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Conjugative transfer of dioxin-catabolic megaplasmids and bioaugmentation prospects of a Rhodococcus sp. Jiao Sun, Yilun Qiu, Pengfei Ding, Peng Peng, Haiyan Yang, and Li Li Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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Environmental Science & Technology

1

Conjugative transfer of dioxin-catabolic megaplasmids and bioaugmentation

2

prospects of a Rhodococcus sp.

3 4

Jiao Sun ⋅ Yilun Qiu ⋅ Pengfei Ding ⋅ Peng Peng§ ⋅ Haiyan Yang ⋅ Li Li*

5 6

Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse,

7

School of Environmental Science and Engineering, Shandong University, Jinan,

8

China

9 10

§

11

Stippeneng 4, 6708WE Wageningen, The Netherlands

Present address: Laboratory of Microbiology, Wageningen University & Research,

12 13

*Corresponding author

14

Phone: +86-531-88364250; fax: +86-531-88364513; e-mail address: [email protected]

15 16 17

ABSTRACT

18

Genetic bioaugmentation, in which bacteria harboring conjugative plasmids provide

19

catabolic functions, is a promising strategy to restore dioxin-contaminated

20

environments. Here we examined the conjugative transfer of the dioxin-catabolic

21

plasmids pDF01 and pDF02 harbored by Rhodococcus sp. strain p52. A mating

22

experiment using strain p52 as a donor showed that pDF01 and pDF02 were

23

concomitantly and conjugatively transferred from strain p52 to a Pseudomonas

ACS Paragon Plus Environment


24

aeruginosa recipient at a conjugation frequency of 3×10−4 colonies per recipient.

25

pDF01 and pDF02 were isolated from the P. aeruginosa transconjugant and identified

26

by Southern hybridization, and they were localized in the transconjugant cells by

27

fluorescence in situ hybridization. Moreover, the catabolic plasmids functioned in the

28

transconjugant, which gained the ability to use dibenzofuran and chlorodibenzofuran

29

for growth, and they were maintained in 50% of the transconjugant cells for 30

30

generations without selective pressure. Furthermore, conjugative transfer of the

31

catabolic plasmids to activated sludge bacteria was detected. Sequencing of pDF01

32

and pDF02 revealed the genetic basis for the plasmids’ conjugative transfer and stable

33

maintenance, as well as their cooperation during dioxin catabolism. Therefore, strain

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p52 harboring pDF01 and pDF02 has potential for genetic bioaugmentation in

35

dioxin-contaminated environments.

36 37

TOC/ABSTRACT

38 39 40

1. INTRODUCTION

41

Dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated

42

dibenzofurans (PCDFs), can potentially induce developmental toxicity, cancers, and


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endocrine disruption, and they are extremely persistent in the environment.1 Thus far,

44

efforts have been made to decrease the industrial production of dioxins; however, the

45

levels of dioxins that have accumulated over the past decades decline very slowly in

46

the environment.2 Accordingly, the lack of an efficient treatment of such dioxins

47

could result in widespread environmental contamination, which has significant public

48

health concerns.1 Thus, efficient techniques are urgently needed to eliminate or

49

detoxify these compounds.

50

Although they are recalcitrant to degradation, previous studies have shown that

51

PCDDs/PCDFs are transformed or used by some bacteria. Anaerobes can

52

dehalogenate numerous chlorinated PCDDs/PCDFs in the environment,3 and

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dehalorespiration by anaerobic bacteria increases ATP synthesis.4 Aerobes can

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mineralize the carbon backbones of PCDDs/PCDFs and transform low-chlorinated

55

PCDDs/PCDFs.5, 6 Most aerobic, dioxin-catabolic processes are initiated by

56

dihydroxylation, which is catalyzed by an angular dioxygenase that attacks a ring

57

adjacent to the ether oxygen bridging the two rings (position 1,10a in

58

dibenzo-p-dioxin and position 4,4a in dibenzofuran).7, 8 This results in the formation

59

of chlorinated 2,2’,3-trihydroxydiphenyl ethers and chlorinated

60

2,2’,3-trihydroxybiphenyl in the case of PCDD and PCDF, respectively.7, 8 This is

61

followed by the meta-cleavage of the dihydroxylated ring, resulting in ring-opening,

62

which is catalyzed by an extradiol dioxygenase.7, 8 Then, the ring-opened products

63

undergo hydrolysis, and the resulting products are metabolized further to chlorinated

64

catechol or chlorinated salicylates in the case of PCDD and PCDF, respectively.7 In



65

some bacteria, the initial activation is mono-oxygenated by cytochrome P450.7 The

66

mineralization of dioxins by aerobic bacteria has the advantage of enabling faster

67

growth rates and transformation kinetics compared with those of anaerobic bacteria,

68

and, therefore, it is a potential solution to dioxin remediation.9 It is interesting that

69

diverse aerobes, either Gram-positive (G+) bacteria or Gram-negative (G–) bacteria,

70

transform aromatic compounds via relatively similar catabolic pathways; however,

71

they use different enzymes to do so.8 In some G+ degraders, there are two different

72

sets of enzyme systems that contribute to dioxin degradation, e.g., DfdA and DbfA

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are both angular dioxygenases that are able to hydroxylate dibenzo-p-dioxin,

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dibenzofuran.6, 10 Genes involved in dioxin catabolism have been found on plasmids,

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as well as on bacterial chromosomes,11-14 and some dioxin-catabolic plasmids have

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been characterized extensively.14

77

Dioxin cleanup in the environment remains a challenge. Because the natural dioxin

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degradation process takes a long time, enhancing the biodegradation process is a

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promising strategy for restoring contaminated environments. Aerobic bacteria are

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considered to be attractive candidates for on-site bioaugmentation and ex situ

81

remediation strategies.9 Nonetheless, bioaugmentation efforts often fail because of a

82

rapid depletion of the introduced bacterial strains, which exhibit low fitness and

83

survival in contaminated environments during competition with indigenous bacteria.15

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An alternative approach is to use genetic bioaugmentation, which introduces bacteria

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harboring conjugative, catabolic plasmids, which encode enzymes that degrade the

