<<

bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 Identification and characterization of a novel pic gene cluster responsible for

2 picolinic acid degradation in faecalis JQ135

3

4 Jiguo Qiu1, Lingling Zhao1, Siqiong Xu1, Qing Chen1,3, Le Chen1, Bin Liu1, Qing

5 Hong1, Zhenmei Lu2, and Jian He1*

6

7 1 Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of

8 Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China

9 2 College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

10 3 College of Life Sciences, Zaozhuang University, Zaozhuang 277100, China

11

12 Running Title: Catabolism of picolinic acid in Alcaligenes faecalis

13

14 Keywords: Alcaligenes faecalis, picolinic acid, bacterial degradation, pathway, pic

15 gene cluster

16

17 *To whom correspondence should be addressed. Email: [email protected]

18

19

1 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

20 Abstract

21 Picolinic acid (PA) is a natural toxic derivative. Microorganisms can

22 degrade and utilize PA for growth. However, the full metabolic pathway and its

23 physiological and genetic foundation remain unknown. In this study, we identified the

24 pic gene cluster responsible for the complete degradation of PA from Alcaligenes

25 faecalis JQ135. PA was initially 6-hydroxylated into 6-hydroxypicolinic acid (6HPA)

26 by PA dehydrogenase (PicA). 6HPA was then 3-hydroxylated by a four-component

27 6HPA monooxygenase (PicB) to form 3,6-dihydroxypicolinic acid (3,6DHPA), which

28 was then converted into 2,5-dihydroxypyridine (2,5DHP) by a decarboxylase (PicC).

29 The 2,5DHP was further degraded into fumaric acid, through PicD (2,5DHP

30 dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid

31 amidohydrolase), and PicG (maleic acid isomerase). Homologous pic gene clusters

32 with diverse organizations were found to be widely distributed in α-, β-, and

33 γ-. Our findings provide new insights into the microbial metabolism of

34 environmental toxic pyridine derivatives.

35

36

2 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

37 Importance

38 Picolinic acid is a common metabolite of L- and some aromatic

39 compounds and is an important intermediate of industrial concern. Although the

40 microbial degradation/detoxification of picolinic acid has been studied for over 50

41 years, the underlying molecular mechanisms are still unknown. Here, we show the pic

42 gene cluster responsible for the complete degradation of picolinic acid into the

43 tricarboxylic acid cycle. This gene cluster was found to be widespread in other α-, β-,

44 and γ-Proteobacteria. These findings provide new perspective for understanding the

45 mechanisms of picolinic acid biodegradation in .

46

3 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

47 Introduction

48 Picolinic acid (PA) is a natural toxic pyridine derivate. PA is a dead-end

49 metabolite of L-tryptophan via the kynurenine pathway in humans and other

50 mammals (1, 2). It has been identified in various biological mediums, such as cell

51 culture supernatants, blood serum, and human milk (3). PA can also be produced in

52 other biological processes, including the microbial degradation of 2-aminophenol,

53 nitrobenzene, catechol, anthranilic acid, and 3-hydroxyanthranilic acid (4-7) (Fig. S1).

54 In industrial applications, PA is an important intermediate in the organic synthesis of

55 pharmaceuticals (e.g. carbocaine), herbicides (e.g. and diquat), and

56 fungicide (e.g. 3-trifluoromethyl picolinic acid) (8, 9). Moreover, due to its chelating

57 properties, PA is added to chromium and iron preparations as a way to treat diabetes

58 and anemia (10, 11). Nevertheless, the toxic or disadvantages to the use of PA have

59 also been found. PA has been shown to inhibit the growth of normal rat kidney cells

60 and T cell proliferation, enhance seizure activity in mice, and induce cell death via

61 apoptosis (12-14). In particular, PA showed high antimicrobial activity at

62 concentrations as low as 8 μg/L, which is enough to inhibit the growth of

63 Gram-positive bacteria (15-17).

64 No reports showed that PA could be metabolized by humans (18). Nevertheless,

65 numerous microorganisms were found to degrade PA, such as Alcaligenes (19),

66 Arthrobacter (20), Burkholderia (21), Streptomyces (22), and an unidentified

67 Gram-negative bacterium (designated as UGN strain) (23). The bacterial degradation

68 pathway of PA was proposed as: PA, 6-hydroxypicolinic acid (6HPA),

69 3,6-dihydroxypicolinic acid (3,6DHPA), and 2,5-dihydroxypyridine (2,5DHP),

70 according to several previous determinations of degradation intermediates and crude

71 enzymes (Fig. 1). However, little is known about the degradation mechanism at

4 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

72 genetic level.

73 Alcaligenes faecalis strain JQ135 can degrade PA and utilize it for cell growth (24)

74 (25). Our previous study demonstrated the first genetic evidence involving PA

75 degradation, in which picC (encoding 3,6-dihydroxypicolinic acid decarboxylase)

76 gene was responsible for the conversion of 3,6DHPA to 2,5DHP in A. faecalis strain

77 JQ135 (26). Nevertheless, the complete degradation mechanism of PA are still

78 unknown. In this study, we reported the pic gene cluster responsible for PA

79 degradation in strain JQ135 (Fig. 1). The enzymes encoded by the pic gene cluster

80 were expressed and functionally characterized using in vitro enzymatic assays, while

81 the pic clusters in other bacteria were also predicted.

82

83

5 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

84 Results and discussion

85 Identification of pic gene cluster for PA degradation

86 Previous results showed that gene AFA_15145 (picC) was responsible for the

87 conversion of 3,6DHPA to 2,5DHP in A. faecalis JQ135 (26). Bioinformatic analysis

88 indicated that picC was located in the gene cluster (designated pic). (Fig. 1). The pic

89 cluster consisted of 13 genes and their predicted functions are summarized in Table 1.

90 RT-PCR analysis showed that all of the pic genes were induced by PA, except for

91 citrate in MSM (Fig. 2). The above results suggest that the pic cluster was involved in

92 PA metabolism in A. faecalis JQ135.

93

94 The picA1A2A3 encodes the PA dehydrogenase (PicA)

95 Previous studies reported that PA was initially 6-hydroxylated to 6HPA (19, 23,

96 24, 27). The hydroxylation of N-heterocyclic aromatic compounds were usually

97 catalyzed by multicomponent molybdopterin-containing dehydrogenase (28, 29). In

98 the pic gene cluster, the picA1, picA2, and picA3 gene products showed the highest

99 similarities (~40%) to the respective subunits of molybdopterin-containing

100 dehydrogenases, such as carbon monoxide dehydrogenase and

101 dehydrogenase (Table 1). PicA1, PicA2, and PicA3 were predicted to contain binding

102 domains for the molybdopterin cytosine dinucleotide (MCD), FAD, and two [Fe-S]

103 clusters, respectively (Fig. 3). Thus, the picA1A2A3 genes were considered to catalyze

104 the first step of PA degradation. When the picA1A2A3 genes were deleted, the

105 acquired mutants JQ135ΔpicA1A2A3 lost the ability to grow on PA but could still

106 grow on 6HPA (Fig. 4). The complementation strain

107 JQ135ΔpicA1A2A3/pBBR-picA1A2A3 restored the phenotype of growth on PA.

108 Moreover, the picA1A2A3 genes were then cloned into pBBR1MCS-5

6 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

109 (pBB-picA1A2A3) and transferred into the PA-non-degrading strain Pseudomonas

110 putida KT2440. The recombinant KT/pBBR-picA1A2A3 was found to acquire the

111 ability to convert PA into 6HPA. When lacking one component, the recombinants

112 KT/pBBR-picA2A3 and KT/pBBR-picA1A2 did not show the transformation ability,

113 indicating that all of these three components were essential.

