Identification of the Amidase BbdA That Initiates Biodegradation of the

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Identification of the amidase BbdA that initiates biodegradation of the groundwater micropollutant 2,6dichlorobenzamide (BAM) in Aminobacter sp. MSH1 Jeroen T'Syen, Raffaella Tassoni, Lars Hestbjerg Hansen, Soren J. Sorensen, Baptiste Leroy, Aswini Sekhar, Ruddy Wattiez, René De Mot, and Dirk Springael Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02309 • Publication Date (Web): 26 Aug 2015 Downloaded from http://pubs.acs.org on August 31, 2015

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Environmental Science & Technology

1

Identification of the amidase BbdA that initiates

2

biodegradation of the groundwater micropollutant

3

2,6-dichlorobenzamide (BAM) in Aminobacter sp.

4

MSH1

5

Jeroen T’Syen1, Raffaella Tassoni1, Lars Hansen2, Søren J. Sorensen2, Baptiste Leroy3,

6

Aswini Sekhar1, Ruddy Wattiez3, René De Mot4 and Dirk Springael1*

7

Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg 20, 3001

8

Leuven, Belgium1;

9

Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 København,

10

Denmark2;

11

Department of Proteomics and Microbiology, Research Institute for Biosciences, University

12

of Mons, Place du Parc 20, 7000 Mons, Belgium3;

13

Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Leuven,

14

Belgium4

15 16 17

ACS Paragon Plus Environment


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ABSTRACT

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2,6-dichlorobenzamide (BAM) is a recalcitrant groundwater micropollutant that poses a major

20

problem for drinking water production in European countries. Aminobacter sp. MSH1 and

21

related strains have the unique ability to mineralize BAM at micropollutant concentrations but

22

no information exists on the genetics of BAM biodegradation. An amidase - BbdA -

23

converting BAM to 2,6-dichlorobenzoic acid (DCBA) was purified from Aminobacter sp.

24

MSH1. Heterologous expression of the corresponding bbdA gene and its absence in MSH1

25

mutants defective in BAM degradation, confirmed its BAM degrading function. BbdA shows

26

low amino acid sequence identity with reported amidases and is encoded by an IncP1-β

27

plasmid (pBAM1, 40.6 kb) that lacks several genes for conjugation. BbdA has a remarkably

28

low KM for BAM (0.71 µM) and also shows activity against benzamide and ortho-

29

chlorobenzamide (OBAM). Differential proteomics and transcriptional reporter analysis

30

suggest the constitutive expression of bbdA in MSH1. Also in other BAM mineralizing

31

Aminobacter sp. strains, bbdA and pBAM1 appear to be involved in BAM degradation.

32

BbdA’s high affinity for BAM and its constitutive expression are of interest for using strain

33

MSH1 in treatment of groundwater containing micropollutant concentrations of BAM for

34

drinking water production.

35 36

Introduction

37

2,6-Dichlorobenzamide (BAM) is a transformation product of the broad spectrum herbicide

38

dichlobenil [2,6-dichlorobenzonitril] and the fungicide fluopicolide [2,6-dichloro-N-[[3-

39

chloro-5-(trifluoromethyl)-2-pyridinyl]methyl]benzamide]1.

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compounds, BAM is highly mobile in soil leading to frequent BAM contamination of

41

groundwater bodies all over Europe.2-5 Concentrations of BAM in groundwater are at the

In


contrast

to

its

parent

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micropollutant level (µg.l-1-ng.l-1 range), but often still exceed the threshold concentration of

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0.1 µg.l-1 for individual pesticide residues in drinking water in EU countries. The frequent

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occurrence of BAM in groundwater resulted into the costly closure of drinking water wells6 or

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inclusion of expensive activated carbon filters in drinking water production plants.7 The use

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of dichlobenil is banned in the European union since 20108 but its presence in a sorbed state

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in soil generates a continuous input of BAM to groundwater in which BAM proved to be

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extremely persistent.9-11

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Aminobacter sp. ASI1, ASI2 and MSH1 have the unique ability to use BAM as sole energy,

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carbon and nitrogen source resulting in complete mineralization of BAM12-14, and have been

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proposed for removal of BAM from BAM contaminated groundwater by bioaugmentation of

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sandfilters applied in drinking water production schemes.7,

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MSH1 mineralizes BAM at the micropollutant concentrations found in groundwater12 and

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shows long-term survival in nutrient-poor groundwater.16 The pathway for BAM

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mineralization and enzymes in BAM mineralizing Aminobacter strains are unknown. In order

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to use those strains for bioremediation, it is crucial to elucidate the BAM degradation

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pathway. In addition to BAM, strain MSH1 is able to degrade two potential metabolites of

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BAM, 2,6-dichlorobenzoate (DCBA) and ortho-chlorobenzamide (OBAM).17 During growth

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on BAM, Aminobacter sp. MSH1 and ASI1 transiently accumulate DCBA as well as an

60

unknown metabolite, suggesting that BAM biodegradation occurs through DCBA involving

61

amide hydrolysis.18 In this paper, we present the genetic identification of an amidase,

62

designated as BbdA, that initiates the degradation of BAM in Aminobacter sp. MSH1. The

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enzyme was characterized in relationship to the ability of strain MSH1 to degrade BAM at

64

trace concentrations.

15

65 66

Materials and methods


Moreover, Aminobacter sp.


67

Bacterial strains, cultivation conditions, and chemicals

68

Strains Aminobacter sp. ASI113, ASI214 and MSH112 were described before. Aminobacter

69

sp. strains were routinely grown at 25°C on either R2A19 or minimal medium MMO20

70

supplemented with 100 to 200 mg.l-1 BAM. Aminobacter sp. M1a100g and A7.100g are

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spontaneous mutants derived from a GFP labeled variant of MSH1 and a RFP labeled variant

72

of ASI2, respectively, after growth on non-selective medium. Strain M1a100g is unable to

73

degrade BAM, but degrades DCBA. Strain A7.100g is unable to degrade BAM and DCBA.

