Identification of Putative Genes Involved in Bisphenol A Degradation

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Identification of putative genes involved in bisphenol A degradation using differential protein abundance analysis of Sphingobium sp. BiD32 Nicolette Angela Zhou, Henrik Kjeldal, Heidi Lois Gough, and Jeppe Nielsen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02987 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 26, 2015

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Environmental Science & Technology

1

Identification of putative genes involved in

2

bisphenol A degradation using differential protein

3

abundance analysis of Sphingobium sp. BiD32

4 5

Nicolette A. Zhou1,2, Henrik Kjeldal1, Heidi L. Gough2, Jeppe L. Nielsen1*

6 7

1

8

Aalborg, Denmark

9

2

Aalborg University, Department of Chemistry and Bioscience, Fredrik Bajers Vej 7H, DK-9220

University of Washington, Department of Civil and Environmental Engineering; More Hall 201

10

Box 352700, Seattle, Washington 98195-2700, USA.

11

*corresponding author, (+45) 99 40 85 06; fax (+45) 98 14 18 08; [email protected]

12 13

Keywords: label-free quantitative proteomics, metabolomics, microbial degradation, wastewater

14

treatment, bisphenol A, PPCP, BPA, TOrC

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Abstract

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Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment

17

plant (WWTP) effluents into surface waters result in deleterious effects on aquatic life.

18

Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to

19

degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-

20

free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a

21

species-specific platform for optimal protein identification. The bacterial proteomes of

22

Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A

23

total of 2155 proteins were identified, 1174 of these proteins were quantified, and 184 of these

24

proteins had a statistically significant change in abundance in response to the presence/absence

25

of BPA (p ≤ 0.05). Proteins encoded for by genes previously identified to be responsible for

26

protocatechuate degradation were upregulated in the presence of BPA. The analysis of the

27

metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated

28

metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was

29

implicated in the metabolic pathway associated with the detected metabolite. This enzyme is

30

hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a

31

future genetic marker for BPA degradation.

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1.

Introduction

33

Wastewater treatment plants (WWTP) discharge endocrine disrupting compounds (EDC) to

34

aquatic environments, resulting in deleterious effects on aquatic life. A common EDC detected in

35

WWTP effluents and surface waters is bisphenol A (BPA). BPA is a widely used plasticizer for

36

polycarbonate plastics and epoxy resins manufacturing 1 with 3.2 million metric tons produced

37

globally in 2003 2. BPA is an estrogen and androgen receptor agonist 3 and has negative effects

38

on aquatic life at concentrations measured in the environment 4–6. While BPA is partially

39

removed during wastewater treatment, residual concentrations enter the receiving aquatic

40

environments 3,7 with BPA detected in 41.2% of US streams tested and a median and maximum

41

concentration of 140 ng/L and 12,000 ng/L, respectively 8. Up to 29 ng/L BPA was detected in

42

groundwater impacted by WWTP effluent 9. New technologies are needed to improve the

43

removal of EDCs, such as BPA, during wastewater treatment.

44 45

Bioaugmentation with EDC-degrading bacteria is a possible solution to improve the removal of

46

EDCs during wastewater treatment 10. Previous studies have isolated many bacteria capable of

47

degrading various contaminants 11–15. Among these is Sphingobium sp. BiD32, which was

48

isolated from activated sludge and is capable of degrading BPA to 5 ng/L 16. As average effluent

49

concentrations range from 4.2 to 1600 ng/L 17,18, bioaugmentation with these EDC-degrading

50

bacteria can likely improve the removal of EDCs in WWTPs. Understanding how the bacteria

51

degrade these contaminants will improve bioaugmentation techniques by potentially allowing for

52

easier monitoring of EDC degradation.

53



54

Identifying genes that could be used as genetic indicators of BPA degradation would enable

55

tracking of biodegradation potential in WWTPs. Proteomic studies have been effective at

56

identifying degradation genes and pathways of other EDCs and pharmaceuticals, as

57

demonstrated by recent work with ibuprofen 19 and 17β-estradiol (E2) 20. Previous proteomic

58

studies have also led to the identification of gene markers for E2 exposure using label-free

59

proteomics 21. However, this approach has not yet been applied for BPA degradation.

60 61

The objective of this study was to identify enzymes involved in BPA degradation by

62

Sphingobium sp. BiD32 and to elucidate the degradation pathway using a combination of

63

genomics, proteomics, and metabolite analysis. Label-free quantitative proteomics was used to

64

investigate the effect of BPA on the proteome of Sphingobium sp. BiD32 and the results were

65

used to identify candidate BPA catabolic genes in the organism’s genome. Analysis of the

66

metabolites from BPA then assisted in confirming potential genes involved and the pathway.

67 68

2.

Material and methods

69

2.1

Chemicals, reagents, and bacterial strain

70

BPA (≥99%) was obtained from Sigma Aldrich (St. Louis, MO, USA). Organic solvents and

71

water used for BPA sample extraction and analysis were high performance liquid

72

chromatography (HPLC) grade. A 35 mg/L BPA aqueous stock was prepared by adding BPA

73

dissolved in acetonitrile (1000 mg/L), evaporating the acetonitrile at 60ºC, adding water, and

74

autoclave sterilizing (121ºC, 20 min). A control of acetonitrile was prepared the same way as the

75

BPA aqueous stock. Sphingobium sp. BiD32 (GenBank accession number JX87937) was

76

previously isolated from activated sludge 16.