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target contaminants.16 In this way, an enhanced degradation potential can be achieved


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by the plasmid-mediated horizontal gene transfer (HGT) of the corresponding

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dioxin-catabolic genes to indigenous microorganisms in polluted sites.16, 17 Whether

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genetic bioaugmentation is successful depends on two critical factors: the successful

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transfer of the catabolic plasmid to as many indigenous bacteria as possible, and an

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active, contaminant-degrading phenotype in all the transconjugants.17 According to a

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genome sequence analysis of plasmids by Smillie et al.18, one-fourth of 1,730

93

plasmids investigated are conjugative (or self-transmissible), and another one-fourth

94

of the plasmids are mobilizable (their transfer still requires a co-resident conjugative

95

plasmid), while the remaining one-half of the plasmids are non-mobilizable. Recently,

96

the transferability of dioxin-catabolic plasmids from some G– bacteria has been

97

addressed. For instance, the 199-kb pCAR1 plasmid from Pseudomonas resinovorans

98

CA10 has been proven to be conjugative,19 and based on a genome sequence analysis,

99

the 223-kb pSWIT02 plasmid from Sphingomonas wittichii RW1 was predicted to be

100

a conjugative, broad-host-range plasmid.20 However, there are only a few reports of

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the transferability of dioxin-catabolic plasmids by G+ bacteria,13 not to mention the

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maintenance/stability of the catabolic plasmids after transfer. Rhodococcus spp. are

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common in diverse environmental niches, and they share with distantly related

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Pseudomonas species a capacity to degrade numerous recalcitrant and toxic pollutants;

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thus, they are ideal candidates for enhancing the bioremediation of contaminated

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sites.21 In our previous studies,13 an isolated dioxin-degrader, Rhodococcus sp. strain

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p52, was shown to catabolize a range of contaminants, such as dibenzo-p-dioxin,

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dibenzofuran, 2-chlorodibenzofuran, 2,8-dichlorodibenzofuran, phenanthrene,



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anthracene, dibenzothiophene, and carbazole. This strain has two distinct gene

110

clusters encoding angular dioxygenases, DfdA and DbfA, for the initial dioxin

111

dihydroxylation, and they are located on two plasmids, pDF01 and pDF02,

112

respectively. Our earlier investigation showed that these two plasmids were

113

transferred concomitantly into a Bacillus cereus strain by conjugation. However, there

114

have not been any subsequent reports concerning the transferability of

115

dioxin-catabolic plasmid by G+ bacteria. Unfortunately, in the aforementioned study,

116

the Bacillus transconjugant was unstable, and the plasmids were reliably cured after a

117

single transfer in Luria–Bertani (LB) broth.13 For the future application of genetic

118

bioaugmentation for dioxin removal, we aimed to answer the following questions: 1)

119

Are pDF01 and pDF02 conjugative/mobilizable plasmids? 2) Are they transferred

120

concomitantly? 3) Can these catabolic plasmids be maintained stably in a

121

transconjugant? 4) Can the catabolic genes function well in a transconjugant? To do

122

so, we first performed a sequencing analysis of the catabolic plasmids to provide the

123

genetic basis for the plasmids’ transfer, stable maintenance, and catabolic functions.

124

Second, mating tests of pDF01 and pDF02 with different recipients were performed,

125

and transfer of the plasmids into a Pseudomonas aeruginosa transconjugant was

126

further confirmed. Third, the plasmids’ maintenance in the P. aeruginosa

127

transconjugant was examined, and the dioxin degradation capacity of the

128

transconjugant was assessed. Moreover, to further judge the potential of strain p52 for

129

genetic bioaugmentation, conjugative transfer of the catabolic plasmids to activated

130

sludge bacteria was also investigated.


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2. MATERIAL AND METHODS

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2.1. Chemicals and bacterial strains. Analytical grade chemicals (> 99% pure)

134

were used in this study. Dibenzofuran was purchased from Sigma-Aldrich (Shanghai,

135

China), and 4-chlorodibenzofuran and 5-chlorodibenzofuran were purchased from

136

AccuStandard (New Haven, CT, USA). Other chemicals were obtained from Sangon

137

Biotech Co., Ltd. (Shanghai, China). Rhodococcus sp. strain p52 harboring pDF01

138

and pDF02 was isolated previously from oil-contaminated soil as a dioxin degrader,13

139

and it has been deposited in the China Center for Type Culture Collection (no.

140

M2011181). Pseudomonas aeruginosa (no. 1.1129) and other recipient strains (Table

141

S1) were obtained from the China General Microbiological Culture Collection Center

142

(CGMCC). All cultures were grown aerobically at 30 °C. LB medium22 and

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carbon-free mineral medium supplemented with dibenzofuran13 were used for growth.

144

2.2. DNA manipulation and Southern hybridization analysis. Genomic and

145

plasmid DNA were prepared as described previously.13 Plasmids were detected by

146

conventional agarose gel electrophoresis under the following conditions: 0.8%

147

agarose, TAE buffer (40 mM Tris–HCl, 40 mM acetate, 2 mM EDTA, pH 8.0), 4.5 V

148

cm−1, and a 5-h running time at 4 °C. DNA separated in an agarose gel was blotted

149

onto positively charged TotalBlot membranes (Amresco, Solon, OH, USA) and

150

subjected to Southern hybridization as described previously.13

151

2.3. Fluorescence in situ hybridization. The catabolic plasmids pDF01 and

152

pDF02 were detected in the transconjugant by fluorescence in situ hybridization



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(FISH) following the method of Niki and Hiraga (1997),23 with a modification. A

154

4.0-kb dfdA1A2A3A4 DNA fragment and a 4.4-kb dbfA1A2 DNA fragment were used

155

as probes to observe the subcellular localization of pDF01 and pDF02, respectively.