74

Additional spontaneous mutants, defective in converting BAM into DCBA, of MSH1 (M1 till

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M38), of ASI1 (A1 and A2) and of ASI2 (ASI2.100g) were derived by cultivating the

76

respective parent strains on the non-selective media R2A or MSN mineral medium18

77

supplemented with 4 g.l-1 glucose. E. coli strains TOP10 (Life Technologies) and

78

BL21(DE3)pLysS were cultivated in lysogeny broth.21 Solid media were prepared using 15

79

g.l-1 agar. Antibiotics were added when appropriate at the following concentrations: ampicillin

80

(Ap), 100 µg.ml-1, chloramphenicol (Cm), 50 µg.ml-1, kanamycin (Km) 50 µg.ml-1. BAM was

81

purchased from Pestanal, UK. Benzamide, benzoic acid, OBAM, ortho-chlorobenzoic acid

82

(OBA) and DCBA were purchased from Sigma-Aldrich, Belgium. [ring-U-14C]-BAM (25.2

83

mCi.mmol-1, radiochemical purity >95%) was obtained from Izotop (Hungary).

84 85

UHPLC analysis

86

Concentrations of BAM, DCBA, OBAM, OBA, benzamide and benzoate were quantified

87

using reverse-phase ultra-high performance liquid chromatography (RP-UHPLC) on a Nexera

88

(Shimadzu Corp) apparatus equipped with a Vision HT C18 HL Column (100x2.0mm,

89

1.5µm; Grace, US) and a UV-Vis spectrometer. Elution consisted of 3 min of isocratic flow of

90

15% acetonitrile + 85% mQ water (acidified with H3PO4 to pH 2.5), followed by a 1 min

91

linear increase up to 30% acetonitrile and 70% acidified water and a linear gradient of 6.5 min


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to a ratio of 40% acetonitrile and 60% acidified water. The column was re-equilibrated to the

93

initial solvent composition for 7.5 min. A flow rate of 0.2 ml.min-1 and injection volume of 5

94

µl was used. Detection of the compounds was at 210 nm. Retention times were 2.3 min for

95

benzamide, 2.7 min for OBAM, 3.4 min for BAM, 5.3 min for DCBA, 6.9 min for benzoate

96

and 8.7 min for OBA. The limits of detection were 41 nM for BAM, 22 nM for DCBA, 25

97

nM for OBAM, 27 nM for OBA, 22 nM for benzamide and 222 nM for BA.

98 99

Purification of BbdA

100

MSH1 was cultured in duplicate in 250 ml R2A to an optical density at 600 nm (OD600) of

101

0.9. One culture was supplemented with 100 mg.l-1 BAM and incubated at 25°C for 5 hours.

102

Crude protein extracts of both cultures were collected using French press as described22 and

103

BAM removal was analyzed by UHPLC. The crude extract of the BAM supplemented culture

104

was fractionated by ammonium sulfate precipitation (30 to 50% ammonium sulfate

105

saturation). The precipitate was resuspended in

106

morpholino)propanesulfonic acid (MOPS), 1 mM dithiothreitol (DDT), 5% glycerol) and the

107

buffer was exchanged to histidine buffer (20 mM, pH 6.0) after spin filtration (Amicon, 10

108

kDa). The resulting protein solution was loaded on an anion exchange column (HiScreen

109

Capto Q ImpRes, Akta Prime ,GE Healthcare) equilibrated with histidine buffer (20 mM, pH

110

6.0) and fractionated using a gradient of NaCl (0 to 1 M) with a flow rate of 1.6 ml.min-1.

111

Fifty µl of the protein fractions were mixed with 700 µl phosphate buffered saline (150 mM

112

NaCl, 7 mM K2HPO4, 2.35 mM KH2PO4) supplemented with 200 mg.l-1 BAM and incubated

113

for 20 h at 25°C. BAM degradation releasing NH4+ was determined using a modified

114

Berthelot reaction23 and confirmed by UHPLC. Sodium dodecyl sulfate (SDS)-PAGE analysis

115

of the protein fractions was done using Mini-PROTEAN TGX Precast Gels (Bio-Rad) in

116

electrophoresis buffer (200 mM glycine, 3.5 mM SDS, 25mM Tris-HCl, pH8.3) with lumitein

MOPS buffer (25 mM 3-(N-



117

(Biotium, CA) staining. Liquid chromatography-electrospray ionization-tandem mass

118

spectrometry (LC-ESI-MS/MS) was used to determine peptide sequences directly in the

119

protein fractions in suspension.24

120 121

DNA isolation

122

Genomic DNA was isolated from Aminobacter cultures grown in R2A to an OD600 of 0.5-

123

1.0 using the Puregene Core kit A (Qiagen) according to the manufacturer’s instructions,

124

except that DNA precipitation was performed with ethanol. Plasmid DNA was isolated from

125

Aminobacter cultures grown in R2A to an OD600 of 0.5-1.0, harvested by centrifugation (15

126

min, 4000 g, 4°C), washed in MgSO4 (10 mM) and cell pellets were frozen. Cells were

127

resuspended in 200 µl TGE buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris),

128

supplemented with 1 mg hen egg white lysozyme (Sigma Aldrich) and incubated at 37°C for

129

45 min. Alkaline lysis was performed by adding 400 µl lysis buffer (0.2 M NaOH, 1% SDS)

130

and incubating on ice for 5 min. Three hundred µl potassium acetate (5 M, pH 5.2) was added

131

before cooling on ice for 15 min and centrifuging (20 min, 13000 g, 4°C). The supernatant

132

was mixed with one volume phenol/chloroform (1/1, pH 8.0), centrifuged (20 min, 13000 g,

133

4°C) and the aqueous phase washed with one volume chloroform (20 min, 13000 g, 4°C).