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77 78

2.2

Library preparation, genome sequencing, assembly, and analysis

79

The whole genome sequence of bacterial strain Sphingobium sp. BiD32 was prepared and

80

completed (details in Supporting Information (SI)). The sequences were trimmed and assembled

81

using de novo assembly by the CLC Genomics Workbench version 5.5.1 (CLC bio, Aarhus,

82

Denmark). During trimming, the quality score limit was set at 0.05 and reads < 50 bp were

83

discarded. Default parameters for assembly were used with the exception of the word size (62),

84

the minimum contig length (1000), and the similarity fraction (0.95). Assembled contigs were

85

curated by CodonCode Aligner v 3.7 (CodonCode Corp.) and analyzed with the Rapid

86

Annotation using Subsystem Technology (RAST) annotation server for subsystem classification

87

and functional annotation 24,23. This whole-genome shotgun project was deposited at

88

DDBJ/EMBL/GenBank under the accession numbers CAVK010000001 to CAVK010000262.

89 90

2.3

Sample preparation for proteomic analysis

91

Samples were prepared for proteomic analysis by growing Sphingobium sp. BiD32 in the

92

presence and absence of BPA (Figure 1). Sphingobium sp. BiD32 was inoculated by transferring

93

a single colony from solid media into R2B media (pre-inoculum culture, PIC), a carbon-rich

94

liquid media prepared based on the composition of the solid media R2A 23. After reaching an

95

optical density of 0.603 at 600 nm (OD600), 5 mL of Sphingobium sp. BiD32 was transferred into

96

a second flask of R2B (inoculum culture, IC). When the IC reached an OD600 of 0.302, 8 mL was

97

inoculated into experimental flasks containing R2B for an estimated initial volatile suspended

98

solids (VSS) concentration of 5 mg/L. Five replicate cultures of Sphingobium sp. BiD32 were

99

grown (100 rpm, 28ºC) with an initial concentration of 1.5 mg/L BPA and five replicates were



101

carbon in the media. OD600 was measured and samples for BPA quantification were collected

102

every one to two hours. An HPLC instrument with ultraviolet detector (HPLC-UV) was used to

103

quantify total BPA concentrations (sorbed and dissolved fraction) using previously reported

104

methods 16. BPA was re-spiked into the cultures at 7.6 hours to ensure that BPA was present and

105

actively being degraded when the cells were harvested. Sphingobium sp. BiD32 cultures (with

106

and without BPA) were harvested when reaching an OD600 between 0.385 and 0.412. At the

107

point of harvest, the cultures were still in exponential growth and approximately 60% of the BPA

108

had been degraded. Cells were harvested by washing the cell pellet twice with 20 mL phosphate-

109

buffered saline (PBS) and collected by centrifugation (10,000 xg, 4ºC, 15 min). The proteins

110

were extracted and digested in-solution (details in SI). 2.5

0.5

2

0.4

1.5

0.3

1

0.2

0.5

0.1

0

0 0

111

Growth (OD600)

grown without BPA. The BPA added to five of the replicates contributed less than 0.6% of the

Bisphenol A (mg/L)

100

5

10

15

Time (hours)

112

Figure 1. Growth and harvesting of Sphingobium sp. BiD32 for differential proteomics analysis.

113

Degradation of BPA is shown as squares. Growth of Sphingobium sp. BiD32 both in the

114

presence and absence of BPA is shown as diamonds; no differences were seen between the two

115

conditions (p=0.45). BPA was re-spiked at 7.6 hours. Cells were harvested for protein extraction

116

at 11.5 hours. Error bars represent the range of measurements from replicates (n=5).

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2.4

LC-MS/MS analyses for proteomics

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Tryptic digests from the cytosolic and membrane fractions were analyzed separately by an

120

automated liquid chromatography electrospray ionization tandem mass spectrometer (LC-ESI-

121

MS/MS) with an UltiMate 3000 RSLCnano system on-line coupled to a Q Exactive mass

122

spectrometer via a Nanospray Flex ion source (Thermo Fisher Scientific, Hvidovre, Denmark) as

123

described elsewhere 24 with the following modifications: survey scans were detected at 350-1850

124

m/z and a dynamic exclusion of 45 sec was used for minimizing repetitive selection of the same

125

ions and normalized collision energy was set to 35. Peptides were eluted using 148 min linear

126

gradient, ranging from 13-50% (v/v) of solvent consisting of 0.1% (v/v) formic acid, 0.005%

127

(v/v) heptafluorobutyric acid, and 90% (v/v) acetonitrile.

128 129

2.5

Label-free quantification

130

Raw data files from the Q Exactive instrument were processed with MaxQuant v.1.5.1.2 (Max

131

Planck Institute of Biochemistry, Martinsried, Germany; Cox and Mann 2008).

132

Carboxymethylation was set as a fixed modification, oxidation of methionine was set as a

133

variable modification, and a false discovery rate of 1% was utilized. Label-free quantification

134

was determined using the label-free quantitation (LFQ) feature of MaxQuant. All other settings

135

were default. The raw data was searched against a sequence database retrieved from UniProt

136

(downloaded October 21, 2014) and comprised of the predicted open reading frames (ORFs) of

137

Sphingobium sp. BiD32. Differences in protein abundances between control and treated (with

138

BPA) were statistically evaluated using a Student's t-test with the statistical significance set at p

139

≤ 0.05. Protein abundances are represented as log2 transformed LFQ values. Identified proteins



140

include proteins with a single peptide in at least one LC-MS run. Quantified proteins include

141

proteins with at leasttwo quantifiable unique peptides in at least two LC−MS runs.

