1

Recovery from natural environments of plasmid pEMB1 whose toxin-1

antitoxin system stabilizes an ampicillin-resistance β-lactamase gene in E. coli 2

Hyo Jung Lee,1 Hyun Mi Jin,1 Moon Su Park,1 Woojun Park,2 Eugene L. Madsen,3 and Che Ok 3

Jeon1,* 4

1Department of Life Science, Chung-Ang University, Seoul, 156-756, Republic of Korea 5

2Division of Environmental Science and Ecological Engineering, Korea University, 6

Seoul 136-701, Republic of Korea 7

3Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA 8

Running title: pEMB1 harboring a β-lactamase gene and a TA system 9

*Corresponding author: Che Ok Jeon. 10

Mailing address: Department of Life Science, Chung-Ang University, 84, HeukSeok-Ro, Dongjak-11

Gu, Seoul, 156-756, Republic of Korea. 12

Tel: +82-2-820-5864, Fax: +82-2-821-8132., E-mail: cojeon@cau.ac.kr 13

14

Key words: Plasmid pEMB1, Toxin-antitoxin system, Plasmid stability, antibiotic resistance, 15

ParD-ParE, Soil, Sewage sludge 16

AEM Accepts, published online ahead of print on 10 October 2014Appl. Environ. Microbiol. doi:10.1128/AEM.02691-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 17

Nonculture-based procedures were used to investigate plasmids showing ampicillin resistance 18

properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge 19

(Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples 20

was directly transformed into E. coli TOP10, and a single and three different plasmids were 21

obtained from the mountain soil and sludge samples, respectively. Interestingly, the RFLP pattern 22

of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of 23

one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid 24

pEMB1(8744 bp) showed the presence of six open reading frames (ORFs), including a β-25

lactamase gene. Using BlastX, the orf5 and orf6 genes were suggested to encode a CopG family 26

transcriptional regulator and a plasmid stabilization system, respectively. Functional 27

characterization of these genes using a knock-out orf5 plasmid (pEMB1parD) and the cloning 28

and expression of orf6 (pET21bparE) indicated that these genes were antitoxin (parD) and toxin 29

(parE) genes. Plasmid stability tests using pEMB1 and pEMB1parDE in E. coli revealed that 30

the orf5-orf6 genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-31

based survey, pEMB1-like plasmids were additionally detected in samples from other human-32

impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that 33

plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly 34

in the environment. This study extends knowledge about the dissemination and persistence of 35

antibiotic-resistance genes in naturally occurring microbial populations. 36

37

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INTRODUCTION 38

Antibiotic treatments have been one of the most effective ways to control infectious diseases, but 39

recently, the frequency of detecting bacteria that are resistant to antibiotics from environmental 40

samples has increased (1). It is generally thought that the use of antibiotics as human and 41

veterinary medicine, or as animal feed additives, is a major driving force in the development of 42

antibiotic-resistant bacteria and the dissemination of antibiotic resistance genes (2-4). Indeed, 43

high levels of antibiotic-resistance genes have been identified in a variety of human-impacted 44

milieus such as wastewater sludge, soils fertilized with manure, and river waters that have been 45

frequently exposed to antibiotics (5–9). Surprisingly, however, many other studies have shown 46

that the widespread occurrence of antibiotic resistance genes are sometimes unrelated to human 47

activities. For example, antibiotic-resistance genes have been documented in pristine habitats 48

such as Alaskan soil, Antarctic marine waters, ancient sediment samples, glacier ice cores, and 49

non-agricultural soil (2, 10–15) -often in abundances well above trace levels. These findings 50

raise questions about the stable maintenance of antibiotic resistance genes in the absence of 51

human-mediated selective pressures. It should also be noted that some antibiotic-resistance genes 52

may have functions alternative to neutralizing antibiotics (16, 17). 53

Antibiotic-resistance traits in bacteria can be acquired via lateral gene transfer (4, 13). In 54

particular, plasmids may play important roles as mobile genetic elements that can disseminate 55

antibiotic-resistance genes to clinically relevant pathogenic bacteria (18, 19). Surprisingly, some 56

recent studies have shown that antibiotic resistance genes encoded by plasmids were stably 57

maintained in the absence of selective pressures (20–22); however in these studies, the 58

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mechanisms of plasmid maintenance were not suggested. 59

Bacterial toxin-antitoxin (TA) systems have been identified in many pathogenic bacteria; TA 60

systems can stably maintain plasmids in their hosts via post-segregational killing of daughter 61

cells that fail to inherit the TA systems because antitoxin degradation occurs faster than toxin 62

degradation (23–27). In the present study, we discovered and analyzed a putative TA system 63

linked to a β-lactamase gene borne by a plasmid that was isolated from both wastewater sludge 64

and pristine mountain soil samples. Although β-lactamase, ampicillin-resistance plasmids have 65

been recovered from environmental samples (28–31), to our knowledge, none of these have been 66

fully sequenced and none have been associated with TA systems. We functionally characterized 67

the putative TA system and showed that it can play an important role in maintaining the plasmid 68

in its host in the absence of antibiotic-selective pressure. This study extends knowledge about the 69

dissemination and persistence of antibiotic-resistance genes in naturally occurring microbial 70

populations. 71

72

MATERIALS AND METHODS 73

Bacterial strains, plasmids and PCR primers. E. coli strains, plasmids, vectors, and PCR 74

primers used in this study are listed in Table 1. All E. coli strains were grown at 37°C in Luria-75