114 After being pre-grown in MSM containing 1.0 mM citrate and 1.0 mM PA for

115 12h, the cell-free extract of KT/pBBR-picA1A2A3 produced PA dehydrogenase

116 activity when phenazine methosulfate (PMS) was used as an electron acceptor. The

117 Km value for PA at pH 7.0 and 25ºC was 0.65 ± 0.14 μM, the vmax was 44.89 ± 2.45

118 mU/mg (Fig. S2). No PA dehydrogenase activity was detected under anaerobic

119 conditions, which was similar to the nicotinate hydroxylase (NicAB) using O2 as the

120 final electron acceptor in P. putida KT2440 (28). All of these results demonstrated

121 that picA1A2A3 genes are responsible for the hydroxylation of PA into 6HPA in A.

122 faecalis JQ135.

123

124 The picB1B2B3B4 encodes the four-component 6HPA monooxygenase (PicB)

125 The second step in the PA metabolic pathway is predicted to be the

126 3-hydroxylation of 6HPA to 3,6DHPA (23). A BLAST homology search against the

127 database sequences revealed that four genes in the same orientation: picB1, picB2,

128 picB3, and picB4, showed high identities (35%~45%) at amino acid level with the

129 respective components of bacterial Rieske non-heme iron aromatic ring-hydroxylating

130 oxygenases (RHO) (Table 1, Fig. 5). RHOs are usually involved in hydroxylation of

131 aromatic compounds (30, 31). Therefore, the picB1B2B3B4 genes were most likely

132 responsible for the degradation of 6HPA to 3,6DHPA. The picB1B2B3B4 genes were

133 deleted from the genome of A. faecalis JQ135. The mutant JQ135ΔpicB1B2B3B4 lost

7 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

134 the ability to grow on PA or 6HPA, but could still grow on 3,6DHPA (Fig. 6). The

135 complementation strain JQ135ΔpicB1B2B3B4/pBBR-picB1B2B3B4 could utilize

136 6HPA for cell growth. These four genes were cloned into pBBR1MCS-5 and

137 transferred into P. putida KT2440. The recombinant KT/pBBR-picB1B2B3B4 was

138 found to convert 6HPA into 3,6DHPA. Moreover, the recombinants

139 KT/pBBR-picB2B3B4 and KT/pBBR-picB1B2B3 did not exhibit transformation

140 ability, suggesting that all of these four components were necessary. These results

141 indicated that picB1B2B3B4 genes are responsible for the conversion of 6HPA into

142 3,6DHPA in A. faecalis JQ135.

143 Interestingly, the cell-free extract of 6HPA-pre-grown KT/pBBR-picB1B2B3B4

144 could not convert 6HPA, even though several factors (e.g. FAD, FMN, NADH, or

145 NADPH) were added. Previous studies have attempted to purify the 6HPA

146 monooxygenase from cell-free extract of Arthrobacter picolinophilus DSM 20665 (32)

147 and the UGN strain (23), but no PicB activities were detected at all. These results

148 suggested that the PicB was commonly fragile.

149

150 PicDEF and PicG (MaiA) converts 2,5-DHP into fumaric acid

151 In A. faecalis JQ135, the 3,6DHPA was found to be degraded into 2,5DHP by

152 3,6-dihydroxypicolinic acid decarboxylase (PicC) (26). 2,5DHP is a central

153 intermediate of numerous pyridine derivatives, including nicotinate (28), nicotine (33),

154 2-hydroxy-pyridine (34), and 5-hydroxypicolinic acid (25). The bacterial metabolism

155 of 2,5DHP usually employs the maleamate pathway, whose key enzymes include

156 2,5DHP dioxygenase, N-formylmaleamate (NFM) deformylase, maleamate amidase,

157 and maleate cis-trans isomerase (28, 33). The primary structure of PicD, PicE, and

158 PicF showed high similarities (40~60%) to 2,5DHP 5,6-dioxygenase, NFM

8 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

159 deformylase, and maleamic acid amidohydrolase, respectively (Fig. S3), which

160 strongly implies that 2,5DHP follows the maleamate pathway in A. faecalis JQ135.

161 Then PicD was overexpressed in E. coli BL21 (DE3). The purified PicD showed

162 2,5DHP degrading ability, which was measured spectrophotometrically by a decrease

163 in absorbance at 320 nm, with a Km value of 65.72±6.27 μM. PicD showed no activity

164 without 1 mM Fe2+, which resembles previous reports of the 2,5DHP dioxygenase

165 from P. putida KT2440 and Ocrobactrum sp. SJY1 (28, 33). These results suggest that

166 PicD, PicE, and PicF catalyze the formation of maleic acid from 2,5DHP in A.

167 faecalis JQ135.

168 Maleic acid could be isomerized into fumaric acid, an intermediate of the Krebs

169 cycle. However, the pic gene cluster lacks an isomerase gene. Previous study showed

170 that AFA_16520 (maiA, also named picG in this study) gene was only the maleic acid

171 cis-trans isomerase gene in A. faecalis JQ135 and PicG was responsible for the last

172 step of PA degradation (25). Furthermore, a DNA fragment containing picEDFG

173 genes was cloned into a 2,5DHP non-degrading bacterium Sphingomonas wittichii

174 DC-6 (35). Recombinant DC-6/pBBR-picEDFG acquired the ability to degrade and

175 utilize 2,5DHP for growth (Fig. S4). While lacking the picG gene, recombinant

176 DC-6/pBBR-picEDF was not able to grow on 2,5DHP. These results also indicated

177 that PicDEF and PicG (MaiA) were responsible for the conversion of 2,5DHP to

178 fumaric acid.

179

180 Diversity of the pic genes in other bacteria

181 Six complete genome sequences from A. faecalis strains are available in NCBI

182 and pic gene clusters were found in all of these genomes (Fig. 7, Table S1). Besides A.

183 faecalis, the orthologous pic genes were also found in other α-, β-, and

9 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

184 γ-Proteobacteria (21 genera and over 160 strains) (Table S1). Most of these strains

185 belong to the order of the class β-Proteobacteria, including genera of

186 Achromobacter, Alcaligenes, Advenella, , Burkholderia, Caballeronia,

187 Cupriavidus, Pandoraea, Paraburkholderia, Polaromonas, Pseudacidovorax,

188 Pusillimonas, Rolstonia, and Variovorax. Interestingly, some of these strains are toxic

189 compound degraders (e.g., Achromobacter xylosoxidans A8 and Variovorax sp.

190 JS1663) (36, 37), human pathogens (e.g., Tohama I and

191 Bordetella bronchiseptica RB50) (38), and nematicidal bacteria (e.g., A. faecalis

192 ZD02) (39), whereas other strains represent typical soil bacteria (e.g., Pseudomonas),

193 plant rhizosphere-associated bacteria (e.g., Bradyrhizobium), arctic bacterium

194 (Octadecabacter arcticus 238) (40), and antarctic bacterium (Hoeflea sp. IMCC20628)

195 (41). Previous reports also showed that bacteria in the genera of Aerococcus,

196 Arthrobacter, and Streptomyces could degrade PA (20, 22, 27). However, no

197 homologues of pic genes were found in these bacteria, suggesting that other new and

198 unknown catabolic genes might exist.