142 143

2.6

Functional annotation of proteins

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Quantified proteins more abundant in the presence of BPA were compared to a database

145

containing all proteins with a Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology

146

(KO) identifier (downloaded from UniProt 2nd of November 2013) using CLC Main Workbench

147

v 6.6.2 (CLCbio, Denmark) and The Basic Local Alignment Search Tool for proteins (BLASTp)

148

with default settings. Quantified proteins more abundant in the presence of BPA were also

149

compared to a database containing all proteins in the Patric database (downloaded from Patric

150

16th of August 2015) 26 using CLC Main Workbench v 6.6.2 (CLCbio, Denmark) and The Basic

151

Local Alignment Search Tool for proteins (BLASTp) with default settings. Matches with the

152

highest similarity scores and an E-value ≤ 1E-05 were recorded for each protein. Proteins were

153

mapped using the KO identifier from the BLASTp search and the Pathway mapping feature at

154

the KEGG (v. 68.0) website (http://www.genome.jp/KEGG/).

155 156

2.7

Pathway prediction of BPA

157

The BPA SMILES (simplified molecular-input line-entry system) line notation

158

(Oc1ccc(cc1)C(c2ccc(O)cc2)(C)C) was submitted to the EAWAG biocatalysis/biodegradation

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database pathway prediction system (EAWAG-BBD PPS, http://eawag-bbd.ethz.ch/predict/) 27.

160 161

2.8

Sample preparation for metabolite analysis


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Samples for metabolite analysis were collected from log-phase growth Sphingobium sp. BiD32

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cultures that were actively degrading BPA. Sphingobium sp. BiD32 was inoculated into R2B

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containing either 0.5 or 2 mg/L BPA by transferring a single colony from solid media. The

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bacteria were grown at 100 rpm and 28ºC. Samples were preserved by adding an equal volume

166

of acetonitrile, vortexing, and centrifuging (10 min, 10,000 ×g) to remove particles. Decanted

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samples were stored at 4ºC in glass vials until concentrated for use. Samples were prepared as

168

described elsewhere with minor modifications 28. To concentrate, 1 mL of the sample was

169

evaporated after centrifugation at 10,000×g, under a gentle stream of nitrogen while at 60ºC. The

170

residue was resuspended in 50 µL of mobile phase A/B (1:1;v:v (A) 10 mM ammonium formate

171

in acetonitrile with 0.1% (v/v) formic acid and (B) acetonitrile with 0.1% (v/v) formic acid.

172 173

2.9

LC/MS analyses for metabolite identification

174

An Ultra High Definition (UHD) Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) coupled

175

with an ultra-HPLC system (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used to

176

determine the presence of BPA and the corresponding metabolites (details in SI).

177 178

3.

Results

179

3.1

Whole genome sequencing

180

Whole genome sequencing of Sphingobium sp. BiD32 was completed to provide an organism-

181

specific genomic template for the MS-based label-free quantitative proteomics. Information

182

obtained from the assembly of the Sphingobium sp. BiD32 genome is summarized in Table 1.

183

169 out of 206 (82%) bacterial essential genes proposed by Gil et al. were identified in the

184

genome, suggesting that the genome is mostly complete (Table S1) 26.



185 186

Table 1. Sphingobium sp. BiD32 whole genome assembly. Parameter

Value

Contigs

262

Reads

2,632,020

Fold coverage

65

Mean read length (bp)

118.31

N50 contig length (Kb)

33.3

Draft genome size (b)

4,788,021

Predicted coding sequences

4,670

GC content (%)

31.3

187 188

3.2

Label-free quantitative MS-based proteomics of Sphingobium sp. BiD32

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Proteins extracted from five biological replicates of Sphingobium sp. BiD32 grown in the

190

presence and absence of BPA were subjected to shotgun-proteomics and label-free

191

quantification. A total of 2180 proteins were identified (full proteins lists available in

192

SI_proteinlist.xlsx), corresponding to approximately 47% of the predicted CDS of Sphingobium

193

sp. BiD32. The number of proteins quantified, differentially abundant in response to the

194

presence/absence of BPA (p ≤ 0.05), and differentially abundant by more than twofold (-1.0 ≤

195

log2 (fold abundance) ≥ 1.0, p ≤ 0.05) in response to BPA are summarized in Table 2. Proteins

196

differentially abundant by more than twofold are shown by the shaded regions of Figure 2 for

197

both the cytosolic and membrane proteins; 43 proteins were upregulated in the cells exposed to


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BPA, while 48 proteins were downregulated in the presence of BPA. The upregulated proteins

199

were further evaluated to identify those potentially involved in BPA degradation.

