TCA: tricarboxlic acid cycle. (B) Organization of
505 the gene cluster hpa. 18530 indicates the locus tag of AFA_18530. Genes have been
506 functionally annotated following the color code indicated in panel A. The lines below
507 the gene cluster show the location and size of the PCR fragments in panel C. (C)
508 Agarose gel electrophoresis of RT-PCR products. + and - indicate that the cells were
509 cultured with 5HPA and glycerol, respectively.
510
511 Fig. 2 Degradation of 5HPA (solid lines) and cell growth (dotted lines) of the
512 wild-type strain JQ135 (■), the mutant Z10 (▲), and the complemented strain
513 Z10-pBBR-hpaM (○).
514
515 Fig. 3 Organization of the hpa cluster for 5HPA degradation in A. faecalis JQ135
516 (A), the nic2 cluster for nicotine degradation in P. putida S16 (B), and the nic cluster
517 for nicotinic acid degradation in P. putida KT2440 (C) (8). AFA_16520 (maiA): locus
518 tag (gene name). Numbers within arrows indicate the protein sequence identity with
519 the orthologous from strain JQ135. hpaM: 5-hydroxypicolinic acid 2-monooxygenase
520 gene; hspB: 6-hydroxy-3-succinoylpyridine 3-monooxygenase gene; nicC:
521 6-hydroxynicotinate 3-monooxygenase gene; hpaX, hpo, and nicX:
522 2,5-dihydroxypyridine 5,6-dioxygenase gene; hpaD, nfo, and nicD:
22 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
523 N-formylmaleamate deformylase gene. hpaF, ami, and nicF: maleamate
524 amidohydrolase; maiA, iso, and nicE: maleic acid cis-trans isomerase gene.
525
526 Fig. 4 Rapid determination of the HpaM activity. (A) Proposed conversion of
527 5HPA to 2,5-DHP catalyzed via HpaM. (B) UV absorption spectrum of authentic 5HPA,
528 2,5-DHP, and NADH in 50 mM PBS (at pH 7.0). (C) Spectrophotometric changes
529 during the transformation of 5HPA via HpaM. The reference cuvette contained HpaM,
530 FAD, and NADH. The reaction was initiated via addition of 5HPA. The spectra were
531 recorded every 90 s. The arrow indicates the direction of spectral changes.
532
533 Fig. 5 Classification analysis of HpaM and several related flavin-dependent
534 monooxygenases. (A) Phylogenetic tree construction based on the alignment of HpaM
535 with related monocomponent flavin-dependent monooxygenases in Group A (which
536 contained one Rossmann fold) and Group B (which contained two Rossmann folds)
537 (22, 23). Multiple-alignment analysis was performed via ClustalX v2.0. The
538 phylogenetic tree was constructed via the neighbor-joining algorithm using MEGA 6.0
539 and bootstrap values (based on 1,000 replications) have been indicated at branch nodes.
540 Bar, 0.20 substitutions per nucleotide position. Each item was arranged in the following
541 order: protein name, UniProtKB/SwissProt accession number, and EC number. (B)
542 Reactions of HpaM and two monooxygenases, catalyzing the 3-decarboxylative
543 hydroxylation of pyridine derivatives. NicC, 6-hydroxynicotinate 3-monooxygenase
544 (EC 1.14.13.114); HspB, 6-hydroxy-3-succinoylpyridine 3-monooxygenase (EC
545 1.14.13.163). (C) Five decarboxylative hydroxylation reactions of the benzene ring.
546 SalM, salicylate 1-monooxygenase (EC 1.14.13.1); pHB1M, p-hydroxybenzoate
547 1-monooxygenase (EC 1.14.13.64); 4AB1M, 4-aminobenzoate 1-monooxygenase (EC
23 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
548 1.14.13.27); HipH, 4-Hydroxyisophthalic acid 3-monooxygenase; PhzS,
549 5-methylphenazine-1-carboxylate 1-monooxygenase (EC 1.14.13.218). The proteins in
550 panels B and C of the phylogenetic tree have been indicated in red and blue,
551 respectively.