199 The genetic organization of the pic gene clusters was highly diverse (Fig. 7). The

200 picA genes usually adjoin picB genes, except in the Cupriavidus strains. In most

201 genera, the picG gene was located between picC and picE. However, in Alcaligenes,

202 Pusillimonas, and some Achromobacter strains, picG was located at a distant site

203 away from the pic gene cluster. In β- and γ-Proteobacteria, PicB consists of four

204 components. Interestingly, in α-Proteobacteria, including genera of Bradyrhizobium,

205 Hoeflea, and Octadecabacter, PicB consists of two components, in which the large

206 subunit PicB123 encodes proteins that correspond to PicB1, PicB2, and PicB3 (Fig.

207 S5).

208

10 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

209 Conclusions

210 PA is generated through cell metabolism or synthesized artificially industry and is

211 thus ubiquitous in the environment. The degradation and detoxification of PA by

212 microorganisms have been studied for more than 50 years (27). This study revealed

213 that the pic gene cluster is responsible for the complete degradation of PA. PicA

214 initially converted PA into 6HPA, then PicB hydroxylated 6HPA into 3,6DHPA, which

215 was further converted into 2,5DHP by PicC. The 2,5DHP was further degraded into

216 fumaric acid by PicD, PicE, PicF, and PicG. The genetic functions of picA and picB

217 genes is reported here for the first time. In addition, the findings of homologous pic

218 gene cluster in other α-, β-, and γ-Proteobacteria would help us to understand the

219 growth, competition, and environmental adaptability of bacteria in the natural

220 environment.

221

222

11 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

223 Methods

224 Chemicals

225 PA, 6HPA, and 2,5DHP were purchased from J&K Scientific Ltd. (Shanghai,

226 China). 3,6DHPA was chemically synthesized (26). Other reagents were purchased

227 from Sangon Biotech Co., Ltd. (Shanghai, China).

228

229 Strains, plasmids, and culture conditions

230 All bacterial strains and plasmids used in this study are listed in Table 2. Mineral

231 salts medium (MSM) and Luria-Bertani medium (LB) have been described previously

232 (25). Alcaligenes faecalis JQ135 was the wild-type PA-degrading strain (24). P.

233 putida KT2440 is a model Gram-negative organism that cannot degrade PA (28).

234 Sphingomonas wittichii DC-6 is a chloroacetanilide herbicide degrader that is not

235 unable to degrade PA or 2,5DHP (35). E. coli DH5α acted as host for the construction

236 of plasmids. E. coli BL21(DE3) was used to over-express proteins. Bacteria were

237 grown in LB medium at 37 ºC (E. coli) or 30 ºC (Alcaligenes, Pseudomonas,

238 Sphingomonas, and their derivates). Antibiotics were added as required at the

239 following concentrations: chloramphenicol (Cm), 34 mg/L; gentamicin (Gm), 50

240 mg/L; kanamycin (Km), 50 mg/L; and streptomycin (Str), 50 mg/L.

241

242 General DNA techniques

243 Routine isolation of genomic DNA, extraction of plasmids, restriction digestion,

244 transformations, PCR, and electrophoresis were performed according to standard

245 procedures in Molecular Cloning-A Laboratory Manual (New York: Cold Spring

246 Harbor Laboratory). Primer synthesis and the sequencing of PCR products or

247 plasmids were performed by Genscript Biotech (Nanjing, China). The primers used in

12 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

248 this study are listed in Table 3.

249

250 Construction of recombinant plasmids and heterologous expression

251 The genes from A. faecalis JQ135 were PCR amplified using corresponding

252 primers (Table 3). The fusion of DNA fragments and cut plasmids were performed

253 with the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co.,Ltd,

254 Nanjing, China).

255 The plasmid pBBR1MCS-5 (42) was used for heterologous expression in P.

256 putida KT2440. PCR products and XhoI/HindIII-digested plasmid were ligated and

257 the resulting recombinant plasmids were transferred into P. putida KT2440 by

258 biparental mating using SM10λpir.

259 To test the components of PA dehydrogenase, pBBR1MCS-5-based plasmid

260 clones containing different genes (picA1A2A3, picA1A2, and picA2A3) were

261 transferred into P. putida KT2440, to yield recombinants KT/pBBR-picA1A2A3,

262 KT/pBBR-picA1A2, and KT/pBBR-picA2A3. Similarly, to test the components of

263 6HPA monooxygenase, the strains KT/pBBR-picB1B2B3B4, KT/pBBR-picB1B2B3,

264 and KT/pBBR-picB2B3B4 were constructed.

265 For the over-expression of picD gene in E. coli BL21(DE3), the complete ORF

266 without its corresponding stop codon was amplified and inserted into the

267 NdeI/XhoI-digested plasmid pET29a(+), resulting in the plasmid pET-PicD. The

268 inductuion and purification of 6×His-tagged PicD from E. coli BL21(DE3)

269 (containing pET-PicD) were same with a previous report (25). The purified 6×

270 His-tagged PicD were then analyzed by 12.5% SDS-PAGE. The protein

271 concentrations were quantified using the Bradford method (43).

272

13 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

273 Gene knockout and genetic complementation of A. faecalis JQ135

274 Deletion mutants of the picA1A2A3 and picB1B2B3B4 genes in A. faecalis JQ135

275 were constructed using a two-step homogenetic recombination method with the

276 suicide plasmid pJQ200SK (44). Using the deletion of picA1A2A3 as an example, two

277 homologous recombination-directing sequences (500 to 1000 bp) were amplified

278 using primers, kopicA1A2A3-UF/-UR and kopicA1A2A3-DF/-DR, respectively. The

279 two PCR fragments were subsequently ligated into SacI/PstI-digested pJQ200SK

280 yielding pJQ-ΔpicA1A2A3. The pJQ-ΔpicA1A2A3 plasmid was then introduced into A.

281 faecalis JQ135 cells. The single-crossover mutants were screened on a LB plate

282 containing Str and Gm. The gentamicin-resistant strains were then subjected to

283 repeated cultivation in LB medium containing 10% sucrose and no gentamicin. The

284 double-crossover mutants, which lost their vector backbone and were sensitive to

285 gentamicin, were selected on LB Str plates. Deletion of the picA1A2A3 genes was

286 confirmed by PCR. This procedure resulted in construction of the deletion mutant

287 strain JQ135ΔpicA1A2A3.

288 Knockout mutants were complemented by the corresponding gene. For example,

289 the intact picA1A2A3 gene was amplified with primers picA1A2A3-F/-R, and then

290 ligated with the XhoI/HindIII-digested pBBR1-MCS5, generating pBBR-picA1A2A3.

291 The pBBR-picA1A2A3 vector was transferred into the mutant strain

292 JQ135ΔpicA1A2A3 to generate the complemented strain

293 JQ135ΔpicA1A2A3/pBBR-picA1A2A3.

294

295 Preparation of resting cells and biodegradation assays

296 The Alcaligenes, Pseudomonas, and Sphingomonas strains and their derivates

297 were grown in LB at 30 ºC. When the late exponential phase was reached, the cells

14 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

298 were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed

299 twice by MSM and finally resuspended with MSM. The OD600 was adjusted to 2.0.

300 These strains were named resting cells and were used for the biodegradation of PA or

301 other related compounds.

302

303 RT-PCR

304 Resting cells of A. faecalis JQ135 were inoculated into 20 mL MSM

305 supplemented with 1 mM PA or citrate and cultured at 30ºC. The initial OD600 of the

306 cultures was adjusted to 0.5. When half of the PA was degraded, the cells were

307 harvested. Total RNA was isolated using an RNA isolation kit (TaKaRa) and reverse

308 transcription-PCR (RT-PCR) was carried out with a PrimeScript RT reagent kit

309 (TaKaRa). The primers used for RT-PCR are listed in Table 3. All samples were run

310 in triplicate.