200 201

Table 2. Sphingobium sp. BiD32 proteomic analysis. Proteins

Value

Identified

2155

Quantified

1174

Differentially abundant

184

in response to BPA Differentially abundant

90

by more than twofold 202

identified, protein was measured in 1 of 5 replicate incubations

203

quantified, protein was measured in 2 or more of 5 replication incubations

204 b) 4

3.5

3.5

3

3 -Log10(p-value)

-Log10(p-value)

a) 4

2.5 2 1.5

2.5 2 1.5

1

1

0.5

0.5 0

0 -5

-4

-3

-2 -1 0 1 2 log2(fold abundance)

3

4

-5

5

-4

-3

-2

-1

0

1

2

log2(fold abundance)


3

4

5


205

Figure 2. Volcano plot of a) cytosolic and b) membrane quantified proteins. Statistically

206

significant (p ≤ 0.05) differentially abundant proteins (-1 ≤ log2 (fold abundance) ≥ 1) are shaded

207

gray.

208 209

3.3

Functional annotation of proteins

210

Proteins were functionally annotated by comparing them to those with KO identifiers and those

211

in the Patric database. Twenty-seven (27) of the 43 upregulated proteins matched to proteins

212

with KO identifiers. These proteins were annotated to the following categories: metabolism,

213

genetic information processing, environmental information processing, cellular processes, and

214

organismal systems. Metabolism included proteins related to xenobiotics biodegradation and

215

metabolism (N1MT47, N1MMX0, N1MNM9, N1MWA7, N1MSB0, and N1MS32), energy

216

metabolism (N1ML71, N1MN00, N1MTK7, N1MRW4, N1MPN2, and N1MLU6), lipid

217

metabolism (N1MTK7), amino acid metabolism (N1MF55, N1MFT4, N1MFX4, N1MK88,

218

N1ML71, N1MMX0, N1MPU3, N1MQ53, N1MS39, N1MWX7, and N1MRW4), metabolism

219

of other amino acids (N1MPU3), metabolism of cofactors and vitamins (N1MH82 and

220

N1MPU3), glycan biosynthesis and metabolism (N1MQ53), biosynthesis of other secondary

221

metabolites (N1MTJ4), metabolism of terpenoids and polyketides (N1MTK7), and carbohydrate

222

metabolism (N1MJQ9, N1MKJ6, N1ML71, N1MN00, N1MNR5, N1MTJ4, N1MTK7, and

223

N1MWX7). Genetic information processing included proteins related to translation (N1MH82).

224

Environmental information processing included proteins related to membrane transport

225

(N1MGE8) and signal transduction (N1MTJ4). Cellular processes included transport and

226

catabolism (N1ML71) and cell growth and death (N1MPU5). Organismal systems included

227

endocrine system (N1MN00). Functional information for four additional proteins was

228

determined by matching upregulated proteins to proteins in the Patric database. These proteins 12 ACS Paragon Plus Environment

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had Enzyme Class (EC) assignments of glycosidases (N1MKW1), enoyl-(acyl-carrier-protein)

230

reductase (NADH) (N1MSL9), and glutamate dehydrogenase (N1MUY1). A protein without an

231

EC assignment, had a Gene Ontology (GO) assignment of translation release factor activity and

232

translation termination (N1MIL6).

233 234

The xenobiotic biodegradation and metabolism orthology were given particular focus as these

235

were most likely to be involved with BPA degradation. These accounted for more of the

236

functionally annotated upregulated proteins than any other orthology. The level of homology

237

between the proteins annotated to xenobiotic biodegradation and metabolism and the entries in

238

the KO database varied, ranging from moderate (44% sequence identity) to high (88% sequence

239

identity) (Table 3). These proteins were characterized as dehydrogenases, a dioxygenase, a

240

hydratase, a hydroxylase, and a cycloisomerase and were related functionally to the degradation

241

of aromatic contaminants (Table 3). The presence of BPA resulted in a three-to-six-fold

242

increased abundance (log2 of 1.6 to 2.7) of these proteins. While two of the proteins were

243

exclusively associated with ‘benzoate degradation’ (N1MT47 and N1MWA7), the remaining

244

four were associated with degradation of multiple aromatic xenobiotics, including polycyclic

245

aromatic hydrocarbons, toluene, and aminobenzoate. Additionally, the hydroxymuconate-6-

246

semialdehyde (N1MMX0), which was almost four-fold upregulated in samples exposed to BPA

247

relative to the control, was found to be highly homologous (98% sequence identity, BLAST

248

against the UniProtKB database) to a predicted oxidoreductase of the aromatic hydrocarbon

249

degrading bacterium Sphingobium sp. Ant17.

250



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K10220

■

1.8

Q2G4H6

86

567

K10219

■

■

2.0

Q2G4H5

85

497

K04101

■

■

■

2.7

B8GZG3

78

184

K00481

■

1.6

Q2G4H4

78

225

K04100

■

■

■

1.6

Q39HW9

44

179

K01856

■

■

proJ

hydratase

N1MMX0

4-carboxy-2-

proC

hydroxymuconate-

Polycyclic aromatic hydrocarbon degradation

638

Toluene degradation

88

Chlorocyclohexane and chlorobenzene degradation

Q2G4H3

Aminobenzoate degradation

KEGG Orthology

2.5

Benzoate degradation

Greatest bit score

4-oxalomesaconate

N1MT47

Accession (Hit)

Greatest identity %

metabolism proteins. Log2 (Fold Abundance)

252

Protein name assigned in genome annotation

Table 3. Proteins upregulated in response to BPA related to xenobiotic degradation and