134

Plasmid DNA was precipitated with 0.7 volumes of isopropanol (30 min, 21000 g, 20°C),

135

washed twice with 70% ethanol (20 min, 16000 g, 4°C) and dissolved in Tris-buffer (10 mM,

136

pH 8.0).

137 138

Sequencing and bioinformatic analysis

139

Sequencing of MSH1 genomic DNA was done on Illumina GAIIx platforms. Sequencing

140

libraries with insert lengths of 400, 500 and 6000 bp were prepared and sequenced paired end

141

with read lengths of 50, 90 and 90 bp, respectively. A draft genome sequence of 219 contigs


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was assembled using SOAPdenovo25 and automatically annotated using RAST (Rapid

143

Annotation using Subsystem technology).26 MSH1 plasmid DNA was sequenced on a Roche

144

454 GS FLX sequencer and assembled into 115 contigs using Newbler 2.6 (Roche). The

145

genome of Aminobacter sp. M1a100g was sequenced (90 bp paired reads with 500 bp inserts)

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on an Illumina GAIIx sequencer, quality trimmed and mapped onto the consensus sequence of

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the MSH1 plasmids assembly using SolexaQA27 and Burrows-Wheeler Aligner (bwa-0.6.2)28

148

and inspected manually for missing regions.

149 150

Expression of BbdA in E. coli

151

Based on LC-ESI-MS/MS peptide sequences, the BAM amidase gene bbdA was identified

152

in the draft genome of strain MSH1. bbdA was amplified using primers bbdA-F1 (5’-

153

ATGCCCAGTGGTGCAAATCTGCCA-3’)

154

GCGCTGACGGTGGACTACTTCTTG-3’) with Platinum Taq DNA Polymerase High

155

Fidelity (Life technologies) from MSH1 genomic DNA and cloned into pEXP-5-CT/TOPO

156

(Life Technologies). A nonsense fragment provided by the pEXP-5-CT/TOPO kit was used

157

for control reactions. The resulting pEXP-5-CT/TOPO_bbdA and pEXP-5-CT/TOPO_ctrl

158

constructs were propagated in One Shot TOP10 Chemically Competent E. coli, isolated and

159

electroporated into E. coli BL21(DE3)pLysS. After growth on LB with ampicillin (100 µg.ml-

160

1

161

induced with various isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (0, 0.01,

162

0.03, 0.1, 0.3, 1.0 mM) and expression allowed to proceed for 4 h at 37°C. An abiotic control

163

and E.coli BL21(DE3)pLysS without vector were included as additional negative controls.

164

Cells resuspended in buffer (0.5 M NaCl, 10 mM imidazole, 20 mM NaH2PO4, pH 7.4) and

165

one volume Laemmli buffer29 were heated for 5 min at 95°C for protein expression analysis

166

by SDS-PAGE using the Precision Plus Protein standard (Bio-Rad) as a reference. BAM

and

bbdA-1551R

(5’-

) and chloramphenicol (50 µg.ml-1) at 37°C until an OD600 of 0.6, BbdA production was



167

degradation was tested by harvesting (15 min, 3000 g centrifugation) and resuspending the

168

cells in MMO supplemented with BAM (20 mg.l-1) followed by incubation at 25°C while

169

monitoring BAM/DCBA concentrations by UHPLC.

170 171

Phylogenetic analysis of trfA and bbdA

172

Multiple alignment of trfA genes of plasmids from different IncP-1 subgroups was

173

performed using Clustal Omega30 and a phylogenetic neighbor joining tree was constructed

174

using SplitsTree431. BlastP was used to identify proteins homologous to BbdA. A

175

phylogenetic tree was constructed from a multiple alignment of the pfam domains (PF01425)

176

of BbdA and members of the amidase signature family with Geneious Pro (version 7.1.7,

177

Biomatters) using PHYML (JTT matrix).32

178 179

BbdA enzyme characterization

180

BbdA substrate range, optimal temperature and Michaelis constants (KM) were determined

181

using crude protein extracts of E. coli BL21(DE3)pLysS carrying pEXP-5-CT/TOPO_bbdA

182

and carrying pEXP-5-CT/TOPO_ctrl as a negative control. The strains were grown at 25°C

183

until an OD600 of 0.4-0.6. Expression was induced with 1 mM IPTG and the cells were

184

incubated overnight at 25°C. After harvest, cells were resuspended in MMO with CelLytic™

185

B Cell Lysis Reagent (Sigma-Aldrich) and lysed while vortexing for 10 min. Cell debris was

186

removed by centrifugation (30 min, 21000 g, 4°C). The BbdA substrate range was determined

187

with 0.1 volume crude protein extract in MMO supplemented with 500 µM of BAM, OBAM

188

or benzamide at 25°C. Michaelis constants of BbdA for BAM and benzamide were estimated

189

in MMO supplemented with the crude protein extract and varying concentrations of BAM

190

(0.12 µM, 0.44 µM, 1.75 µM, 7.19 µM and 30.36 µM) or benzamide (0.20 µM, 1.66 µM,

191

23.45 µM, 248.9 µM and 2432.8 µM). Reactions were performed in triplicate in 300 µl


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volumes at 25°C and stopped by adding 4 µl HCl (11.65 M). The temperature optimum of

193

BbdA for BAM degradation was determined by monitoring BAM degradation by the crude

194

protein extract in 200 µl MMO with 100 µM BAM at temperatures between 20 and 74°C in a

195

Biometra thermocycler. Reactions performed in triplicate, were stopped after 10 min

196

incubation by adding 3 µl HCl (11.65 M). In all assays, residual substrate or product was

197

analyzed by UHPLC. Nonlinear regression analysis of the kinetic data using the Michaelis-

198

Menten model was performed with GraphPad Prism 6.05 software (GraphPad Software) to

199

estimate the KM. Initial conversion rates of the different substrates were determined by

200

calculating the derivative at time zero of the exponential fit made by non-linear regression of

201

the initial product formation rate using GraphPad Prism 6.05 software (GraphPad Software).