311

312 Enzymatic assays

313 PA dehydrogenase (PicA) assays were performed using the cell-free extracts of

314 KT/pBBR-picA1A2A3. Resting cells of KT/pBBR-picA1A2A3 were added into MSM

315 containing 1 mM citrate and 1 mM PA. Following incubation for 24 h, the cells were

316 harvested by centrifugation at 6000 rpm for 10 min at 4 ºC. Cell pellets were then

317 resuspended in 50 mM PBS (pH 7.0) and disrupted by sonication in an ice/water bath.

318 Cell-free extracts were obtained by centrifuging the cell lysates at 16 000 g for 30 min

319 at 4 ºC. The supernatant was later used for PA dehydrogenase assays. PicA activity

320 was analyzed spectrophotometrically at 25 ºC by the formation of 6HPA at 310 nm

321 (ε=4.45 cm-1 mM-1) using a UV2450 spectrophotometer (Shimadzu) in 1 cm path

322 length quartz cuvettes (23). The 1 mL reaction mixture contained 50 mM PBS (pH

15 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

323 7.0), 1 to 10 μg of total protein from cell extract, 1 mM PA, and 1 mM electron

324 acceptor PMS and the reaction was started by adding PA. One unit of activity was

325 defined as the amount of enzyme that catalyzed the formation of 1 μmol 6HPA in 1

326 min.

327 For the 6HPA monooxygenase (PicB) assays, the cell-free extracts of

328 KT/pBBR-picB1B2B3B4 were used. The preparation of cell-free extracts was the

329 same as that of KT/pBBR-picA1A2A3. PicB activity was analyzed

330 spectrophotometrically by looking for the formation of 3,6DHPA at 360 nm (ε=4.4

331 cm-1 mM-1) (26). The 1 mL reaction mixture contained 50 mM PBS (pH 7.0), 1 to 10

332 μg of total protein from cell extract, 1 mM 6HPA, and 1 mM electron donor (NADH

333 or others) and the reaction was started by adding 6HPA. One unit of activity was

334 defined as the amount of enzyme that catalyzed the formation of 1 μmol 3,6DHPA in

335 1 min.

336 The 2,5-DHP dioxygenase (PicD) assays was performed similarly to those

337 previously performed for NicX from P. putida KT2440 (28) and VppE in

338 Ochrobactrum sp. strain SJY1 (33). A 1 mL reaction mixture contained 50 mM PBS

339 (pH 7.0), 0.2 mM 2,5DHP, 0.1 μg purified PicD, and 1 mM Fe2+ and was incubated at

340 25 ºC. Activity was assayed spectrophotometrically by measuring the disappearance

341 of 2,5DHP at 320 nm (ε=5.2 cm-1 mM-1). One unit of activity was defined as the

342 amount of enzyme that catalyzed the consumption of 1 μmol 2,5DHP in 1 min.

343

344 Analytical methods

345 The determination of PA and 6HPA, 3,6DHPA, and 2,5DHP were performed by

346 Ultraviolet-visible spectroscopy and High performance liquid chromatography

347 analysis were same with a previous study (26).

16 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

348

349 Sequence data and bioinformatic analysis

350 The pic cluster sequence and the complete genome sequence of Alcaligenes

351 faecalis JQ135 have been deposited in the GenBank/DDBJ/EMBL database under

352 accession numbers KY264362 and CP021641, respectively.

353 Comparisons of the Pic proteins were performed against the non-redundant

354 protein sequences (nr) database using Blastp (protein-protein BLAST) programs on

355 the NCBI website, employing an Expect (E)-value inclusion threshold of 10. The

356 conserved protein domains of each gene were analyzed using the Conserved Domain

357 Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The genome

358 sequence accession numbers of other strains, and the corresponding locus tags of pic

359 gene clusters, are listed in Table S1.

360

361

17 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

362 Acknowledgments

363 This work was supported by the National Science and Technology Major Project

364 (2018ZX0800907B-002), the National Natural Science Foundation of China (Nos.

365 41630637, 31870092, 31770117, and 31600080). Key R&D Program Project in

366 Jiangsu Province (BE2016374).

367

368 Conflict of interest

369 The authors declare no conflict of interest.

370

371 References

372

373 1. Heyes MP, Eugene O, Saito K. 1997. Different kynurenine pathway enzymes limit quinolinic 374 acid formation by various human cell types. Biochem J 326:351-356. 375 2. Bryleva EY, Brundin L. 2017. Kynurenine pathway metabolites and suicidality. 376 Neuropharmacology 112:324-330. 377 3. Esquivel DG, Ramirez-Ortega D, Pineda B, Castro N, Rios C, de la Cruz VP. 2017. 378 Kynurenine pathway metabolites and enzymes involved in reactions. 379 Neuropharmacology 112:331-345. 380 4. Nishino SF, Spain JC. 1993. Degradation of nitrobenzene by a Pseudomonas 381 pseudoalcaligenes. Appl Environ Microbiol 59:2520-2525. 382 5. Mehler AH. 1956. Formation of picolinic and quinolinic acids following enzymatic oxidation 383 of 3-hydroxyanthranilic acid. J Biol Chem 218:241-254. 384 6. Asano Y, Yamamoto Y, Yamada H. 1994. Catechol 2, 3-dioxygenase-catalyzed synthesis of 385 picolinic acids from catechols. Biosci Biotechnol Biochem 58:2054-2056. 386 7. Chirino B, Strahsburger E, Agullo L, Gonzalez M, Seeger M. 2013. Genomic and functional 387 analyses of the 2-aminophenol catabolic pathway and partial conversion of its substrate into 388 picolinic acid in Burkholderia xenovorans LB400. Plos One 8:e75746. 389 8. Abdullaev M, Klyuev M, Abdullaeva ZS, Kurbanov B, Idrisova A. 2008. Preparation of 390 lidocaine, bipuvacaine, mepivacaine, trimecaine, and pyromecaine by reductive acylation on 391 palladium catalysts. Pharm Chem J 42:357-359. 392 9. McCall PJ, Agin GL. 1985. Desorption kinetics of picloram as affected by residence time in 393 the soil. Environ Toxicol Chem 4:37-44. 394 10. Broadhurst CL, Domenico P. 2006. Clinical studies on chromium picolinate supplementation 395 in diabetes mellitus—a review. Diabetes Technol Therapeut 8:677-687. 396 11. Martin J, Wang ZQ, Zhang XH, Wachtel D, Volaufova J, Matthews DE, Cefalu WT. 2006. 397 Chromium picolinate supplementation attenuates body weight gain and increases insulin 398 sensitivity in subjects with type 2 diabetes. Diabetes Care 29:1826-1832. 399 12. Ogata S, Inoue K, Iwata K, Okumura K, Taguchi H. 2001. Apoptosis induced by picolinic 400 acid-related compounds in HL-60 cells. Biosci Biotechnol Biochem 65:2337-2339. 401 13. Cioczek-Czuczwar A, Czuczwar P, Turski WA, Parada-Turska J. 2016. Influence of 402 picolinic acid on seizure susceptibility in mice. Pharmacol Rep 69:77-80. 403 14. Prodinger J, Loacker LJ, Schmidt RL, Ratzinger F, Greiner G, Witzeneder N, Hoermann 404 G, Jutz S, Pickl WF, Steinberger P. 2015. The tryptophan metabolite picolinic acid suppresses 405 proliferation and metabolic activity of CD4+ T cells and inhibits c-Myc activation. J Leukocyte 406 Biol 99:429-431. 18 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