UniProtKB accession number and gene

251

6-semialdehyde dehydrogenase N1MNM9

Protocatechuate

proB

4,5-dioxygenase beta chain

N1MWA7

p-hydroxybenzoate hydroxylase

N1MSB0

Protocatechuate

proA

4,5-dioxygenase alpha chain

N1MS32

Muconate

■

■

cycloisomerase

253 254

Four (4) of the proteins categorized as xenobiotic biodegradation and metabolism (N1MMX0,

255

N1MNM9, N1MSB0, and N1MT47) were homologous respectively to the genes proC, proB,

256

proA, and proJ, which are part of a proposed protocatechuate degradation pathway for BPA


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257

(Table 4). Previous descriptions of the protocatechuate degradation pathway have suggested two

258

major structural organizations of the involved genes: the Comamonas-type, in which the genes of

259

the pathway are confined to a single operon, and the Sphingobium-type, in which the genes

260

constitute several transcriptional units 29. The structural organization of the protocatechuate

261

degradation genes of Sphingobium sp. BiD32 (Figure 3) appeared to be analogous to the

262

Sphingobium-type (SI_operon.xlsx) and the genes were confined to possibly three operons:

263

proFNTEL, proCBAJXR, and proKUI (Figure 3).

264 265

The proteins related to the proCBAJ operon were all highly upregulated (~four-fold) in response

266

to BPA exposure. Genes proB and proA encode for the small and large subunit of the

267

protocatechuate 4,5-dioxygenase, respectively. This enzyme mediates the first reaction in the

268

protocatechuate 4,5-cleavage pathway, catalyzing the oxygenolytic fission of protocatechuate

269

and its conversion to 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (CHMS).

270

The proC gene of Sphingobium sp. BiD32 encoded for a protein that is highly homologous (~78-

271

85% sequence identity) to the CHMS genes of other protocatechuate degraders (2811, ligC, and

272

pmdC) (SI_operon.xlsx) 29. CHMS catalyzes the oxidation of the hemiacetal form of CHMS

273

resulting in 2-pyrone-4,6-dicarboxylate (PDC) 29. PDC is then converted to the kenol-enol

274

tautomers of 4-oxalomesaconate (OMA) by the enzyme 2-pyrone-4,6-dicarboxylate hydrolase

275

(PDC hydrolase), which is encoded for by proI in Sphingobium sp. BiD32. proU is believed to

276

encode for an OMA tautomerase that catalyzes the tautomerization of the enol form of OMA to

277

the keto form. Previous studies performed on OMA tautomerase mutants indicates that this gene

278

is essential for OMA conversion 30. OMA is then converted to 4-carboxy-4-hydroxy-2-

279

oxoadipate (CHA) by the enzyme 4-oxalomesaconate hydratase (OMA hydratase), which is



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280

encoded for by proJ in Sphingobium sp. BiD32. proJ is highly homologous to ligJ (~85%

281

sequence identity) from Sphingobium sp. strain SYK-6, an organism with the ability to grow

282

using a wide variety of lignin-derived mono- and diaromatic compounds 31. CHA is converted to

283

pyruvate and oxaloacetate catalyzed by the enzyme 4-carboxy-4-hydroxy-2-oxoadipate aldolase

284

(CHA aldolase), which is encoded for by proK in Sphingobium sp. BiD32. proK shows a high

285

degree of sequence similarity (~91% sequence identity) with the CHA aldolase of Sphingomonas

286

sp. LB126 (fldZ), an organism with the ability to grow on the polycyclic aromatic hydrocarbon

287

fluorene as the sole source of energy and carbon 32. Pyruvate and oxaloacetate are then readily

288

assimilated and used in the Krebs cycle. Upstream of proK in the genome of Sphingobium sp.

289

BiD32 is a LysR-type transcriptional regulator (proR), which likely modulates transcription of

290

the proRKUI operon through positive regulation 33.

291 292

Table 4. Gene cluster containing the protocatechuate transformation genes encoding for proteins

293

upregulated by more than twofold in response to bisphenol A and close proximity genes. Gene

UniProtKB

Protein name assigned in genome

Length

log2

accession

annotation

(bp)

(fold

number EBBID32_29090

p-value

abundance)

N1MMW4

p-hydroxybenzoate hydroxylase

404

NQ

N1MNM4

Uncharacterized protein

186

NQ

N1MSA4

Major facilitator superfamily

435

ND

290

ND

(proF) EBBID32_29100 (proN) EBBID32_29110 (proT) EBBID32_29120 (proE)

MFS_1 N1MT38

Transcriptional regulator, AraC family


Page 17 of 31


EBBID32_29130

N1MP91


75

ND

N1MMX0

4-carboxy-2-hydroxymuconate-6-

312

1.8

3.0E-04

299

2.0

2.7E-03

140

1.6

2.0E-04

2.5

1.2E-02

(proL) EBBID32_29140 (proC) EBBID32_29150

semialdehyde dehydrogenase N1MNM9

(proB) EBBID32_29160

beta chain N1MSB0

(proA) EBBID32_29170

Protocatechuate 4,5-dioxygenase

Protocatechuate 4,5-dioxygenase alpha chain

N1MT47

4-oxalomesaconate hydratase

341

N1MP96

3-hydroxyisobutyrate

315

NQ

383

NQ

224

NQ

(proJ) EBBID32_29180 (proX)

dehydrogenase and related betahydroxyacid dehydrogenases

EBBID32_29190

N1MMX5

(proR) EBBID32_29200

Transcriptional regulator, LysR family

N1MNN2

(proK)