202 203

bbdA expression analysis

204

A 484 bp fragment containing the region directly upstream of bbdA carrying the putative

205

bbdA promoter and the first 108 bp of bbdA was amplified by PCR using primers PbbdA_F

206

(5’-AAGCGATTAATTAAGTATTCGCCGTGACCTGGTTTCC-3’) and PbbdA_R (5’-

207

AAGCGAGGTACCCATGAGGGCCTGATACGACTTCAC-3’), digested with PacI and

208

KpnI and cloned directionally into vector pRU109733 in front of the promoterless gfp-mut3.1

209

gene composing recombinant plasmid pRU1097-PbbdA. pRU1097-PbbdA was electroporated

210

into E. coli TOP10 cells. The construct was introduced into the 2,6-diaminopimelic acid

211

(DAP) auxotrophic E. coli BW2942734 by electroporation and transferred to Aminobacter sp.

212

MSH1 by conjugation and selection on MMO with glucose (500 mg.l-1) and gentamycin (20

213

mg.l-1). The effect of the presence of BAM on GFP expression in MSH1 (pRU1097-PbbdA)

214

was determined at the culture level by fluorometric analysis in a Fluoroskan Ascent FL 2.5

215

apparatus (Thermo Scientific). MSH1 (pRU1097-PbbdA) and a negative control carrying

216

pRU1097 were cultivated for 4 days in MMO with glucose (500 mg.l-1) and gentamycin (10



-1

217

mg.l

) to an OD600 of 0.5. Cells were washed (10 min, 5500 g centrifugation) and

218

resuspended in MMO to a final OD600 of 1.0. Fifty µl cell suspension was added to 200 µl

219

R2A or MMO with glucose (500 mg.l-1) both supplemented with gentamycin (10 mg.l-1) and

220

without or with BAM (concentrations of 1 µg.l-1, 100 µg.l-1 or 10 mg.l-1) in transparent 96-

221

well microtiter plates (No. 655180, Greiner Bio-One). The plates were incubated in the

222

Fluoroskan Ascent FL 2.5 apparatus at 25°C with intermittent shaking (2 min at 900 rpm with

223

5 min intervals) with GFP fluorescence measured every 10 min (excitation at 485 nm,

224

emission at 520 nm). OD600 measurements were taken at 130, 254 and 1245 min of incubation

225

and used to normalize expression. The effect of BAM on GFP expression in MSH1

226

(pRU1097-PbbdA) at the single cell level was analyzed by flow cytometry in a DB Accuri C6

227

(DB Biosciences) apparatus. Precultures of MSH1 (pRU1097-PbbdA) and MSH1 (pRU1097)

228

were prepared as reported above for the Fluoroskan analysis. Cells were incubated at final cell

229

densities of 104 cells ml-1 in 300 µl MMO supplemented with glucose (500 mg.l-1) without or

230

with BAM (100 mg.l-1) and of 105 cells.ml-1 in 300 µl cultures in MMO without and with

231

BAM (100mg.l-1) in transparent microtiter plates incubated at 25°C while gently shaking.

232

After 165 min of incubation, 25 µl volumes were analyzed on the flow cytometer using a

233

fluorescence cutoff set at 1000. Fluoroskan and flow cytometric analyses were performed on

234

independently initiated cultures.

235 236

Proteomic analysis

237

Aminobacter sp. MSH1 cultivated in MMO with glucose (500 mg.l-1) and BAM (200 mg.l-

238

1

) for 3 days, was inoculated in MSN supplemented with glucose (4 g.l-1) and grown to an

239

OD600 of 0.5. After washing in MMO (15 min, 3400 g centrifugation) test tubes containing 10

240

mL MMO with either glucose (110 mg.l-1) or BAM (100 mg.l-1) were inoculated at an initial

241

OD600 of 0.5. Five replicates were prepared for each condition. BAM degradation was


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monitored by UHPLC and cells were harvested by centrifugation (5 min, 15000 g, 4°C) after

243

9 h, i.e., when 50% of the BAM was degraded, and stored at -80°C. Label free quantitative

244

differential

245

chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). To this end

246

proteins were extracted and prepared as described.35 Subsequent peptide separation and

247

identification was done on an Eksigent 2D ultra liquid chromatography apparatus combined

248

with a TripleTOF® 5600 System (AB Sciex) as reported previously.35 Proteinpilot 4.0 was

249

used for protein identification using a concatenated database containing both annotated

250

genomic and plasmidic sequences. Proteins identified at 1% false discovery rate, were

251

quantified using skyline 2.5 taking into account the three first isotope peaks. In order to

252

decrease the effect of peak picking errors, only peptides presenting an idotp higher than 0.75

253

and a standard deviation of retention time less than one min across all 10 injections were

254

taken into account. T-test was used to calculate the statistical relevance of the modification of

255

protein abundances.

proteomic

analysis

was

performed

by

ultra-high-performance

liquid

256 257

PCR analysis

258

PCR detection of orf1 (trbE homologue), bbdA, and orf4 (nfnB homologue) was performed

259

using primer pairs orf1_F (5’-AGCTGAAGCTCAAGAAGCATCGGT-3’)/orf1_R (5’-

260

GGTTGATGCGGAAGGACACCATTT-3’),

261

ATATCACGGCCGGTACTATGCCAA-3’)/bbdA_R

262

TCTTCCAAGATCGAACAACCCGGA-3’)

263

GTACGGCCTTCTCAACATCGCAAA-3’)/orf4_R

264

CAGGAACATGCCGTAGTCCAGAAA-3’), respectively in 50 µl reactions containing 1.25

265

U of DreamTaq polymerase in 1X PCR buffer (Thermo scientic), 200 µM of each dNTP, 0.1

266

mg.ml-1 BSA, 0.1 µM of each primer and 1 µl of template DNA. PCR reactions consisted of 5

bbdA_F

(5’(5’-

and


orf4_F

(5’(5’-


267

min initial denaturation at 94°C, 30 cycles of 20 sec at 94°C, 20s at 56°C and 30s at 72°C and

268

a final extension for 10 min at 72°C. PCR products were separated by agarose gel

269

electrophoresis (1.5 %) in Tris-acetate/EDTA buffer and visualized using ethidium bromide.