407 15. Nakata HM, Halvorson HO. 1960. Biochemical changes occurring during growth and 408 sporulation of Bacillus cereus. J Bacteriol 80:801-810. 409 16. Tamer Ö, Tamer SA, İdil Ö, Avcı D, Vural H, Atalay Y. 2017. Antimicrobial Activities, DNA 410 interactions, spectroscopic (FT-IR and UV-Vis) characterizations, and DFT calculations for 411 pyridine-2-carboxylic acid and its derivates. J Mol Struct 1152:399-408. 412 17. Suksrichavalit T, Prachayasittikul S, Nantasenamat C, Isarankura-Na-Ayudhya C, 413 Prachayasittikul V. 2009. Copper complexes of pyridine derivatives with superoxide 414 scavenging and antimicrobial activities. Euro J Med Chem 44:3259-3265. 415 18. Smythe GA, Poljak A, Bustamante S, Braga O, Maxwell A, Grant R, Sachdev P. 2003. 416 ECNI GC-MS analysis of picolinic and quinolinic acids and their amides in human plasma, CSF, 417 and brain tissue, p. 705-712, Developments in Tryptophan and Serotonin Metabolism. Springer. 418 19. Kiener A, Glockler R, Heinzmann K. 1993. Preparation of 419 6-oxo-1,6-dihydropyridine-2-carboxylic acid by microbial hydroxylation of 420 pyridine-2-carboxylic acid. J Chem Soc Perk T:1201-1202. 421 20. Siegmund I, Koenig K, Andreesen JR. 1990. Molybdenum involvement in aerobic 422 degradation of picolinic acid by Arthrobacter picolinophilus. FEMS Microbiol Lett 423 67:281-284. 424 21. Zheng C, Wang Q, Ning Y, Fan Y, Feng S, He C, Zhang TC, Shen Z. 2017. Isolation of a 425 2-picolinic acid-assimilating bacterium and its proposed degradation pathway. Bioresource 426 Technol 245:681-688. 427 22. Zheng C, Zhou J, Wang J, Qu B, Wang J, Lu H, Zhao H. 2009. Aerobic degradation of 428 2-picolinic acid by a nitrobenzene-assimilating strain: Streptomyces sp. Z2. Bioresource 429 Technol 100:2082-2084. 430 23. Orpin CG, Knight M, Evans WC. 1972. The bacterial oxidation of picolinamide, a photolytic 431 product of Diquat. Biochem J 127:819-831. 432 24. Qiu J, Zhang J, Zhang Y, Wang Y, Tong L, Hong Q, He J. 2017. Biodegradation of picolinic 433 acid by a newly isolated bacterium Alcaligenes faecalis strain JQ135. Curr Microbiol 434 74:508-514. 435 25. Qiu J, Liu B, Zhao L, Zhang Y, Cheng D, Yan X, Jiang J, Hong Q, He J. 2018. A novel 436 degradation mechanism for pyridine derivatives in Alcaligenes faecalis JQ135. Appl Environ 437 Microbiol 84:e00910-00918. 438 26. Qiu J, Zhang Y, Yao S, Ren H, Qian M, Hong Q, Lu Z, He J. 2019. Novel 439 3,6-dihydroxypicolinic acid decarboxylase mediated picolinic acid catabolism in Alcaligenes 440 faecalis JQ135. J Bacteriol Accepted. (JB00665-18). 441 27. Dagley S, Johnson PA. 1963. Microbial oxidation of kynurenic, xanthurenic and picolinic 442 acids. Biochim Biophys Acta 78:577-587. 443 28. Jimenez JI, Canales A, Jimenez-Barbero J, Ginalski K, Rychlewski L, Garcia JL, Diaz E. 444 2008. Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic 445 cluster from Pseudomonas putida KT2440. Proc Natl Acad Sci USA 105:11329-11334. 446 29. Grether‐Beck S, Igloi GL, Pust S, Schilz E, Decker K, Brandsch R. 1994. Structural 447 analysis and molybdenum ‐ dependent expression of the pAO1 ‐ encoded nicotine 448 dehydrogenase genes of Arthrobacter nicotinovorans. Mol Microbiol 13:929-936. 449 30. Chakraborty J, Ghosal D, Dutta A, Dutta TK. 2012. An insight into the origin and functional 450 evolution of bacterial aromatic ring-hydroxylating oxygenases. J Biomol Struct Dyn 451 30:419-436. 452 31. Vaitekūnas J, Gasparavičiūtė R, Rutkienė R, Tauraitė D, Meškys R. 2016. A 453 2-hydroxypyridine catabolism pathway in Rhodococcus rhodochrous strain PY11. Appl 454 Environ Microbiol 82:1264. 455 32. Tate RL, Ensign JC. 1974. Picolinic acid hydroxylase of Arthrobacter picolinophilus. Can J 456 Microbiol 20:695-702. 457 33. Yu H, Tang H, Zhu X, Li Y, Xu P. 2015. Molecular mechanism of nicotine degradation by a 458 newly isolated strain, Ochrobactrum sp. strain SJY1. Appl Environ Microbiol 81:272-281. 459 34. Petkevičius V, Vaitekūnas J, Stankevičiūtė J, Gasparavičiūtė R, Meškys R. 2018. 460 Catabolism of 2-hydroxypyridine by Burkholderia sp. strain MAK1: a 2-hydroxypyridine 461 5-monooxygenase encoded by hpdABCDE catalyzes the first step of biodegradation. Appl 462 Environ Microbiol 84:AEM.00387-00318. 463 35. Chen Q, Wang C-H, Deng S-K, Wu Y-D, Li Y, Yao L, Jiang J-D, Yan X, He J, Li S-P. 2014. 464 Novel three-component Rieske non-heme iron oxygenase system catalyzing the N-dealkylation 465 of chloroacetanilide herbicides in sphingomonads DC-6 and DC-2. Appl Environ Microbiol 466 80:5078-5085. 19 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

467 36. Strnad H, Ridl J, Paces J, Kolar M, Vlcek C, Paces V. 2011. Complete genome sequence of 468 the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8. J Bacteriol 469 193:791-792. 470 37. Mahan KM, Zheng H, Fida TT, Parry RJ, Graham DE, Spain JC. 2017. Iron-dependent 471 enzyme catalyzes the initial step in biodegradation of N-nitroglycine by Variovorax sp. strain 472 JS1663. Appl Environ Microbiol 83:e00457-00417. 473 38. Parkhill J, Sebaihia M, Preston A, Murphy LD, Thomson N, Harris DE, Holden MT, 474 Churcher CM, Bentley SD, Mungall KL. 2003. Comparative analysis of the genome 475 sequences of Bordetella pertussis, and Bordetella bronchiseptica. Nat 476 Genet 35:32. 477 39. Ju S, Lin J, Zheng J, Wang S, Zhou H, Sun M. 2016. Alcaligenes faecalis ZD02, a novel 478 nematicidal bacterium with an extracellular protease virulence factor. Appl Environ 479 Microbiol 82:2112-2120. 480 40. Vollmers J, Voget S, Dietrich S, Gollnow K, Smits M, Meyer K, Brinkhoff T, Simon M, 481 Daniel R. 2013. Poles apart: Arctic and Antarctic Octadecabacter strains share high genome 482 plasticity and a new type of xanthorhodopsin. Plos One 8:e63422. 483 41. Cubillas C, Miranda-Sánchez F, González-Sánchez A, Elizalde JP, Vinuesa P, Brom S, 484 García-De LSA. 2017. A comprehensive phylogenetic analysis of copper transporting P1B 485 ATPases from bacteria of the Rhizobiales order uncovers multiplicity, diversity and novel 486 taxonomic subtypes. MicrobiologyOpen 6:e452. 487 42. Kovach M, Elzer P, Hill D, Robertson G, Farris M, Roop R, Peterson K. 1995. Four new 488 derivatives of the broad-hostrange cloning vector pBBR1MCS, carrying different 489 antibioticresistance cassettes. Gene 166:175-176. 490 43. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram 491 quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. 492 44. Quandt J, Hynes M. 1993. Versatile suicide vectors which allow direct selection for gene 493 replacement in gram-negative bacteria. Gene 127:15-21. 494