Putative siderophore biosynthesis protein,related to 2demethylmenaquinone methyltransferase

EBBID32_29210

N1MSB6

2-methylaconitate isomerase

351

NQ

N1MT55

2-pyrone-4,6-dicarboxylic acid

302

NQ

(proU) EBBID32_29220 (proI)

hydrolase

EBBID32_29230

N1MPA3

Carboxylic ester hydrolase

487

ND

EBBID32_29240

N1MMY1


117

ND

EBBID32_29250

N1MNN8


37

ND

294

NQ, not quantified; proteins were not quantified, but were identified

295

ND, not detected; protein was not identified or quantified 17 ACS Paragon Plus Environment


296

297 298

Figure 3. Gene organization of the proposed protocatechuate operons (proCBAJ and proRKUI)

299

and close proximity genes on contig 143 (CAVK010000143.1) of Sphingobium sp. BiD32.

300

Proposed gene names for Sphingobium sp. BiD32 were based upon their similarity to genes of

301

the protocatechuate pathway from other organisms (SI_operon.xlsx). A black arrow indicates

302

that proteins for the corresponding gene were more abundant by more than twofold in response

303

to BPA exposure (Table 4), a grey arrow indicates that the protein was detected, but not

304

quantified, and a white arrow indicates that the gene was detected but the protein was not present

305

in the proteome.

306 307

Located outside the protocatechuate gene cluster, another protein (N1MN05) was functionally

308

annotated (K00100) to bisphenol degradation (SI_proteinlist.xlsx). This protein was found to be

309

more abundant in cells exposed to BPA relative to the control, though by less than twofold (log2

310

= 0.72). It was functionally annotated to the transformation of several bisphenol metabolites,

311

including the transformation of bis(4-hydroxyphenyl)-methanol to 4,4’-dihydroxybenzophenone

312

and transformation of 1-(4’hydroxyphenyl)-ethanol to 4’-hydroxyacetophenone.

313 314

Thirty (30) out of 48 of the downregulated proteins matched to proteins with a KO identifier.

315

These proteins were functionally annotated to the following categories: metabolism, genetic

316

information processing, environmental information processing, cellular processes, and human 18 ACS Paragon Plus Environment

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317

diseases. Metabolism included proteins related to carbohydrate metabolism (N1MKW5,

318

N1MJA6, N1MGN1, N1MG01, N1MIP5, N1MKD6, N1MHY2, N1MSX0, and N1MLE2),

319

nucleotide metabolism (N1MHY2, N1MJA6, and N1MMB5), energy metabolism (N1MKW5,

320

N1MSC3, N1MG01, N1MIP5, N1MSX0, and N1MLE2), lipid metabolism (N1MLC6 and

321

N1MKW5), amino acid metabolism (N1MWN4, N1MIR7, N1MKD6, N1MQT8, N1MN31,

322

N1MS44), metabolism of other amino acids (N1MLE2), glycan biosynthesis and metabolism

323

(N1MI43), metabolism of terpenoids and polyketides (N1MKW5), metabolism of cofactors and

324

vitamins (N1MRQ2, N1MT40, N1MNZ6, N1MIR7, and N1MY92) and biosynthesis of other

325

secondary metabolites (N1MPK8). Genetic information processing included proteins related to

326

translation (N1MMK2, N1MMZ3, N1MNQ3 and N1MQ71) and folding, sorting, and

327

degradation (N1MTE1). Environmental information processing included proteins related to

328

membrane transport (N1MSV8), cellular processes included transport and catabolism

329

(N1MLE2), and human diseases included drug resistance (N1MPK8 and N1MI43) and infectious

330

diseases (N1MJ91).

331 332

3.4

Metabolite identification

333

The parent compound (BPA) and one metabolite (BPA-M; 1,2-Bis(4-hydroxyphenyl)-2-

334

propanol) were detected during metabolite analysis (Table 5). BPA-M was detected in the

335

samples with an initial BPA concentration of 2 mg/L, but not in the samples with an initial BPA

336

concentration of 0.5 mg/L. This suggests that BPA-M did not reach concentrations above the

337

method detection limits at the lower initial BPA concentration. Pure chemicals of the

338

intermediate were not available so that standards for quantification could not be prepared.

339



340

Page 20 of 31

Table 5. BPA metabolites identified during BPA degradation by Sphingobium sp. BiD32. Identified BPA metabolites

Retention time (min)

m/z

BPA

20.6

227.10775

15

243.10267

BPA-M (1,2-Bis(4-hydroxyphenyl)-2propanol)

341 342

3.5

Catabolic pathway of bisphenol A

343

The catabolic pathway of BPA by Sphingobium sp. BiD32 was predicted using the online

344

prediction tool EAWAG-BBD PPS (Figure 4, BPA to BPA-M1). It was predicted that BPA-M

345

was a potential product of the first step in BPA degradation (Figure 4, top panel). More than one

346

pathway was predicted by EAWAG-BBD PPS, however metabolites and enzymes were observed

347

only for the pathway shown in Figure 4. Smaller compounds predicted as intermediates are

348

unlikely to be detected even if present as they have molecular masses too small for detection by

349

the HPLC-TOF method used for metabolite analysis, and these metabolites are likely to be

350

readily transformed.