270 271

Genbank accession numbers

272

The nucleotide sequence of plasmid pBAM1 was deposited in Genbank under accession

273

number KP792998.

274 275

Results

276

Identification of a BAM amidase in Aminobacter sp. MSH1

277

Protein fractions showing conversion of BAM into DCBA were obtained by ammonium

278

sulfate precipitation and anion exchange chromatography of Aminobacter sp. MSH1 cell

279

extracts. The protein fractions showing BAM conversion consisted almost entirely of one

280

protein of approximately 50 kDa (Figure S1A). The peptide sequences determined by mass

281

spectrometry (Table S1) corresponded to a putative amidase of 55.7 kDa encoded by a 1560

282

bp open reading frame in the MSH1 draft genome sequence. The enzyme was designated

283

BbdA and the corresponding gene bbdA. To confirm BAM degradation by BbdA, bbdA was

284

cloned and expressed in E. coli BL21(DE3)pLysS using different IPTG concentrations. A

285

protein of the expected size of around 55 kDa was visible on the SDS-PAGE gel in all

286

induced cell extracts albeit without difference in expression level between the used IPTG

287

concentrations. A protein was visible at the same height in the non-induced control but

288

appears to be an E. coli protein since the corresponding band also appeared in the

289

BL21(DE3)pLysS negative control without vector (Figure S1B). Stoichiometric conversion of

290

BAM into DCBA was recorded for resting cells of E. coli BL21(DE3)pLysS containing

291

pEXP-5-CT/TOPO_bbdA upon induction with IPTG. Minor BAM conversion was also


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observed with the non-induced control, probably due to leaky expression of BbdA36 while E.

293

coli BL21(DE3)pLysS carrying pEXP-5-CT/TOPO_ctrl containing a cloned nonsense

294

fragment did not convert BAM (Figure 1).

295 296

Sequence analysis of bbdA and its genomic context

297

Based on amino acid sequence, BbdA belongs to the amidase signature (AS) family of

298

amidases. BbdA shows moderate amino acid sequence homology to other members of the AS

299

family (Figure 2). The closest characterized homologue is the enantioselective amidase

300

AmdA of Agrobacterium tumefaciens d3 (52 % amino acid identity).37 In the MSH1 draft

301

genome sequence, bbdA was located on a 8240 bp contig (designated C1) that contains eight

302

open reading frames (ORFs) (Table S2). Two ORFs encode for putative proteins that show

303

homology with proteins involved in nitrogen metabolism: a periplasmic binding protein of a

304

nitrile hydratase/amidase (orf7) and a nitroreductase (orf4). orf2 and orf3 encode for putative

305

transposases. Finally, orf1 encodes a truncated mating pair formation protein TrbE involved

306

in plasmid transfer indicating that bbdA is located on a plasmid. To verify this, plasmids

307

isolated from Aminobacter sp. MSH1, ASI1 and two spontaneous ASI1 mutants (A1, A2)

308

defective in BAM conversion were analyzed, revealing that the ASI1-mutants lacked one

309

plasmid compared to the wild type strain. A plasmid showing similar migration was present in

310

MSH1 (Figure S2). 454 pyrosequencing of MSH1 plasmidic DNA, allowed the compilation

311

of a circular map of a 40.6 kb plasmid designated as pBAM1, that contains the complete C1

312

contig carrying bbdA (Figure 3). Sequence analysis showed that pBAM1 is an IncP-1 plasmid.

313

The plasmid belongs to the IncP-1β-2 subgroup as shown by a neighbor joining tree

314

constructed from trfA gene sequences from IncP-1 plasmids of different subgroups (Figure

315

S3).38 The backbone of pBAM1 (from traC till the truncated trbE gene) shows 99%

316

nucleotide sequence identity with other IncP-1β-2 plasmids but lacks several mating pair



317

formation genes (trbF-P). The accessory genes of pBAM1 comprises 10.8 kb including a

318

complete IS1071 element.

319 320

bbdA appears indispensable for BAM degradation in strain MSH1

321

PCR analysis showed that the MSH1 mutants defective in BAM conversion lacked bbdA,

322

indicating that indeed BbdA can be linked to the conversion of BAM into DCBA in MSH1.

323

Moreover, all mutants lacked orf4 (nfnB homologue) and orf1 (trbE homologue) suggesting

324

that loss of the BAM degradative phenotype is due to loss of pBAM1. However, analysis of

325

the draft genome sequence of one BAM degradation defective MSH1 mutant (M1a100g)

326

revealed that only an 11 kb region of plasmid pBAM1 (from gene trbA till IS1071 including

327

bbdA) was absent compared to the wild type MSH1 while other pBAM1 backbone genes were

328

still present.

329 330

Biochemical characterization of BbdA

331

Strain MSH1 degrades the BAM structurally related benzamide and OBAM.12 MSH1

332

mutant M1a100g, deficient in BAM conversion, also lacked the ability to degrade OBAM and

333

benzamide, indicating that BbdA is also responsible for benzamide and OBAM degradation.