156

The probe DNA was amplified with the primer pairs dfdADetect-F (5′–

157

AGGCAACAATGCTGACTGTG–3′)/dfdADetect-R

158

ATCTGGCTTCGTGATGAGCG–3′)

159

dbfAFISH-F

160

TCGTCCCTCCGTGGTTAAGTC–3′)

161

Approximately 5 ng of total DNA was used as a template for probe preparation, using

162

the Mirus Label It Nucleic Acid Labeling Kit (Fisher Scientific Corp., Pittsburgh, PA,

163

USA), according to the manufacturer’s instructions. The probe DNA was labeled with

164

fluorescein for dfdA detection and cyanine-3 (Cy3) for dbfA detection. The probes

165

were incubated at 95 °C for 10 min, and then kept on ice to prevent self-annealing

166

before hybridization. To display the profile of a cell, 4’,6-diamidino-2-phenylindole

167

(DAPI; 0.5 µg ml−1) was used to stain intracellular DNA.

targeting

(5′– dfdA-harboring

pDF01,

(5′–AGAACAGTTGGACCAGCAATGAC–3′)/dbfAFISH-R targeting

dbfA-harboring

and (5′–

pDF02.

168

2.4. Conjugation experiment. A filter-mating procedure was performed according

169

to Shintani et al.24, using Rhodococcus sp. strain p52 as the donor and P. aeruginosa

170

CGMCC 1.1129 and other bacterial strains (Table S1) as the recipients. Before the

171

mating experiments, the natural tolerance to antibiotics and use of dibenzofuran by

172

the recipients were tested. Unlike the recipient P. aeruginosa strain (which is

173

erythromycin-resistant and unable to grow on dibenzofuran), because strain p52 is

174

sensitive to erythromycin, mineral medium containing dibenzofuran (300 mg l−1) as


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the sole carbon source and 50 µg ml−1 erythromycin served as the selective medium

176

for the P. aeruginosa transconjugant. The number of transconjugants was determined

177

by colony counting on selective medium 2 d post-mating. The transfer frequency was

178

calculated as the ratio of transconjugants to recipients.

179

Conjugative transfer of the catabolic plasmids to activated sludge bacteria was

180

performed as follows: the experiments were conducted in 250-ml Erlenmeyer flasks

181

containing 50 ml of mineral medium supplemented with 300 mg l−1 dibenzofuran.

182

Strain p52 was precultured in LB medium to the mid-exponential phase, and then it

183

was washed with mineral medium and inoculated as the donor strain at a final

184

concentration of approximately 108 colony-forming units (CFU) ml−1. Activated

185

sludge was collected from a municipal wastewater treatment plant in Jinan, Shandong,

186

China. The sludge was inoculated into LB medium (0.1%, v/v) and cultured to an

187

optical density at 600 nm (OD600) of approximately 4.0. Then, the cells were collected,

188

washed with mineral medium, and inoculated into flasks at approximately 108 CFU

189

ml−1. Flasks inoculated with only strain p52 or only sludge bacteria, as well as

190

non-inoculated flasks, served as controls. All the flasks were incubated at 30 °C, with

191

shaking at 180 rpm for approximately 100 h. Then, they were sampled, and the

192

bacteria were spread onto 300 mg l−1 dibenzofuran-supplemented mineral medium

193

plates. After 2 d of incubation, colonies that differed from those of strain p52 (those

194

that formed orange-red circles with a diameter less than 1 mm) were selected as

195

transconjugant candidates. To confirm the transconjugants, colony polymerase chain

196

reactions were performed. A 4.0-kb dfdA1A2A3A4 fragment of pDF01 and a 1.8-kb



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dbfA1A2 fragment of pDF02 were amplified from the same colony, using the primer

198

pairs

199

GCTCATGACCAGCATTAGCG–3′)/dbfADetect-R

200

GGGCCTCAGAAGAAGATGGAG–3′),

201

transconjugant was determined by 16S rRNA gene amplification from the same

202

colony (as described above) and DNA sequencing. The catabolic plasmids were

203

extracted from an individual transconjugant, and a Southern hybridization analysis

204

was performed as described above.

dfdADetect-F/dfdADetect-R

and

respectively.

dbfADetect-F

(5′– (5′–

The

identity

of

the

205

2.5. Measurement of plasmid maintenance and the dibenzofuran-degrading

206

ability of the transconjugant. Plasmid maintenance was measured using a method

207

modified from De Gelder et al. (2007).25 The newly obtained P. aeruginosa

208

transconjugant was pre-cultured in mineral medium containing dibenzofuran (500 mg

209

l−1) and erythromycin (50 µg ml−1) to the mid-exponential phase. Then, it was

210

harvested, washed twice with phosphate-buffered saline, and transferred into 250-ml

211

Erlenmeyer flasks containing 50 ml of LB medium to an OD600 of approximately 0.01.

212

For the first generation of the plasmid maintenance test, the flasks were incubated at

213

30 °C with shaking at 180 rpm. Subsequently, the cultures were transferred to fresh

214

LB medium every 8 h while keeping the initial OD600 at 0.01. According to the

215

growth curves, eight generations were achieved during 8 h of exponential growth. For

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each transfer, cells were collected, washed twice with phosphate-buffered saline, and

217

then diluted and spread onto LB agar plates and dibenzofuran-supplemented mineral

218

medium plates. After incubation at 30 °C for 48 h, total cell counts were determined


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on LB plates, and plasmid-bearing cell counts were determined on selective mineral

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medium plates containing 300 mg l−1 dibenzofuran and 50 µg ml−1 erythromycin.

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Thus, the plasmid maintenance rate (%) could be calculated for different generations

222

using the formula: (1−NP/Nt)×100, where NP and Nt are the plasmid-bearing cell

223

counts and total cell counts, respectively. Simultaneously, the colonies on each

224

dibenzofuran selective plate were picked randomly for amplification of the dfdA- and

225

dbfA-containing DNA fragments using the primer sets dfdADetect-F/R and

226

dbfADetect-F/R, and a sequencing analysis was performed to confirm the presence of

227

pDF01 and pDF02, respectively.