351 352

Enzymes predicted to be involved in the transformation of BPA to BPA-M were compared to the

353

proteins upregulated in Sphingobium sp. BiD32. This transformation involves substitution by a

354

hydroxyl group. The top two matches to enzymes predicted by the EAWAG-BBD PPS were

355

N1MMX0 (83% identity similarity to Q9KWL3) and N1MWA7 (68% identity similarity to

356

Q03298). N1MWA7, identified as a p-hydroxybenzoate hydroxylase by genome annotation

357

(Table 3), was identified as a protein likely involved in the degradation of BPA by Sphingobium


Page 21 of 31


358

sp. BiD32 (Figure 4). Following hydroxylation of BPA to BPA-M, it was predicted that BPA-M

359

was dehydrated forming 4,4'-dihydroxy-alpha-methylstilbene (BPA-M1). No enzyme catalyzing

360

this step is known, and hence no candidate was found for BiD32. The middle steps (BPA-M2 to

361

BPA-M5), suggested that BPA-M1 is broken into two aromatic constituents (BPA-M2 and BPA-

362

M4) based upon proteomics data. BPA-M2 is oxidized to BPA-M3 and BPA-M4 is processed

363

via the protocatechuate pathway. It remains unclear if BPA-M3 is processed by Sphingobium sp.

364

BiD32, though a previous study proposed the demethylation of BPA-M3 resulting in 4-

365

hydroxybenzaldehyde, which was further converted to benzoic acid and eventually to carbon

366

dioxide and water 34. The bottom part of the pathway (BPA-M5 to BPA-M12) represents the

367

protocatechuate pathway described from other organisms, and the Sphingobium sp. BiD32

368

enzymes catalyzing each step.

369



370 371

Figure 4. Proposed pathway for BPA degradation by Sphingobium sp. BiD32 based on measured

372

metabolites (bold) and metabolites and proteins predicted to be involved in BPA degradation by

373

the EAWAG Biocatalysis/Biodegradation Pathway Prediction System (top panel). The


Page 22 of 31

Page 23 of 31


374

degradation pathway of BPA metabolites (bottom panels) was proposed based upon previous

375

findings35. Solid arrows indicate identification of a candidate enzyme upregulated in

376

Sphingobium sp. BiD32 by more than twofold in response to BPA relative to the control. Grey

377

arrows indicate identification of a candidate enzyme only quantified in the proteome of BPA-

378

exposed cells. Thin arrows indicate that the step is not catalyzed enzymatically. Metabolites are

379

as follows: BPA, bisphenol A; BPA-M, 1,2-bis(4-hydroxyphenyl)-2-propanol; BPA-M1, 4,4’-

380

(1E)-prop-1-ene-1,2-diyldiphenol; BPA-M2, 1-(4’-hydroxyphenyl)-ethanol; BPA-M3, 4’-

381

hydroxyacetophenone; BPA-M4, 4-hydroxybenzoate; BPA-M5, protocatechuate (PCA); BPA-

382

M6, 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS); BPA-M7, 4-carboxy-2-

383

hydroxymuconate-6-semialdehyde (CHMS hemiacetal form); BPA-M8, 2-pyrone-4,6-

384

dicarboxylate (PDC); BPA-M9, 4-oxalomesacconate (OMA) enol form; BPA-M10, OMA

385

ketoform; BPA-M11, OMA ketoform; BPA-M12, 4-carboxy-4-hydroxy-2-oxoadipate (CHA).

386 387

4.

Discussion

388

In this study, the metabolism of BPA by Sphingobium sp. BiD32 was investigated using three

389

different tiers of biochemistry (genome, proteome, and metabolome) and was successfully

390

employed to identify previously undocumented proteins putatively involved in bacterial BPA

391

degradation. The overarching goal was to identify genes involved in BPA degradation for use as

392

potential biomarkers in future work. The gene involved in the initial transformation of BPA may

393

also enable identification of other new BPA-degrading bacteria.

394 395

A p-hydroxybenzoate hydroxylase (N1MWA7) was identified as involved in BPA degradation

396

based on proteomics results and metabolite analysis. This protein was significantly more

397

abundant in the presence of BPA (Table 3), was functionally annotated to proteins involved in 23 ACS Paragon Plus Environment


398

xenobiotics biodegradation and metabolism (Table 3), and closely matched enzymes predicted to

399

be involved in the transformation of BPA to BPA-M (Figure 4). Upregulated Sphingobium sp.

400

BiD32 proteins that were functionally annotated to xenobiotics biodegradation and metabolism

401

were distantly related to each other.

402 403

Proteins involved in protocatechuate transformation are also likely to be involved in the BPA

404

degradation pathway (N1MMX0, N1MNM9, N1MSB0, and N1MT47). These proteins were

405

more abundant in the presence of BPA (Table 4), were functionally annotated to proteins

406

involved in xenobiotics biodegradation and metabolism (Table 3), and matched genes previously

407

identified to be involved in protocatechuate degradation in a variety of organisms including

408

Sphingobium sp. strain SYK-6 36, Sphingomonas sp. strain LB126 37, Novosphingobium

409

aromaticivorans DSM 12444 37, Comamonas sp. strain E6 35, Comamonas testosterone BR6020

410

38

411

protocatechuate degradation genes in Sphingobium sp. BiD32 most notably resembles that of

412

Sphingobium sp. strain SYK-6, a bacterium capable of degrading a wide variety of lignin-derived

413

biaryls and monoaryls 36.