334

Degradation experiments with heterologously expressed crude BbdA protein extracts

335

confirmed that BbdA converts benzamide and OBAM with nearly stoichiometric production

336

of BA and OBA, respectively. Maximum rates of degradation of OBAM and benzamide were

337

respectively around 45 and 90 times higher compared to BAM degradation (Figure S4). KM

338

values of BbdA for BAM and benzamide were estimated as 0.71 ± 0.09 µM and 2.51 ± 0.35

339

µM (Figure S5), respectively while the optimal temperature (Topt) for BAM hydrolysis by

340

BbdA was 62.5°C ± 3.2°C (Figure S6).

341


Page 14 of 38

Page 15 of 38


342

Expression of bbdA in Aminobacter sp. MSH1

343

Based on BAM degradation experiments in which BAM induced and non-induced cells of

344

MSH1 displayed similar degradation kinetics, Simonsen et al.18 previously suggested that

345

BAM degradation in MSH1 is constitutive indicating that BbdA is also constitutively

346

expressed in MSH1. Comparison of BbdA expression levels in Aminobacter sp. MSH1 by

347

differential proteomic analysis corroborates this hypothesis: the abundance of BbdA and other

348

pBAM1 encoded proteins in MSH1 grown on glucose or on BAM was highly similar (BbdA

349

data shown in Figure S7). To examine transcriptional regulation, a 484 bp fragment

350

containing the region upstream of bbdA was cloned in front of the promoterless gfpmut3.1

351

gene in vector pRU109733 and introduced into Aminobacter sp. MSH1. GFP expression of the

352

derivative strain, MSH1 (pRU1097-PbbdA), was analyzed in the presence and absence of

353

BAM by recording GFP fluorescence at the culture level (by Fluoroskan) and at the single cell

354

level (by flow cytometry). Strong and similar GFP fluorescence was observed on all media,

355

independent of the presence of BAM, suggesting indeed the constitutive character of bbdA

356

expression at the transcriptional level (see Figure S8).

357 358

Presence of bbdA in other BAM degrading Aminobacter strains

359

The presence of bbdA, orf4 (nfnB homologue) and orf1 (truncated trbE) was examined by

360

PCR in the BAM degrading Aminobacter sp. ASI1 and ASI214, as well as in mutants of ASI1

361

(A1 and A2) and ASI2 (ASI2.100g and A7.100g) that lacked the BAM degrading ability in

362

order to interrogate whether BbdA was also essential for BAM conversion in other BAM

363

degrading Aminobacter strains. Both wild type strains but not their mutants, tested positive for

364

all 3 markers, suggesting that bbdA and pBAM1 homologues are also responsible for BAM

365

conversion in ASI1 and ASI2. This result also strongly indicates that the missing plasmid in

366

the BAM degradation defective mutants of strain AS1 is indeed a pBAM1 homologue.



367 368

Discussion

369

Characteristics of BbdA

370

BbdA is the first reported enzyme that catalyzes the degradation of the groundwater

371

micropollutant BAM. BbdA is a member of the amidase signature (AS) protein family and

372

displays the highly conserved amidase signature domain. However, BbdA shows only

373

moderate amino acid sequence homology with other characterized AS signature amidases

374

(Figure 2).39 Using Phyre2, a putative structure of BbdA can be predicted based on the

375

structure of a benzamide degrading amidase of Rhodococcus sp. N-771 that shows 43%

376

amino acid identity with BbdA.40 It suggests that BbdA has a N-terminal α helical domain,

377

potentially involved in dimerization and formation of a narrow substrate binding tunnel, that

378

is not present in other amidase signature family enzymes (data not shown).41

379

In addition to BAM, BbdA converts OBAM and benzamide at rates that were substantially

380

higher than for BAM indicating that BbdA is better tuned to degrade benzamides that are less

381

chlorinated than BAM. Such a broad substrate range is common amongst amidases but only

382

some AS family members catalyze the conversion of cyclic and aromatic amides.37, 42-50 As

383

such, the ability of BbdA to convert the aromatic substrates BAM, OBAM and benzamide,

384

agrees with the substrate range of AS family amidases. Several other amidases convert

385

benzamide (Table 1), including enzymes from Rhodococcus, Pseudomonas, Pseudonocardia

386

and Sulfolobus and expressed from a metagenomics library from activated sludge.49 Those are

387

all AS family members, show broad substrate specificity, and often display higher activity

388

towards other substrates than benzamide.50-53 Amino acid sequence identities between those

389

enzymes and BbdA range between 28 to 46%.53,

390

tested for BAM or OBAM conversion. Interestingly, BbdA and the mentioned other AS

391

family signature proteins that degrade benzamide, show relatively high Topt values of around

54

However none of these enzymes were


Page 16 of 38

Page 17 of 38


392

60°C and higher (Table 1). Such a high Topt is not common for all AS family members since

393

LibA that catalyzes the conversion of the phenyl urea linuron in Variovorax sp. SRS16, shows

394

a Topt between 22 and 30°C.22

395 396

Genetics of BbdA

397

Our data suggest that there exists no functional redundancy in strain MSH1 to degrade

398

BAM and related compounds. Apparently, MSH1 recruited this enzyme to acquire the ability

399

to convert BAM to DCBA and use BAM as sole source of carbon and energy by cooperation

400

with yet to be identified DCBA catabolic genes. The recruitment of bbdA is likely due to

401

horizontal gene transfer, since bbdA is located on an IncP-1β-2 plasmid, which are postulated

402

to play a crucial role in recruitment and evolution of xenobiotic catabolic pathways and are

403

often encoding them.55,

404

pBAM1 are located between the trb and tra operons. Next to bbdA, those accessory genes

405

include ORFs whose function is not clear but cannot be excluded to have a function in BAM

406

metabolism since in mutants affected in BAM degradation, they were missing along with

407

bbdA. Variovorax paradoxus B4 contains a gene cluster whose deduced protein sequences

408

show substantial similarity to the sequences of orf6, orf7 and bbdA in MSH1. The orf6

409

homologue of strain B4, encoding a periplasmic binding protein, appears to be part of gene

410

cluster encoding the additional components of an ABC transporter, along with an amidase.