228

2.6. Degradation experiments. Biodegradation tests of dibenzofuran and

229

chlorinated dibenzofurans were conducted in 250-ml Erlenmeyer flasks containing 50

230

ml of mineral medium with the tested substrates as the sole carbon source. When

231

testing for the

232

4-chlorodibenzofuran, and 100 mg l−1 5-chlorodibenzofuran, the P. aeruginosa

233

recipient and transconjugant were pre-cultured in LB medium to the exponential

234

phase, collected, washed twice with mineral medium, and finally inoculated into

235

flasks at an initial OD600 of approximately 0.04. The same procedure was used to test

236

the degradation of 300 mg l−1 dibenzofuran by strain p52. When testing for the

237

degradation of 300 mg l−1 dibenzofuran by activated sludge bacteria and its mixture

238

with strain p52, the sludge was inoculated into LB medium and cultured to an OD600

239

of approximately 4.0. Then, the cells were collected, washed twice with mineral

240

medium, and inoculated directly into the flasks, or they were mixed 1:1 (by cell

degradation

of

500 mg l−1

dibenzofuran,


100

mg

l−1


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241

numbers) with strain p52, which was treated as described above, and then inoculated.

242

The inocula were adjusted to the identical cell numbers (OD600=0.04) in all the tested

243

systems. All the flasks were incubated aerobically at 30 °C with vigorous shaking at

244

180 rpm. To confirm bacterial growth on dibenzofuran, 4-chlorodibenzofuran, and

245

5-chlorodibenzofuran, the cellular protein content was monitored according to

246

Fortnagel et al. (1990) by Bradford assays.26 The residual compounds were extracted

247

for a gas chromatography analysis as described previously.13 Each test was conducted

248

in triplicate using a control without a bacterial inoculum. Data are reported as the

249

means of the triplicate experiments.

250

2.7 Complete genome sequencing and plasmid accession numbers. Complete

251

genome sequencing was performed using a HiSeq4000 system (Illumina, San Diego,

252

CA, USA) combined with the PacBio RSII sequencing platform (Pacific Biosciences,

253

Menlo Park, CA, USA). A de novo assembly was conducted using HGAP 2.3.0.27

254

Open reading frames were predicted by Glimmer3.0.28 The National Center for

255

Biotechnology

256

(http://www.ncbi.nlm.nih.gov/genome/annotation_prok) was used for annotation.

257

Potential protein-coding sequences were also analyzed manually using the Basic

258

Local Alignment Search Tool (BLAST) suite of programs, including BLASTN,

259

BLASTP, BLASTX, as well as clusters of orthologous groups, and the conserved

260

domain database.

261 262

Information

Prokaryotic

Genome

Annotation

Pipeline

The sequences of the pDF01 and pDF02 plasmids of strain p52 were deposited in GenBank under the accession numbers CP016821 and CP016820, respectively.


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3. RESULTS

265

3.1. General features of the catabolic plasmids pDF01 and pDF02. The exact

266

size and form of the catabolic plasmids pDF01 and pDF02, which were separated and

267

identified previously by pulsed-ﬁeld gel electrophoresis,13 were defined by complete

268

genome sequencing. The plasmid harboring the dfdA gene cluster6 that is required for

269

the initial dihydroxylation of dioxin, previously termed pDF01, is a 170,002-bp

270

circular plasmid, while pDF02 is a 242,246-bp linear plasmid harboring the dbfA gene

271

cluster6 that is also involved in the initial dihydroxylation of dioxins. pDF01 and

272

pDF02 contain 167 and 227 protein-encoding gene sequences, respectively, which are

273

responsible for plasmid replication/maintenance/partition, conjugative transfer,

274

catabolism, transport, transcriptional regulation, and transposition/recombination

275

(Figure 1). Additionally, pDF01 encodes heavy metal (e.g., copper and arsenic)

276

resistance proteins. Nine percent and 23.6% of the genes in pDF01 and pDF02,

277

respectively, encode proteins with unknown functions that were annotated as

278

hypothetical proteins. The mean G+C content is 65.7% for pDF01 and 66.2% for

279

pDF02. Comparing the two plasmids, pDF02 contains more complex components for

280

plasmid replication/partition and conjugation transfer. Specifically, pDF02 contains

281

three genes for plasmid replication, including repA and repB.20 It is worth mentioning

282

that the amino acid sequence of RepB encoded by pDF02 shares 28.9% identity with

283

the replicon of IncP-6 family plasmids. Additionally, pDF02 contains three parA

284

genes and one parB gene for plasmid partition,20 while pDF01 encodes only one



285

replication protein and one ParA. Regarding the stable maintenance of plasmids, both

286

plasmids contain more than one copy of genes that encode a toxin–antitoxin system. A

287

previous study indicated that pDF01 and pDF02 may adopt the “plasmid addiction”

288

mechanism for efficient plasmid maintenance, i.e., the host cell is killed selectively if

289

it has not received any copy of the plasmid.29 Regarding genes related to conjugative

290

plasmid transfer,18, 30 both plasmids contain genes encoding the components required

291

for plasmid mobilization, i.e., oriT, relaxases, and the type IV secretion system (T4SS)

292

coupling protein VirD4 (which overlaps with traG), and genes related to the T4SS

293

machinery, such as virB4, virB6, and virB11. Specifically, pDF02 has three relaxase

294

genes (traA),18, 30 two of which are identical but incomplete because of missing start

295

or stop codons, and the complete TraA shared 35% identity with the one in pDF01. In

296

addition, approximately 3% of the genes on pDF01 and 3.5% of genes on pDF02

297

encode T4SS components.

298

Regarding the dioxin-catabolic genes, pDF01 contains two copies of dfdB and

299

dfdC,10 encoding an extradiol dioxygenase and a hydrolase, respectively, which are

300

located downstream and upstream of dfdA1A2A3A4 (encoding a complete angular

301

dioxygenase with components of a terminal oxygenase, ferredoxin, and ferredoxin

302

reductase),6,

303

reductase components were not found in the neighboring region of dbfA1A2

304

(encoding a terminal oxygenase component)6, 11 of pDF02, and there are no genes

305

encoding the corresponding extradiol dioxygenase and hydrolase for subsequent

306

catalysis after the initial dihydroxylation of dibenzofuran.