, Pseudomonas straminea NGJ1 39, and Arthrobacter keyseri 12B 40. The organization of the

414 415

While several studies have documented microbial degradation of BPA and predicted BPA

416

degradation pathways, few proteins involved in BPA degradation have been reported 41. Included

417

amongst these was a cytochrome P450 monooxygenase reported to catalyze the transformation

418

of BPA to BPA-M by Sphingomonas sp. AO1 42. This cytochrome P450 monooxygenase was

419

found to be involved in BPA degradation by using a cytochrome P450 inhibitor 43 and then

420

confirmed by purifying the components of the cytochrome P450 monooxygenase system and


Page 24 of 31

Page 25 of 31


421

testing their ability to degrade BPA 42. The Sphingomonas sp. AO1 cytochrome P450 (B1Q2N5)

422

was related to a Sphingobium sp. BiD32 putative cytochrome P450 hydroxylase (N1MI62) that

423

was identified in the genome, but not detected based on the proteomics portion of this study

424

(Figure S1; 30% identity match; E-value = 5.1e-45). Also, a Sphingomonas sp. AO1 ferredoxin

425

that is part of the cytochrome P450 monooxygenase system (B1Q2N4) was closely related to a

426

Sphingobium sp. BiD32 ferredoxin gene (N1MLS9) detected in the genome and proteome

427

(Figure S1; 47% identity match; E-value = 4.4e-23), but not differentially expressed. Lack of

428

increased proteome expression of these enzymes by Spingobium sp. BiD32 suggested that

429

differing pathways may be involved in BPA degradation by these two organisms.

430 431

Other enzymes previously reported to be involved in BPA degradation included a second

432

cytochrome P450, an ammonia monooxygenase, and an extracelluar laccase. The cytochrome

433

P450 was reported to catalyze the ipso substitution as the initial step in BPA degradation by

434

Sphingomonas sp. TTNP3 44. An ammonia monooxygenase in Nitrosomonas europaea 45 and an

435

extracellular laccase in Pseudomonas sp. LBC1 46 were also reported to be involved in BPA

436

degradation, though specific degradation steps mediated were not identified. No proteins similar

437

to these three were identified in the proteome of Sphingobium sp. BiD32.

438 439

Investigation of the metabolome resulted in the identification of BPA-M (Table 5). BPA-M has

440

also been detected during BPA degradation by other isolated bacteria 41, generally resulting in

441

complete mineralization. For example, BPA degradation by Sphingomonas sp. MV1 resulted in

442

the transformation of 85% of the BPA to BPA-M47. BPA-M was further degraded and finally

443

oxidized to carbon dioxide and cell biomass, as determined by HPLC. BPA degradation by



444

unidentified strain WH1 followed the main degradation pathway that occurred in Sphingomonas

445

sp. MV1, resulting in transformation to BPA-M and, ultimately, mineralization 48. BPA-M was

446

also detected during BPA degradation by Sphingomonas sp. AO1, though in that study further

447

investigation of the degradation products was not conducted, as it was not the focus of the study

448

42

449

Sphingobium sp. BiD32; other metabolites were not detected suggesting that they were either too

450

small to be detected or they were transformed quickly, resulting in too low concentrations to be

451

detected. While the same metabolite, BPA-M, was identified during BPA degradation by

452

Sphingobium sp. BiD32 and by Sphingomonas sp. AO1, different enzymes were predicted to be

453

involved in the transformation 42 suggesting that the mechanisms responsible for BPA

454

degradation by these organisms likely differ.

. The current study detected BPA-M during degradation of high concentrations of BPA by

455 456

This is the first study using a combination of genomics, proteomics, and metabolite analysis to

457

identify BPA metabolites and enzymes responsible for BPA degradation. Previous studies

458

identified enzymes responsible for the initial transformation of BPA using various inhibitors.

459

This study identified a novel enzyme responsible for the initial transformation of BPA. It also

460

identified a novel BPA transformation pathway with the increased abundance in the

461

protocatechuate transformation genes. The results further suggest how Sphingobium sp. BiD32

462

degrades BPA beyond 4-hydroxybenzoate. Additional research is needed to elucidate the

463

function of multiple enzymes in BPA degradation. Identification of these enzymes may allow for

464

future monitoring of bioaugmented bacteria in WWTPs and identifying other similar BPA-

465

degrading bacteria.

466


Page 26 of 31

Page 27 of 31


467

Acknowledgements

468

This work was funded by the Danish Research Council for Strategic Research via the Research

469

Centre “EcoDesign-MBR” (Grant No. 26-03-0250) and the National Science Foundation (NSF

470

CBET-0829132). Support was provided to N.A. Zhou by the Valle Scholarship and

471

Scandinavian Exchange and the King County Technology Transfer Fellowship Programs at the

472

University of Washington.

473 474

Supporting Information Available

475

Tables and Figures addressing the full protein quantitation lists, protocatechuate transformation

476

gene cluster, the gene sequences of the proteins of interest, and the phylogenetic relationship of

477

identified proteins of interest. This material is available free of charge via the Internet at

478

http://pubs.acs.org.

479



480

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