411

This observation might point to a possible co-evolution of orf6, orf7 and bbdA and hints to a

412

possible common origin with the Variovorax genes.

56

Like in other catabolic IncP-1 plasmids, the accessory genes in

413

pBAM1 is clearly an IncP-1 plasmid but its structure is rather unusual since it lacks part of

414

the mating pair formation genes (trbF-P). trbE-F encode part of the envelope-spanning

415

secretion apparatus for the conjugational transfer of IncP-1 plasmids.57 The IncP-1δ plasmid

416

pEST4011 encoding the catabolism of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D),



417

lacks the same mating pair formation genes.58 pEST4011 is a derivate of an unstable plasmid,

418

generated during laboratory manipulations of the host strain to increase stability of the 2,4-D

419

degradation phenotype.58 The positions where the trbE-F genes were deleted are remarkably

420

similar in plasmids pBAM1 and pEST4011, suggesting that this genetic organization is not a

421

coincidence. Therefore, the mating pair formation genes in pBAM1 could also have been lost

422

during laboratory cultivation of MSH1. However, pBAM1 still showed instability. In the

423

BAM degradation deficient mutant M1a100g, another fragment containing the remaining trb

424

and accessory genes of pBAM1 is lost without loss of the rest of the plasmid backbone.

425

Plasmid instability, gene content variability and mosaicism in the extrachromosomal elements

426

appear common within Rhizobiales bacteria to which Aminobacter belongs.59

427

Although strains ASI1, ASI2 and MSH1 were isolated from different geographical locations

428

in Denmark and despite the existence of various benzamide converting AS family amidases

429

with broad substrate range, all 3 strains contain a bbdA homologue on a similar plasmid.

430

Strains MSH1, ASI1, ASI2 belong to the genus Aminobacter indicating a specialization for

431

BAM biodegradation within this genus. Moreover, Sjoholm et al.60 showed a correlation

432

between BAM mineralization capacity and the number of Aminobacter bacteria in dichobenil

433

contaminated soil. These observations indicate a close connection between BAM degradation,

434

Aminobacter spp. and bbdA and insinuate a role of the background genetic context of

435

Aminobacter in this specialization.

436 437

BbdA characteristics in relation to biodegradation of BAM as a micropollutant

438

The KM of BbdA determined for BAM was 0.71 µM. A comparison of reported KM values

439

of enzymes catalyzing the conversion of xenobiotic compounds and of benzamide converting

440

amidases (Table 1), shows that the KM value of BbdA for BAM is one of the lowest KM values

441

listed. Amongst the AS amidases, only this of the hyperthermophylic Sulfolobus solfataricus,


Page 18 of 38

Page 19 of 38


442

SsAH (Table 1) shows a lower KM for several substrates including benzamide (KM,benzamide is

443

0.674 µM).61 Other KM values for benzamide degradation of amidases are substantially higher

444

than those of BbdA for both BAM and benzamide.52 We conclude that the KM,BAM of BbdA of

445

0.71 µM is extremely low and in fact approximates the lowest values of Michaelis constants

446

reported for enzymes in general, as for most enzymes KM lies between 10-1 and 10-7 M.62 For

447

many enzymes, experimental evidence suggests that KM is an approximation of in vivo

448

substrate concentration.62 As such, BbdA’s low KM,BAM value translates the low BAM

449

concentrations that are expected in soil contaminated with dichlobenil that MSH1

450

experienced. Dichlobenil strongly sorbs to soil and its transformation to BAM might be

451

limited by desorption. Therefore, bioavailability of BAM in soil is probably low and the

452

recruitment of BbdA for BAM catabolism might be linked with its low KM, reflecting the rare

453

ability of Aminobacter sp. MSH1 to degrade BAM at nanomolar concentrations.17 However, a

454

too low KM value can result into a slower reaction velocity due to the tight binding of

455

substrate to the enzyme63, which might explain the difference in rate between BAM, OBAM

456

and benzamide. Nevertheless the low KM,BAM of BbdA is of high importance for applying the

457

strain to remove BAM at the micropollutant concentrations found in groundwater for drinking

458

water production.

459

Furthermore, differential proteomics as well as transcriptional analysis provided evidence

460

that bbdA is constitutively expressed as previously suggested.18 Although the expression of

461

many xenobiotic catabolic pathways are under transcriptional control64, for some catabolic

462

pathways this is not the case. For instance the first step of the catabolic pathway of atrazine

463

degradation in Pseudomonas sp. strain ADP and of carbofuran degradation in

464

Novosphingobium sp. KN65.2 is constitutively expressed.65, 66 Also for linuron a constitutive

465

mineralization activity at low substrate concentrations was reported.67 The constitutive

466

expression of bbdA is also of interest for applying the strain for treating groundwater with



467

trace levels of BAM since this means that its expression does not depend on BAM-mediated

468

induction which could form a problem at trace level concentrations.7

469


Page 20 of 38

Page 21 of 38

470


FIGURES

471 472

Figure 1. Conversion of BAM into DCBA by recombinant BbdA expressed in E. coli

473

BL21(DE3)pLysS carrying pEXP-5-CT/TOPO_bbdA after (A) induction with 1 mM IPTG

474

(induction with other IPTG concentrations gave identical conversion kinetics) or (B) without

475

induction (no IPTG) and incubation at 25°C of 24 and 72 hours. Ctrl indicates the negative

476

control, i.e., E. coli BL21(DE3)pLysS carrying vector pEXP-5-CT/TOPO_ctrl containing a

477

cloned non-sense fragment and incubated with (1 mM) and without (0 mM) IPTG. n.d.: not

478

detected. DCBA production was detected in both the induced and non-induced cultures

479

containing E. coli BL21(DE3)pLysS carrying pEXP-5-CT/TOPO_bbdA but was below the

480

detection limit in the negative control (Ctrl).