10

respectively. However, genes encoding ferredoxin and ferredoxin


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Figure 1. Genetic map of plasmids pDF01 (inner) and pDF02 (outer). Predicted

309

coding regions are shown by arrows indicating the direction of transcription. Different

310

colors represent different putative functions: white, hypothetical proteins; dark purple,

311

plasmid

312

metabolism; orange, heavy metal resistance; yellow, transposition and recombination;

313

blue, others including DNA processing, regulatory protein, transporter, and membrane

314

proteins.

replication/maintenance/partition;

green,

plasmid

conjugation;

red,

315 316

3.2. Transferability of dioxin-catabolic plasmids. Conjugative transfer of pDF01

317

and pDF02 from Rhodococcus sp. strain p52 to different bacterial strains was

318

investigated. Rhodococcus, Bacillus, and Pseudomonas transconjugants were obtained

319

with transconjugation frequencies ranging from 1.8×10−7 to 3.5×10−4 colonies per

320

recipient (Table S2). The highest transconjugation frequency was achieved when



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321

using a G– P. aeruginosa strain as a recipient. However, except the P. aeruginosa

322

transconjugant, the other transconjugants did not grow in dibenzofuran-supplemented

323

mineral medium after they were transferred one or two times in LB broth, or even in

324

dibenzofuran-supplemented

325

transconjugant was further examined. The P. aeruginosa transconjugant was

326

confirmed primarily by the presence of the 4.04-kb dfdA or 1.8-kb dbfA target

327

fragments corresponding to pDF01 or pDF02, respectively (Figure 2), and its identity

328

was confirmed simultaneously by sequencing its 16S rRNA gene. Interestingly, when

329

the mating experiments were repeated, the transconjugants always contained both the

330

dfdA and dbfA genes (Figure 2), which is consistent with our previous mating

331

experiment using a B. cereus strain as a recipient.13 The results indicate that pDF01

332

and pDF02 were transferred concomitantly from the Rhodococcus sp. to the P.

333

aeruginosa strain. It should be mentioned that similar transconjugation frequencies

334

were achieved when the mating process was performed in liquid medium or by filter

335

mating.

mineral

medium.

Therefore,


the

P.

aeruginosa

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336 337

Figure 2. Transconjugant confirmation by polymerase chain reaction amplification of

338

the dfdA and dbfA fragments. Amplicons of the 4.04-kb dfdA fragment and the 1.8-kb

339

dbfA fragment from the donor Rhodococcus sp. strain p52 (1), the P. aeruginosa

340

transconjugant (2), and the P. aeruginosa recipient (3) are shown in A and B,

341

respectively, with a corresponding molecular mass standard (M). All the amplicons

342

were further confirmed by sequencing.

343 344

To confirm the transfer of plasmids pDF01 and pDF02 from the Rhodococcus

345

donor into the P. aeruginosa strain, plasmids were isolated from the transconjugant

346

and separated by conventional agarose gel electrophoresis. A Southern hybridization



347

analysis was performed using probes targeting the dfdA1 gene on pDF01 and the

348

dbfA1 gene on pDF02. As shown in Figure 3, in contrast to the recipient, the

349

electrophoretic profile revealed three bands in the transconjugant, which corresponded

350

to plasmids of the donor strain p52. A Southern hybridization analysis of the separated

351

plasmids demonstrated that the upper two bands corresponded to pDF01 and pDF02,

352

respectively. The third band in the transconjugant also showed a weakly positive

353

signal corresponding to the probe targeting dfdA1; this band might be related to the

354

circular form of pDF01, although its migration in the gel was similar to that of pDF03

355

of strain p52 (as determined by a genome sequence analysis, pDF03 does not contain

356

any catabolic genes such as dfdA or dbfA (data not shown)). Therefore, the results

357

indicate that the P. aeruginosa transconjugant contained pDF01 and pDF02.

358 359

Figure 3. Detection of the catabolic plasmids in the transconjugant following alkaline

360

lysis, and identification of the plasmids by Southern hybridization. Plasmid profiles of

361

the donor Rhodococcus sp. strain p52 (1), the P. aeruginosa transconjugant strain (2),

362

and the P. aeruginosa recipient strain (3), which were obtained by conventional


Page 18 of 47

Page 19 of 47


363

agarose gel electrophoresis, are shown in A. Southern hybridization patterns of the

364

plasmids from the donor strain p52 (1), the P. aeruginosa transconjugant strain (2),

365

and the recipient strain (3) using probes targeting dfdA and dbfA are displayed in B

366

and C, respectively.

367 368

The catabolic plasmids pDF01 and pDF02 coexisting in the P. aeruginosa

369

transconjugant cell were visualized by FISH. As shown in Figure 4, the presence of

370

pDF01 in the transconjugant cells was indicated by a fluorescein-labeled probe

371

targeting the dfdA1A2A3A4 cluster (Figure 4A), while the presence of pDF02 was

372

indicated by a Cy3-labeled probe targeting dbfA1A2 and its flanking region (Figure

373

4B). Using DAPI staining of the transconjugant cells (Figure 4C) as a background, the

374

co-existence of pDF01 and pDF02 was observed in P. aeruginosa transconjugant cells

375

(Figure 4D).

376



Page 20 of 47

377 378

Figure 4. Observation of pDF01 and pDF02 in P. aeruginosa transconjugant cells. A

379

FISH assay using a fluorescein-labeled probe for pDF01 detection is shown in A, and

380

a Cy3-labeled probe for pDF02 detection is shown in B. DAPI-stained transconjugant

381

cells are shown in C. An overlap image of the fluorescein-labeled, Cy3-labeled, and

382

DAPI-stained transconjugant cells is shown in D.

383 384

3.3 Dioxin-degrading ability of the P. aeruginosa transconjugant. To determine

385

whether the catabolic plasmids function in their new host, the ability of the P.

386

aeruginosa transconjugant to degrade dibenzofuran, 4-chlorodibenzofuran, and 5-

387

chlorodibenzofuran was investigated. The results showed that the transconjugant

388

gained

389

5-chlorodibenzofuran as sole sources of carbon and energy, compared with the P.