552
553
24 / 26
bioRxiv preprint
doi:
554 certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/171595
555 Table 1 Sequence comparisons of hpa gene cluster of A. faecalis JQ135 with database entries.
Gene locus Product Database b Homologous protein c GenBank Identity Proposed function size a accession no. (%) AFA_18535 369 NR Branched-chain amino acid ABC transporter, substrate-binding protein livK CUI69837 99 Swiss-Prot Leu/Ile/Val/Thr/Ala-binding protein P21175 30 AFA_18540 291 NR Branched-chain amino acid ABC transporter, substrate-binding protein LivH WP_035269511 100 Swiss-Prot High affinity branched chain amino acid transport system permease protein LivH P0AEX8 35 AFA_18545 319 NR Branched-chain amino acid ABC transporter, substrate-binding protein LivM WP_083055103 99 ;
5-Hydroxypicolinic acid transporters this versionpostedAugust14,2017. Swiss-Prot High affinity branched chain amino acid transport system permease protein LivM P22729 30 AFA_18550 252 NR Branched-chain amino acid ABC transporter, ATP-binding protein LivG WP_042485129 100 Swiss-Prot High affinity branched chain amino acid transport system permease protein LivG P0A9S8 41 AFA_18555 242 NR Branched-chain amino acid ABC transporter, ATP-binding protein LivF WP_035269507 100 Swiss-Prot High affinity branched chain amino acid transport system permease protein LivF P0A191 46 AFA_18560 (hpaD) 275 NR alpha/beta hydrolase WP_042485331 98 N-Formylmaleamic acid deformylase HpaD Swiss-Prot N-Formylmaleamate deformylase NicD Q88FY3 57 AFA_18565 (hpaX) 346 NR 2,5-Dihydroxypyridine 5,6-dioxygenase WP_083055106 98 2,5-Dihydroxypyridine 5,6-dioxygenase HpaX Swiss-Prot 2,5-Dihydroxypyridine 5,6-dioxygenase NicX Q88FY1 53 AFA_18570 (hpaF) 205 NR N-Carbamoylsarcosine amidase WP_060186798 100 Maleamic acid amidohydrolase HpaF Swiss-Prot Maleamate amidohydrolase NicF Q88FY5 41
AFA_18575 (hpaM) 405 NR salicylate 1-monooxygenase ARP55611 100 5-Hydroxypicolinic acid 2-monooxygenase HpaM The copyrightholderforthispreprint(whichwasnot Swiss-Prot 6-Hydroxynicotinate 3-monooxygenase NicC Q88FY2 35 AFA_18580 291 NR DUF2236 domain containing protein WP_086060754 99 Unknown function Swiss-Prot Ubiquitin activating enzyme E1C Q99MI7 25 AFA_18585 235 NR TetR family transcriptional regulator WP_035269502 100 Transcriptional regulator Swiss-Prot HTH type transcriptional regulator BetI Q13NG5 41 556 a Number of amino acids. 557 b NR, NCBI Nonredundant Protein Sequences Database. 558 c The top BLASTP hit was selected. 559 560
25 / 26
bioRxiv preprint
doi:
561 certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/171595
562 Table 2 Strains and vectors used in this study.
Strains or vectors Description Source Strains A. faecalis JQ135 Strr; wild type; 5HPA-degrading strain; G – (16) Z10 Strr, Kmr; transposon mutant of JQ135; 5HPA growth-deficient This study Z10-pBBR-hpaM Strr, Kmr, Gmr; Z10 containing pBBR-hpaM ; JQ135ΔhpaM Strr; hpaM-deletion mutant of JQ135 This study this versionpostedAugust14,2017. JQ135ΔhpaM-pBBR-hpaM Strr, Gmr; JQ135ΔhpaM containing pBBR-hpaM This study
Escherichia coli DH5α F− recA1 endA1 thi-1 hsrdR17 supE44 relA1 deoRΔ(lacZYA-argF) U169 φ80lacZ ΔM15 TaKaRa
SM10λpir Donor strain for biparental mating Lab stock BL21(DE3) F− ompT hsdS(rB− mB−) gal dcm lacY1(DE3) Novagen Vectors pET29a(+) Kmr, expression vector Novagen The copyrightholderforthispreprint(whichwasnot pSC123 Cmr, Kmr; suicide vector, mariner transposon Lab stock pJQ200SK Gmr, mob+, orip15A, lacZα+, sacB; suicide vector Lab stock pBBR1-MCS5 Gmr; broad-host-range cloning vector Lab stock pJQΔhpaM Gmr; hpaM gene deletion vector; the upstream and downstream regions of hpaM gene fused into SacI/PstI-digested pJQ200SK This study pBBR-hpaM Gmr; hpaM gene complementation vector; the hpaM gene fused into XhoI/HindIII-digested pBBR1-MCS5 This study pET-hpaM Kmr; NdeI-XhoI fragment containing hpaM inserted into pET29a(+) This study 563
26 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.