495

496

20 / 20 bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

A PicA PicB OH PicC OH PicD COOH PicE COOH PicF COOH PicG COOH CHO HO N O N N COOH HO N COOH HO N COOH H CONH2 COOH HOOC PA 6HPA 3,6DHPA 2,5DHP N-formylmaleamic acid Maleamic acid Maleic acid Fumaric acid

B R C E D F B4 B3 B2 B1 A1 A2 A3 G

1 2 3 4 5 6 7 8 9 10 11 12 13

Fig. 1 PA degradation in A. faecalis JQ135. (A) The complete catabolic pathway and corresponding enzymes. (B) Genetic organization of the pic gene cluster. Genes are annotated following the color scheme in A.

A M 16S 1 2 3 4 5 6 7 8 9 10 11 12 13 M bp 750 500 250 100

B 750 500 250 100

Fig. 2 Agarose gel electrophoresis of RT-PCR products generated using RNA of A. faecalis JQ135. when cells grow with PA (A) or citrate (B). M, DNA marker. 16S, positive control. 1 to 13 were corresponding to the positions in Fig. 1B. bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

A Subunit I Subunit II Subunit III

MPT1 MPT2 MPT3 FAD [FeS]1 [FeS]2 SRPBCC PicA A1 A2 A3 Spm A B C

CDH A B C NDH L M S

KDH L M S CODH L M S Xdh A B C Qox L M S

B a 100 PicA1 90 SpmA 53 CDHA 56 NDHL 98 KDHL CODHL XdhA QoxL

0.1 Moco binding subunit b c 46 CDHB 48 KDHS 37 KDHM 38 NDHS CDH NDHM C CODHM XdhC

62 XdhB CODHS QoxS QoxM 34 72 PicA2 43 PicA3 100 95 SpmB SpmC

0.1 0.1 FAD binding subunit Fe-S binding subunit

Fig. 3 Bioinformatic analysis of PicA. (A) Molecular architecture of several multicomponent molybdenum-containing hydroxylases. Subunits I, II, and III are molybdopterin cytosine

dinucleotide (MCD), FAD, and two [Fe-S] clusters containing components, respectively. SpmABC (GenBank accession numbers AEJ14617 and AEJ14616), 3-succinoylpyridine dehydrogenase

from P. putida; CDHABC (D7REY3, D7REY4, and D7REY5), caffeine dehydrogenase from

Pseudomonas sp. strain CBB1; NDHLMS (CAA53088, CAA53087, and CAA53086), nicotine

dehydrogenase from Athrobacter nicotinovorans; KDHLMS (WP_016359451, WP_016359456, and

WP_016359457), ketone dehydrogenase from A. nicotinovorans; CODHLMS (P19913, P19914,

and P19915), carbon monoxide dehydrogenase from Hydrogenophaga pseudoflava; XDHABC

(Q46799, Q46800, and Q46801), xanthine dehydrogenase from E. coli; QoxLMS (CAD61045, bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

CAD61046, and CAD61047), quinaldine 4-oxidase from A. ilicis. The letters depicted below the proteins indicate the subunit names of the corresponding proteins. The conserved domains are as follows: MPT, domains for binding to the molybdopterin cytosine dinucleotide cofactor (MoCo); FAD, FAD-binding domain; [FeS], ferredoxin-like [2Fe-2S]-binding domain; SRPBCC, SRPBCC ligand-binding domain. (B) Phylogenetic analysis of PicA and related molybdenum containing hydroxylases. (a) Phylogenetic analysis of PicA1 and the Moco binding subunit of other enzymes. (b) Phylogenetic analysis of PicA2 and the FAD binding subunit of other enzymes. (c) Phylogenetic analysis of PicA3 and the [2Fe-2S] binding subunit of other enzymes. The phylogenetic trees were constructed by using the neighbor-joining method (with a bootstrap of 1000) with software MEGA 6.0. The bar represents amino acid substitutions per site.

Fig. 4 Growth phenotype of wild type A. faecalis JQ135 (WT), mutant JQ135ΔpicA1A2A3 (ΔA), and the complemented strain JQ135ΔpicA1A2A3/pBBR-picA1A2A3 (ΔA/A) on MSM plates with 1.0 mM PA and 6HPA as sole carbon sources.

bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

A Fd Red OL OS B OH PicB B1 B2 B3 B4 HO N COOH

OH OH H H BphA A1 A2 A3 A4 38% 18% 40% 33%

Hca A1 A2 C D 27% 19% 43% 35%

AndA Ac Ad Ab Aa 42% 35% 41% 34%

Nag Aa G H Ab

14% 45% 45% 34%

CmtA Aa Ab Ac Ad 35% 24% 24% 34%

Fig. 5 Genetic organizations of the PicB encoding genes as compared with similar Rieske non-heme iron aromatic ring-hydroxylating oxygenase genes (A) and the corresponding enzyme reactions (B). The arrows in A indicate the size and direction of each gene. Homologous genes are shown in the same color. Fd, ferredoxin; Red, reductase; OL, oxygenase large component; OS, oxygenase small component. Double vertical gray lines indicate discontinuous genes. Numbers below the arrows indicate the percent amino acid sequence identity with the ortholog picB gene product. BphA1A2A3A4 (GenBank accession numbers Q53121, Q53122, Q53123, and Q0S032), biphenyl 2,3-dioxygenase from Rhodococcus jostii. HcaA1A2CD (P0ABR5, Q47140, P0ABW0, and P77650), 3-phenylpropionate dioxygenase from E. coli. AndAaAbAcAd (Q84BZ0, Q84BZ1, Q84BZ3, and Q84BZ2), anthranilate 1,2-dioxygenase from Burkholderia cepacia. NagAaGHAb (O52379, O52380, and O52381), salicylate 5-hydroxylase from Ralstonia sp. CmtAaAbAcAd (Q51973, Q51974, Q51975, and Q51978), p-cumate 2,3-dioxygenase from Pseudomonas putida.

bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

Fig. 6 Growth phenotype of wild type A. faecalis JQ135 (WT), mutant JQ135ΔpicB1B2B3B4 (ΔB), and the complemented strain JQ135ΔpicB1B2B3B4/pBBR-picB1B2B3B4 (ΔB/B) on MSM plates with 1.0 mM PA, 6HPA, and 3,6DHPA as sole carbon sources.

bioRxiv preprint doi: https://doi.org/10.1101/530550; this version posted January 25, 2019. 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.