390

aeruginosa recipient (Figure 5). Growth of the recipient on dibenzofuran,

391

4-chlorodibenzofuran, and 5-chlorodibenzofuran was indicated by increases of its

392

cellular protein content that occurred simultaneously with decreases in the levels of

393

these compounds. Dibenzofuran (500 mg l−1) was degraded completely within 80 h by

394

the P. aeruginosa transconjugant, which is comparable to that of the donor strain p52

395

within 48 h.13 In addition, more than 60% of 100 mg l−1 4-chlorodibenzofuran and at

396

least 80% of 100 mg l−1 5-chlorodibenzofuran were removed by the P. aeruginosa

397

transconjugant within 70 h. It should be noted that the losses of different substrates

398

due to the substrates’ adherence to the flask were similar between the recipient culture

the

ability

to

use

dibenzofuran,

4-chlorodibenzofuran,


and

Page 21 of 47


399

and the non-inoculation control. In contrast, no significant increase in protein content

400

of the P. aeruginosa recipient was observed during the process. Thus, the results

401

indicate that the catabolic plasmids functioned after they were transferred into the P.

402

aeruginosa host.

403 404

Figure

405

5-chlorodibenzofuran (C) as sole sources of carbon and energy by the P. aeruginosa

406

transconjugant, compared with the recipient strain. Decreases in the substrate levels in

5.

Use

of

dibenzofuran

(A),

4-chlorodibenzufuran


(B),

and


407

the transconjugant culture (■), recipient culture (□), and non-inoculation control (▲)

408

were monitored by gas chromatography. Growth is shown as an increase in the protein

409

contents of the transconjugant (●) and the recipient (○). Data are the means and

410

standard deviations of independent triplicates.

411 412

3.4. Plasmid stability in the P. aeruginosa transconjugant. Growth of the P.

413

aeruginosa harboring pDF01 and pDF02 was compared to that of the recipient stain

414

in LB medium (Figure 6A). The results demonstrated that harboring the catabolic

415

plasmids slightly decreased the growth rate of the transconjugant, although its growth

416

rate was still comparable to that of the recipient strain. The generation time of both P.

417

aeruginosa strains in LB medium was approximately 1 h, and both strains entered the

418

stationary phase after a 10-h incubation.

419

To examine whether the P. aeruginosa transconjugant could stably maintain the

420

catabolic plasmids, losses of dibenzofuran catabolic activity were monitored without

421

selective pressure (e.g., during culturing in LB medium). As shown in Figure 6B,

422

approximately 50% of the transconjugant cells maintained the catabolic plasmids after

423

propagation in LB medium for 30 generations. However, less than 5% of the

424

transconjugant cells maintained pDF01 and pDF02 after propagation for 50

425

generations without selective pressure, indicating that selective pressure played an

426

important role in the stability of the catabolic plasmids in the transconjugant.


Page 22 of 47

Page 23 of 47


427 428

Figure 6. Growth and plasmid loss of the P. aeruginosa transconjugant in LB medium.

429

Growth in LB medium of the P. aeruginosa transconjugant (□) was compared to that

430

of the recipient (●) in A. The loss of plasmids in the P. aeruginosa transconjugant in

431

LB medium is shown in B. Data are the means and standard deviations of independent

432

triplicates.

433 434

3.5. Transfer of pDF01 and pDF02 to activated sludge bacteria. For potential

435

application purposes, the transferability of the catabolic plasmids to activated sludge

436

bacteria was tested. The activated sludge bacteria were subjected to enrichment, and

437

they were collected as recipients. Before the mating test, the recipients were spread

438

onto a mineral medium plate supplemented with dibenzofuran (as a selective plate),

439

and occasionally a few colonies were observed after a 7-d incubation. In contrast,



440

hundreds of colonies (except those of strain p52) were obtained after mating on the

441

selective plate within 2 d of incubation. The dfdA and dbfA fragments were amplified

442

successfully from these colonies (Figure 7), and this was confirmed by sequencing.

443

The recipients were Klebsiella spp., Pseudomonas spp., Arthrobacter sp. and

444

Glutamicibacter sp., as determined by sequencing the 16S rRNA gene (Table S2). The

445

results indicate that there were some transconjugants among the sludge bacteria after

446

mating. To confirm this, dibenzofuran degradation by the activated sludge bacteria

447

before and after mating (after mixing with strain p52) was examined, and it was

448

compared to that of strain p52. As shown in Figure 8, after mating, the sludge bacteria

449

exhibited an enhanced ability to degrade dibenzofuran, compared with their

450

counterparts before mating. Considering the presence of strain p52 among the sludge

451

bacteria after mating, it is encouraging that the degradation ability of the sludge

452

bacteria after mating surpassed that of strain p52. However, the obtained

453

transconjugants were not as robust as the aforementioned P. aeruginosa

454

transconjugant, in terms of the stable maintenance of the catabolic plasmids without

455

selective pressure (data not shown).


Page 24 of 47

Page 25 of 47


456 457

Figure 7. Confirmation of transconjugants from activated sludge bacteria by colony

458

polymerase chain reaction amplification of the dfdA and dbfA fragments. Amplicons

459

of the 4.04-kb dfdA fragment and the 1.8-kb dbfA fragment from the same colony are

460

shown in lanes in A and B, respectively, with a molecular mass standard (M). All the

461

amplicons were further confirmed by sequencing

462



463

Figure 8. Removal of dibenzofuran by activated sludge bacteria before and after

464

mating, compared to that of the donor Rhodococcus sp. strain p52. Dibenzofuran

465

degradation by the sludge bacteria before mating (○), the sludge bacteria mated with

466

strain p52 (●), strain p52 (□), and the inoculation control (▲) was monitored by gas

467

chromatography. Data are the means and standard deviations of independent

468

triplicates

469 470 471

4. DISCUSSION Horizontal transfers of catabolic genes contribute to the ability of bacterial

472

communities to degrade xenobiotics in environment.31 As an application of HGT for

473

the bioremediation of contaminated sites, genetic bioaugmentation prevails over

474

traditional bioaugmentation, which depends on exogenous microorganisms, by

475

disseminating catabolic functions to indigenous microorganisms via conjugative

476

plasmids.17 In the present study, the transferability of two dioxin-catabolic plasmids,