481 482



483 484

Figure 2. Phylogenetic analysis of BbdA and selected members of the amidase signature

485

family. Multiple alignment of the pfam domains (PF01425) was used to construct a

486

maximum-likelihood tree. Both biochemically characterized and hypothetical amidases are

487

included. The host, substrate and accession number of characterized amidases are: AmdA

488

Atum d3 (Agrobacterium tumefaciens d3, 2-phenylpropionamide, AAK28498), AmdA Brev

489

R312 (Brevibacterium sp. R312, 2-aryl and 2-aryloxy propionamides, AAA62721), AmdA

490

Rhod MP50 (Rhodococcus erythropolis MP50, 2-aryl propionamides, AAK11724), AmdA

491

Rhod N-774 (Rhodococcus erythropolis N-774, propionamide, P22984), MG-6H10

492

(expressed from metagenome, benzamide, BAJ23980), MG-10A5 (expressed from


Page 22 of 38

Page 23 of 38


493

metagenome, benzamide, BAJ23981) and Pchl B23 (Pseudomonas chlororaphis B23,

494

propionamide, P27765). Several hypothetical amidases were included: Aaur TC1

495

(Arthrobacter aurescens TC1, ABM10653), Acin ADP1 (Acinetobacter sp. ADP1,

496

YP_046288), Bdil WSM3556 (Burkholderia dilworthii WSM3556, WP_027802717), Bmet

497

PB1 (Bacillus methanolicus PB1, ZP_10131529), Brad Tv2a-2 (Bradyrhizobium sp. Tv2a-2,

498

WP_024514442), Citr 357 (Citreicella sp. 357, ZP_10019207), Meth 73B (Methylopila sp.

499

73B, WP_020186309), Pseu P1 (Pseudonocardia sp. P1, WP_010244091), Rcer ATCC

500

49957 (Roseomonas cervicalis ATCC 49957, ZP_06898729), Rxyl DSM 9941 (Rubrobacter

501

xylanophilus DSM 9941, YP_644473), Sfre HH103 (Sinorhizobium fredii HH103,

502

YP_005193109), Sisl REY15A (Sulfolobus islandicus REY15A, YP_005647652), Vpar B4

503

(Variovorax paradoxus B4, WP_021004516). The scale bar represents 0.3 substitutions per

504

site and bootstrap values are indicated at the branches.

505 506



507 508

Figure 3. Schematic representation of the IncP-1β plasmid pBAM1 carrying bbdA. Putative

509

coding sequences are indicated and their direction of transcription shown by the arrows. The

510

IncP-1 backbone genes are indicated in blue and accessory genes in red. orf1 (trbE*) is

511

truncated compared to trbE of complete IncP-1 plasmids. The region between relA and trfA in

512

pBAM1 corresponds to the oriV region of IncP-1 plasmids and shows 99% nucleotide

513

sequence similarity with the same region in pA81 (CP002288.1), pLME1 (JF274988.1), pC1-

514

1 (HQ891317.1), pNB8c (JF274990.2) and pTB30 (JF274987.1).


Page 24 of 38

Page 25 of 38


515

TABLES

516

Table 1. Summary of Michaelis constants (KM) and optimal temperatures (Topt) of different

517

AS family amidases converting benzamide and of different enzymes transforming organic

518

pollutants with emphasis on pesticides. Protein

Protein function

Substrate

KM (µM)

Topt (°C)

Host organism

Reference

BbdA

AS amidase

BAM

0.71

62.5

Aminobacter sp. MSH1

This work

BbdA

AS amidase

benzamide

2.51

62.5

Aminobacter sp. MSH1

This work

SsAH

AS amidase

benzamide

0.674

95

Sulfolobus solfataricus

53, 61

Amidase

AS amidase

benzamide

40

55

Rhodococcus erythropolis No. 7

52

Amidase

AS amidase

benzamide

100

NR

Rhodococcus sp. Strain R312

46

Amidase

AS amidase

benzamide

150

55

Rhodococcus rhodochrous J1

44, 68

RhAmidase

AS amidase

benzamide

1030

55

Rhodococcus sp. N-771

41

Amidase

AS amidase

benzamide

4900

70

Pseudonocardia thermophila

50

Amidase

AS amidase

benzamide

NR

50

Pseudomonas chlororaphis B23

47

Amidase

AS amidase

benzamide

NR

55-60

Rhodococcus rhodochrous M8

51

Amidase

AS amidase

benzamide

NR

75

Sulfolobus tokodaii strain 7

54

LibA

AS amidase

linuron

5.8

22-30

Variovorax sp. SRS16

22

HylA

Amidohydrolase

linuron

14.97

35

Variovorax sp. WDL1

69

PuhA

Amidohydrolase

linuron

6.8

30-35

Arthrobacter globiformis D47

70

PuhA

Amidohydrolase

isoproturon

136

30-35

Arthrobacter

70



Page 26 of 38

globiformis D47

519

PuhB

Amidohydrolase

linuron

7.6

30-35

Mycobacterium brisbanense JK1

70

PuhB

Amidohydrolase

isoproturon

499

30-35

Mycobacterium brisbanense JK1

70

AtzA

Amidohydrolase

atrazine

149

NR

Pseudomonas sp. ADP

71

TrzN

Amidohydrolase

atrazine

23.8

37

Arthrobacter sp. MCM B-436

72

TOD

Dioxygenase

o-cresol

0.8

NR

Pseudomonas putida

73

TOD

Dioxygenase

trichloroethyl ene

12

NR

Pseudomonas putida

74

TfdA

Monooxygenase

2,4-D

17.5

Identification of the Amidase BbdA That Initiates Biodegradation of the

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