R C E D F B4 B3 B2 B1 A1 A2 A3 G I: Afa

II: Pus

III: Ax y

IV: Var , Pac

V: A ka β VI: Ama , Api, Bbr, Bpa, Bpe

VII: Ban , Bps

VIII: Cup, Ral

IX: Var2, Pol X: Cab , Bce, Pfu, Psp

γ XI: Pfl

XII: Hoe , Lve, Oar

XIII: Pse α

XIV: Abr, Bra

XV: Bsp

Fig. 7 Predicted PA catabolism gene clusters in bacterial genomes. α, β, and γ indicate the alpha-, beta-, and gamma-Proteobacteria. I to XV: The 15 different types of PA catabolism loci. Abbreviations and representative strains: Afa, Alcaligenes faecalis JQ135; Pus, Pusillimonas sp. T2; Axy, Achromobacter xylosoxidans A8; Var, Variovorax paradoxus S110; Pac, Pseudacidovorax sp. RU35E; Aka, Advenella kashmirensis W13003; Ama, Achromobacter marplatensis B2; Api, Achromobacter piechaudii ATCC 43553; Bbr, Bordetella bronchiseptica RB50; Bpa, Bordetella parapertussis ATCC BAA-587; Bpe, Bordetella pertussis Tohama I; Ban, Bordetella ansorpii NCTC13364; Bps, Bordetella pseudohinzii HI4681; Cup, Cupriavidus necator N-1; Ral, Ralstonia eutropha JMP134; Var2, Variovorax sp. JS1663; Pol, Polaromonas sp. OV174; Cab, Caballeronia arvi LMG 29317; Bce, Burkholderia cepacia JBK9; Pfu, Paraburkholderia fungorum NBRC 102489; Psp, Pandoraea sputorum DSM 21091; Pfl, Pseudomonas fluorescens C8; Hoe, Hoeflea sp. IMCC20628; Lve, Loktanella vestfoldensis SMR4r; Oar, Octadecabacter arcticus 238; Pse, Paracoccus seriniphilus DSM 14827; Abr, Afipia broomeae GAS525; Bra, Bradyrhizobium canariense GAS369; Bsp, Bradyrhizobium sp. C9. The detailed genomic accession numbers and the gene locus tags are listed in Table S1. Identities (percent) of amino acid sequences between Pic proteins of strain A. faecalis JQ135 and representative homologs are listed in Table S2.

bioRxiv preprint

Table 1 The pic genes and assigned function in Alcaligenes faecalis JQ135. was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission.

doi:

Gene (Locus tag) Product Assigned function Sequence Homologous proteins in UniProtKB/SwissProt (accession no.) https://doi.org/10.1101/530550 (aa) identity (%) picR (AFA_15150) 186 MarR-type negative regulator PicR 33 mexR (P52003) picC (AFA_15145) 323 3,6DHPA decarboxylase 37 2,3-Dihydroxybenzoate decarboxylase (P80402) picE (AFA_15140) 274 N-formylmaleamic acid deformylase 60 NFM deformylase (Q88FY3) picD (AFA_15135) 350 2,5DHP 5,6-dioxygenase 55 2,5-DHP dioxygenase (Q88FY1) ; picF (AFA_15130) 216 Maleamic acid amidohydrolase 43 Maleamic acid amidohydrolase (Q88FY5) this versionpostedJanuary25,2019. picB4 (AFA_15125) 172 6HPA monooxygenase subunit IV 35 Anthranilate 1,2-dioxygenase small subunit (Q84BZ2) picB3 (AFA_15120) 429 6HPA monooxygenase subunit III 45 Salicylate 5-hydroxylase, oxygenase large subunit (O52379) picB2 (AFA_15115) 425 6HPA monooxygenase subunit II 36 Benzene 1,2-dioxygenase ferredoxin reductase (B1LNJ8) picB1 (AFA_15110) 104 6HPA monooxygenase subunit I 46 Naphthalene 1,2-dioxygenase ferredoxin (Q51493) picA1 (AFA_15100) 800 PA dehydrogenase large subunit 39 Caffeine dehydrogenase large subunit (D7REY3) picA2 (AFA_15095) 278 PA dehydrogenase small subunit 33 Carbon monoxide dehydrogenase medium chain (P19914) picA3 (AFA_15090) 395 PA dehydrogenase medium subunit 46 Carbon monoxide dehydrogenase small chain (P19915) picG (AFA_16520) 253 Maleic acid cis-trans isomerase 99 Maleic acid isomerase (O24766) The copyrightholderforthispreprint(which

bioRxiv preprint

was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. doi: Table 2 Strains used in this study. https://doi.org/10.1101/530550

Strains or plasmids Description Source Alcaligenes faecalis strains JQ135 Strr; PA-degrading bacterium. Gram-negative. Wild type. CCTCC M 2015812 JQ135ΔpicA1A2A3 Strr; picA1A2A3-deletion mutant of JQ135 This study JQ135ΔpicA1A2A3/pBBR-picA1A2A3 Strr, Gmr; JQ135ΔpicA1A2A3 complementation with pBBR-picA1A2A3 This study

r ;

JQ135ΔpicB1B2B3B4 Str ; picB1B2B3B4-deletion mutant of JQ135 This study this versionpostedJanuary25,2019. JQ135ΔpicB1B2B3B4/pBBR-picB1B2B3B4 Strr, Gmr; JQ135ΔpicB1B2B3B4 complementation with pBBR-picB1B2B3B4 This study Pseudomonas putida strains KT2440 Cmr; Metabolically versatile saprophytic soil strain; PA-non-degrading bacterium ATCC 47054 KT/pBBR-picA1A2A3 Cmr, Gmr; KT2440 containing plasmid pBBR-picA1A2A3 This study KT/pBBR-picA1A2 Cmr, Gmr; KT2440 containing plasmid pBBR-picA1A2 This study KT/pBBR-picA2A3 Cmr, Gmr; KT2440 containing plasmid pBBR-picA2A3 This study KT/pBBR-picB1B2B3B4 Cmr, Gmr; KT2440 containing plasmid pBBR-picB1B2B3B4 This study KT/pBBR-picB1B2B3 Cmr, Gmr; KT2440 containing plasmid pBBR-picB1B2B3 This study

KT/pBBR-picB2B3B4 Cmr, Gmr; KT2440 containing plasmid pBBR-picB2B3B4 This study The copyrightholderforthispreprint(which Sphingomonas wittichii DC-6 Strr; Chloroacetanilide herbicide degrader; PA-non-degrading bacterium KACC 16600 DC-6/pBBR-picEDFG Strr; Gmr; S. wittichii DC-6 containing pBBR-picEDFG This study DC-6/pBBR-picEDF Strr; Gmr; S. wittichii DC-6 containing pBBR-picEDF This study E. coli strains DH5α F− recA1 endA1 thi-1 hsrdR17 supE44 relA1 deoRΔ(lacZYA-argF) U169 φ80lacZ ΔM15 TaKaRa BL21(DE3) F− ompT hsdS(rB− mB−) gal dcm lacY1(DE3) TaKaRa

SM10λpir Donor strain for biparental mating Lab stock bioRxiv preprint

Plasmids was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. r pET29a(+) Km , expression plasmid Novagen doi: r + + pJQ200SK Gm , mob , orip15A, lacZα , sacB; suicide plasmid Lab stock https://doi.org/10.1101/530550 pBBR1MCS-5 Gmr; broad-host-range cloning plasmid Lab stock pJQ-ΔpicA1A2A3 Gmr; picA1A2A3 genes deletion plasmid; the upstream region of picA1 gene and downstream region of This study picA3 gene fused into SacI/PstI-digested pJQ200SK pJQ-ΔpicB1B2B3B4 Gmr; picB1B2B3B4 genes deletion plasmid; the upstream region of picB1 gene and downstream region of This study picB4 gene fused into SacI/PstI-digested pJQ200SK pBBR-picA1A2A3 Gmr; the fragment containing the picA1A2A3 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 This study r Gm ; the fragment containing the picA1A2 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 ; pBBR-picA1A2 This study this versionpostedJanuary25,2019. pBBR-picA2A3 Gmr; the fragment containing the picA2A3 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 This study pBBR-picB1B2B3B4 Gmr; the fragment containing the picB1B2B3B4 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 This study pBBR-picB1B2B3 Gmr; the fragment containing the picB1B2B3 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 This study pBBR-picB2B3B4 Gmr; the fragment containing the picB2B3B4 gene inserted into XhoI/HindIII-digested pBBR1MCS-5 This study pET-PicD Kmr; NdeI-XhoI fragment containing picD gene inserted into pET29a(+) This study