477

pDF01 and pDF02, was confirmed experimentally, and its genetic basis was

478

determined. A complete sequencing analysis of the two catabolic plasmids indicated

479

that both plasmids contain genes required for plasmid mobilization and the T4SS

480

machinery, which contributes to the conjugative transfer of pDF01 and pDF02.18, 30

481

Interestingly, we observed the concomitant transfer of the circular plasmid pDF01

482

(170 kb) harboring dfdA and the linear plasmid pDF02 (242 kb) harboring dbfA from

483

the donor strain p52 to the recipient strains. In G+ bacteria, the dbfA and dfdA clusters

484

involved in dihydroxylation of dioxins are highly conserved, and they have been


Page 26 of 47

Page 27 of 47


485

detected in different genera, such as Terrabacter10, 32 and Rhodoccocus.6, 13 Previous

486

studies have demonstrated that two initial angular dioxygenases, DfdA and DbfA,

487

attack different chlorodibenzofurans,6 and that DfdA exhibits a broader substrate

488

range than DbfA.10 The coexistence of dbfA and dfdA clusters in G+ bacteria

489

contributes to the broad substrate range of dioxin-degraders.6, 10, 13, 32 However, there

490

is no experimental proof of the transferability of catabolic plasmids harboring dbfA

491

and dfdA clusters, except pDF01 and pDF02 in strain p52. The transferability of

492

pDF01 and pDF02 in the present study proved that plasmid-mediated HGT plays an

493

important role in disseminating dioxin-catabolic genes among actinomycetes. It

494

should be noted that genes encoding extradiol dioxygenase and hydrolase, which are

495

crucial enzymes for the dioxin-catabolic pathway that follows the initial

496

hydroxylation of dioxins, were found on pDF01 and pDF02, although the genes

497

differed between the two plasmids. There are two copies of dfdB and dfdC located

498

upstream and downstream, respectively, from dfdA1A2A3A4 in pDF01, while pDF02

499

contains genes encoding an extradiol dioxygenase (edi4/flnD1)33, 34 and a hydrolase

500

(flnE),34 which are involved in fluorene metabolism and are ineffective for

501

dibenzofuran degradation,33, 34 downstream from dbfA1A2. Therefore, the concomitant

502

transfer of pDF01 and pDF02 (under selective pressure) was ascribed to the catabolic

503

cooperation of two sets of catabolic genes harbored on the two plasmids, which

504

promotes dibenzofuran utilization by recipients.

505 506

The indigenous microbial communities of polluted soils and waters comprise diverse bacterial strains that could serve as potential recipients for a broad-host-range



507

catabolic plasmid, which provides the basis for plasmid-mediated genetic

508

bioaugmentation.35, 36 The presence of such diversified, indigenous hosts for a

509

catabolic plasmid enhances the catabolic potential of these microorganisms and

510

alleviates the risk of failure of genetic bioaugmentation.17 Although the host range of

511

a plasmid is thought to be defined by its replication determinants,37 studies have

512

shown that the donor strain of a conjugative plasmid influences the host range of the

513

plasmid.38, 39 In the present study, pDF01 and pDF02 were transferred conjugatively

514

between phylogenetically distant bacteria, i.e., from a G+ Rhodococcus strain to a G–

515

Pseudomonas strain. Additionally, we demonstrated the conjugative transfer of

516

pDF01 and pDF02 from strain p52 to sludge bacteria, with transconjugants belonging

517

to Klebsiella, Pseudomonas, Arthrobacter, and Glutamicibacter. Therefore, strain p52,

518

the natural host of pDF01 and pDF02, has great promise for genetic bioaugmentation.

519

Conjugative, catabolic plasmids and a suitable donor make it possible to implement

520

a genetic bioaugmentation strategy. The transconjugation frequency is an index used

521

to assess the bioremediation potential of such a strategy. In laboratory studies, the

522

transfer of broad-host-range plasmids occurs at variable frequencies (generally in the

523

range of 10−3 to 10−6) depending on the plasmid and the mating-pair genotype.40 In the

524

present study, pDF01 and pDF02 were transferred from Rhodoccocus sp. strain p52

525

into a P. aeruginosa strain at a frequency of 3×10−4 colonies per recipient by a

526

filter-mating experiment. In a previous report, the presence of the bacterial

527

community impacted the ability of a recipient strain to acquire a conjugative

528

plasmid.41 In the present study, conjugative transfer of pDF01 and pDF02 to sludge


Page 28 of 47

Page 29 of 47


529

bacteria occurred at a frequency that was greater than approximately 10−6 per recipient.

530

Until now, few studies have compared laboratory results using pure cultures for

531

mating with those of field tests. An early study by Neilson et al.42 showed a high

532

conjugation frequency of approximately 10−3 per donor/recipient in a pure culture

533

decreased significantly to 10−5 per donor/recipient after the introduction of an abiotic

534

material (sterile soil), and it decreased to 10−6 per donor/recipient under biotic

535

(nonsterile soil) stresses. The study revealed that the frequency of plasmid transfer in

536

the environment may be several orders of magnitude lower than that in laboratory

537

mating tests. In the environment, a detectable transfer of a catabolic plasmid does not

538

ensure a successful bioremediation. Even if the catabolic plasmid is transferred

539

successfully into a group of indigenous bacteria during the bioremediation, there is

540

only a small fraction of transconjugants among the total indigenous bacteria at the

541

initial time period, which makes it difficult to degrade pollutants, especially

542

considering the decrease in the donor population.16, 17 Although conjugative plasmid

543

transfer can occur in a few minutes to 1 h,43 it takes days to detect transconjugants,36,

544

42

545

contaminant levels in polluted soils and waters.16, 35, 36 In an example of a successful

546

genetic bioaugmentation of soil, Top et al. 16 illustrated that low numbers (101–104

547

CFU g−1 soil) of transconjugants were often observed during the first several days

548

after the donor inoculation, while significant removal of 2,4-dichlorophenoxyacetic

549

acid (2,4-D) was achieved when the transconjugant numbers were approximately 106

550

CFU g−1 soil or higher, depending on the proliferation of the transconjugants.