The copyrightholderforthispreprint(which bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. Table 3 Primers used in this study. doi:

https://doi.org/10.1101/530550 Primers Sequence(5’-3’) Description kopicA1A2A3-UF AGCTTGATATCGAATTCCTGCAGCTGATTTTGCCAAGATCTATC To construct plasmid pJQ-ΔpicA1A2A3 kopicA1A2A3-UR TCTAGAACTAGTGGATCCTTCCATCGGCAACATGCACTGC kopicA1A2A3-DF GGATCCACTAGTTCTAGACCGTCGTGGCCGAGTTCAATCC kopicA1A2A3-DR AGGGAACAAAAGCTGGAGCTCGGCAGGCAACATGAACAGCAC ; kopicB1B2B3B4-UF AGCTTGATATCGAATTCCTGCAGACTCGGAGCGCGACTGCTCAC To construct plasmid pJQ-ΔpicB1B2B3B4 this versionpostedJanuary25,2019. kopicB1B2B3B4-UR TCTAGAACTAGTGGATCCGTATAAGTCCGTGGTTTGCATC kopicB1B2B3B4-DF GGATCCACTAGTTCTAGAGTTTCGGAAATTTTTATCCAGT kopicB1B2B3B4-DR AGGGAACAAAAGCTGGAGCTCTCTGCGCGTAAGGCACCAGTTC picA1A2A3-F CAGGAATTCGATATCAAGCTTTACGACAAGTCTCAGGAGCTGTGG To construct plasmid pBBR-picA1A2A3 picA1A2A3-R GGTACCGGGCCCCCCCTCGAGGTACCATTGCACTTCCAGCCAGG picA1A2-F CAGGAATTCGATATCAAGCTTTACGACAAGTCTCAGGAGCTGTGG To construct plasmid pBBR-picA1A2 picA1A2-R GGTACCGGGCCCCCCCTCGAGGTCGCGGCTGGTGGCCAACATGC The copyrightholderforthispreprint(which picA2A3-F CAGGAATTCGATATCAAGCTTATCGCCCAATACGGAGTTTGG To construct plasmid pBBR-picA2A3 picA2A3-R GGTACCGGGCCCCCCCTCGAGGTACCATTGCACTTCCAGCCAGG picB1B2B3B4-F GGTACCGGGCCCCCCCTCGAGGTTGCGGCCGTTGCCGATTTCAG To construct plasmid pBBR-picB1B2B3B4 picB1B2B3B4-R CAGGAATTCGATATCAAGCTTGGAATCAGAAGACCTTCTGACTC picB1B2B3-F GGTACCGGGCCCCCCCTCGAGGTTGCGGCCGTTGCCGATTTCAG To construct plasmid pBBR-picB1B2B3 bioRxiv preprint

picB1B2B3-R CAGGAATTCGATATCAAGCTTACGATCCAACGTGGCACAGTAC was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. picB2B3B4-F GGTACCGGGCCCCCCCTCGAGGTAGAAACTGGCCCTGCACAAACTGG To construct plasmid pBBR-picB2B3B4 doi: https://doi.org/10.1101/530550 picB2B3B4-R CAGGAATTCGATATCAAGCTTGGAATCAGAAGACCTTCTGACTC picEDF-F CAGGAATTCGATATCAAGCTTCATCGGCAGCAATATTCTGATCACC To construct plasmid pBBR-picEDF picEDF-R GGTACCGGGCCCCCCCTCGAGGTCACATCGTTCTTCTCAAATAC picEDFG-F1 CAGGAATTCGATATCAAGCTTCATCGGCAGCAATATTCTGATCACC To construct plasmid pBBR-picEDFG picEDFG-R1 CCAAGCATCGTCGCACATTTCCCACATCGTTCTTCTCAAATAC

picEDFG-F2 GGAAATGTGCGACGATGCTTGG ; this versionpostedJanuary25,2019. picEDFG-R2 GGTACCGGGCCCCCCCTCGAGGTGGCAGGGCTGTGTACTGCTAAG expPicD-F CTTTAAGAAGGAGATATACATATGAGCACATTTTTGTATGGC To construct plasmid pET-PicD expPicD-R GTGGTGGTGGTGGTGGTGCTCGAGCACCAAGCGCTGGCCCAG RT-16S F CGCGGTAATACGTAGGGTG To amplify fragment 16S in Fig. 2 RT-16S R AACTTCACGCGTTAGCTGCG RT-1 F TCGTCGGGGTTGATACGACG To amplify fragment 1 in Fig. 2

RT-1 R GGCCGACATCCTGGGTCCAGTG The copyrightholderforthispreprint(which RT-2 F GTACGTCGTGCTGTCGCAGACT To amplify fragment 2 in Fig. 2 RT-2 R ATAGAACGGCACATCCAGCTT RT-3 F GGCAGTAACTTGGGGTTTTGTGG To amplify fragment 3 in Fig. 2 RT-3 R AGAAGGCGCGCATATCCTCAG RT-4 F AGATTGCCGCTCAGTCCATGG To amplify fragment 4 in Fig. 2 bioRxiv preprint

RT-4 R TCACGAATCGCAGGGAATTC was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission.

RT-5 F GCAACGGCACGTGAGCAG To amplify fragment 5 in Fig. 2 doi:

https://doi.org/10.1101/530550 RT-5 R CAACCGCTGGTAACCGCAC

RT-6 F ATACCGGGAACACCAGATCGTTCG To amplify fragment 6 in Fig. 2 RT-6 R ATTGAGGCCGAGGGTAATTTCATGC

RT-7 F GCATACTTGTCCTCGGACTTGG To amplify fragment 7 in Fig. 2

RT-7 R ACCAGTGGTCTTACAGCCTGAAGG

RT-8 F ATCCTGCCCGACAATGGCG To amplify fragment 8 in Fig. 2 ;

this versionpostedJanuary25,2019. RT-8 R CACTGTCATTGAAGCGAATG

RT-9 F AGGCCTGGCTAAAGTCAGCC To amplify fragment 9 in Fig. 2

RT-9 R AAATTTCCGAAACCGATGC RT-10 F CGCCGCCCGTGCTGACCGC To amplify fragment 10 in Fig. 2 RT-10 R GCAGGCGGCCTTCATTGC RT-11 F CCCACGCTCAGATTGAAGA To amplify fragment 11 in Fig. 2

RT-11 R ACATCCACAGACTCAATAAAC The copyrightholderforthispreprint(which RT-12 F GAACGGCTTTACGACGTTTG To amplify fragment 12 in Fig. 2 RT-12 R TCCAACGTGGCATTGCCCTTGAAGG RT-13 F CCGCATGAGCGTGATGGC To amplify fragment 13 in Fig. 2 RT-13 R TCGTGGTTCAGGCGCTTGAC