, and it takes weeks after the donor inoculation to see any effect of the plasmid on



551

Therefore, a high conjugation frequency of a catabolic plasmid results in relatively

552

high numbers of transconjugants after mating, which ensures the subsequent, rapid

553

increase of the transconjugant population, which is required for sufficient catabolic

554

activity. Therefore, researchers have paid attention to the growth and function of

555

transconjugants during genetic bioaugmentation.44 A pilot field study showed that the

556

establishment of stable, indigenous plasmid hosts enhanced 2,4-D degradation.38 In

557

the present study, pDF01 and pDF02 were maintained in the P. aeruginosa

558

transconjugant without selective pressure for 30 generations, while selective pressure

559

played an important role in the long-term (>50 generations) maintenance of the

560

plasmids. Sequencing of pDF01 and pDF02 revealed genes encoding proteins,

561

including toxin–antitoxin systems, partition systems, and multiple site-specific

562

recombinases, which are required for their stable maintenance.29 The effect of

563

selective pressure (e.g., the presence of a substrate to be degraded) on transconjugant

564

proliferation in the environment was highlighted in previous studies.16, 17 A previous

565

study indicated that biological conditions, such as the phylogenetic relationship

566

between a donor and recipient, influence the expression of catabolic genes in a

567

transconjugant.17 In the present study, the catabolic genes were expressed and

568

functioned in dibenzofuran degradation in a Pseudomonas transconjugant, as well as

569

sludge bacterial transconjugants. This suggests that barriers between donor and

570

recipient strains, such as phylogenetic distance, do not prevent plasmid transfer and

571

catabolic gene expression. However, the underlying mechanism requires further

572

studies. Overall, the present study indicates that strain p52 harboring pDF01 and


Page 30 of 47

Page 31 of 47


573

pDF02 has great potential for genetic bioaugmentation applications, such as dioxin

574

removal.

575 576

ACKNOWLEDGMENTS

577

Funding was provided by the Natural Science Foundation of China (grant no.

578

21377069).

579

ASSOCIATED CONTENT

580

Supporting Information. This material is available free of charge via the internet at

581

http://pubs.acs.org

582 583

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584

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84x47mm (300 x 300 DPI)



Figure 1. Genetic map of plasmids pDF01 (inner) and pDF02 (outer). Predicted coding regions are shown by arrows indicating the direction of transcription. Different colors represent different putative functions: white, hypothetical proteins; dark purple, plasmid replication/maintenance/partition; green, plasmid conjugation; red, metabolism; orange, heavy metal resistance; yellow, transposition and recombination; blue, others including DNA processing, regulatory protein, transporter, and membrane proteins. 177x165mm (300 x 300 DPI)


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Figure 2. Transconjugant confirmation by polymerase chain reaction amplification of the dfdA and dbfA fragments. Amplicons of the 4.04-kb dfdA fragment and the 1.8-kb dbfA fragment from the donor Rhodococcus sp. strain p52 (1), the P. aeruginosa transconjugant (2), and the P. aeruginosa recipient (3) are shown in A and B, respectively, with a corresponding molecular mass standard (M). All the amplicons were further confirmed by sequencing. 171x349mm (300 x 300 DPI)



Figure 3. Detection of the catabolic plasmids in the transconjugant following alkaline lysis, and identification of the plasmids by Southern hybridization. Plasmid profiles of the donor Rhodococcus sp. strain p52 (1), the P. aeruginosa transconjugant strain (2), and the P. aeruginosa recipient strain (3), which were obtained by conventional agarose gel electrophoresis, are shown in A. Southern hybridization patterns of the plasmids from the donor strain p52 (1), the P. aeruginosa transconjugant strain (2), and the recipient strain (3) using probes targeting dfdA and dbfA are displayed in B and C, respectively. 105x62mm (300 x 300 DPI)


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Figure 4. Observation of pDF01 and pDF02 in P. aeruginosa transconjugant cells. A FISH assay using a fluorescein-labeled probe for pDF01 detection is shown in A, and a Cy3-labeled probe for pDF02 detection is shown in B. DAPI-stained transconjugant cells are shown in C. An overlap image of the fluorescein-labeled, Cy3-labeled, and DAPI-stained transconjugant cells is shown in D 177x133mm (300 x 300 DPI)



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Figure 5. Use of dibenzofuran (A), 4-chlorodibenzufuran (B), and 5-chlorodibenzofuran (C) as sole sources of carbon and energy by the P. aeruginosa transconjugant, compared with the recipient strain. Decreases in the substrate levels in the transconjugant culture (■), recipient culture (□), and non-inoculation control (▲) were monitored by gas chromatography. Growth is shown as an increase in the protein contents of the transconjugant (●) and the recipient (○). Data are the means and standard deviations of independent triplicates. 76x176mm (300 x 300 DPI)


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Figure 6. Growth and plasmid loss of the P. aeruginosa transconjugant in LB medium. Growth in LB medium of the P. aeruginosa transconjugant (□) was compared to that of the recipient (●) in A. The loss of plasmids in the P. aeruginosa transconjugant in LB medium is shown in B. Data are the means and standard deviations of independent triplicates. 107x150mm (600 x 600 DPI)



Figure 7. Confirmation of transconjugants from activated sludge bacteria by colony polymerase chain reaction amplification of the dfdA and dbfA fragments. Amplicons of the 4.04-kb dfdA fragment and the 1.8kb dbfA fragment from the same colony are shown in lanes in A and B, respectively, with a molecular mass standard (M). All the amplicons were further confirmed by sequencing 106x148mm (300 x 300 DPI)


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Figure 8. Removal of dibenzofuran by activated sludge bacteria before and after mating, compared to that of the donor Rhodococcus sp. strain p52. Dibenzofuran degradation by the sludge bacteria before mating (○), the sludge bacteria mated with strain p52 (●), strain p52 (□), and the inoculation control (▲) was monitored by gas chromatography. Data are the means and standard deviations of independent triplicates 76x58mm (300 x 300 DPI)


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