Bertani (LB) medium in a rotary incubator (180 rpm). 76

Isolation of plasmids harboring ampicillin resistance genes. To isolate plasmids harboring 77

ampicillin resistance genes from environmental samples, activated sludge and pristine mountain 78

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soil samples were collected from the Tancheon municipal wastewater treatment plant (WWTP) 79

(Seoul) and a remote area of Mt. Jeombong (38°02′17″N,128°27′40″E, altitude approximately 80

900–950 m, Gangwon-do) in Korea, respectively. Approximately 200-g soil was sampled from 81

the upper 1 to 5 cm of the mountain soil using a sterile spatula and aseptic plastic bags (Fisher 82

Scientific, USA) and transported immediately to the laboratory on ice. Total DNA from sludge 83

and soil samples was extracted using a FastDNA Spin kit (MP Biomedicals, USA) according to 84

the manufacturer’s instructions with a modification in the homogenization step: homogenization 85

for the cell lysis was performed by Mini-Beadbeater 3110BX (BioSpec, USA) for 30 sec at 4800 86

rpm. Approximately 250 ng DNA was directly transformed into 50 μl of competent E. coli 87

TOP10 cells (Invitrogen, USA) by the electroporation method using a Micropulser system (Bio-88

Rad, USA). The transformed suspension was spread onto LB agar containing ampicillin (50 89

μg/ml) and incubated at 37°C overnight. Colonies were randomly picked and cultivated 90

overnight in 5 ml of LB broth containing ampicillin. Plasmid DNA from each culture was 91

extracted using the Plasmid Mini Extraction kit (Bioneer, Korea) following the manufacturer’s 92

instructions. To evaluate the diversity of the plasmids, a restriction fragment length 93

polymorphism (RFLP) technique using Sau3AI was used. The digested plasmids were analyzed 94

on a 1.5% (w/v) agarose gel and their fragment patterns were compared. Plasmids showing the 95

same RFLP patterns were considered to be the same plasmids, and this was verified by partial 96

sequencing of the plasmids using BlaParD-OF and BlaParD-OR primers (Table 1): the primers 97

were designed after the complete sequencing of plasmid pEMB1. 98

Sequencing of plasmid DNA. To obtain complete sequences of the three plasmids, designated 99

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as pEMB1, pEMB2, and pEMB3, showing unique RFLP patterns, 25 µg of each purified 100

plasmid DNA in 50 µl TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) was mixed with 750 µl 101

shearing buffer (10% glycerol in TE buffer) and fragmented by mechanical shearing using a 102

Nebulizer (Invitrogen, USA) at 13 psi (nitrogen) for 2 min on ice to obtain a mean fragment size 103

of 1.5–2.0 kb. Each of the fragmented plasmids was precipitated by centrifugation after the 104

addition of one volume of ice-cold isopropanol and end-repaired using DNA End Repair Mix 105

(Invitrogen, USA) according to the manufacturer’s instructions. The DNA fragments of 106

approximately 1.5–2.0 kb in size were extracted using a gel purification kit (Bioneer, Korea) 107

following agarose gel electrophoresis, and ligated into the EcoRV site of pCR2.1-TOPO 108

(Invitrogen, USA). This was followed by electrotransformation into E. coli TOP10 to construct 109

clone libraries for each of the three plasmids. Plasmid DNA from 25 colonies of each clone 110

library were extracted, and then sequenced using the M13 reverse and T7 primers and a 3730xl 111

DNA Analyzer. The resulting sequences were assembled using SeqMan in DNAStar Lasergene 112

(DNASTAR, USA) and the assembled contigs were gap-closed using a primer walking approach. 113

Regions with poor sequencing qualities in each plasmid were resequenced, and finally, the 114

complete sequences for plasmids pEMB1, pEMB2, and pEMB3 were obtained. 115

Sequence analysis of plasmids. The origin of replication in plasmid pEMB1 was predicted by 116

Ori-Finder (http://tubic.tju.edu.cn/Ori-Finder/) (32). Putative open reading frames (ORFs) were 117

predicted using the web-based ORF Finder program at the National Center for Biotechnology 118

Information (NCBI) and potential protein-coding sequences were subsequently analyzed using 119

BlastX searches. Conserved domains and motifs of the putative proteins were searched using the 120

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Conserved Domain search service at NCBI (33). Multiple amino acid sequences were aligned 121

using ClustalX in DNAStar Lasergene. Restriction enzyme sites on plasmid pEMB1 were 122

identified using the NEB cutter Version 2.0 program 123

(http://tools.neb.com/NEBcutter2/index.php). Transcriptional promoters and termination 124

sequences in plasmid pEMB1 were predicted by using a web-based program 125

(http://www.softberry.com/; http://www.fruitfly.org/seq.tools/promoters.html). 126

Determination of minimum inhibitory concentration (MIC). MICs of some β-lactam 127

antibiotics including ampicillin, penicillin G, cefotaxime, meropenem, and aztreonam aganist E. 128

coli TOP10 harboring pEMB1 were determined using the serial twofold dilution method of 129

antibiotics based on the Clinical and Laboratory Standards Institute (CLSI) guidelines (34). 130

Briefly, overnight cultures of E. coli TOP10 harboring pEMB1 in LB broth containing ampicillin 131

(50 μg/ml) at 37°C were diluted to be 1 106 cells/ml in LB broth. The diluted cells of 100 μl 132

were dispensed into 96-well plates with 100 μl of LB broth containing twofold diluted antibiotics 133

(2–2048 μg/ml) and incubated at 37°C for 18 hrs. The growth was assayed using a microtiter 134

ELISA reader (SynergyMx; BioTek, USA) at 660 nm and the MICs were determined as the 135

lowest concentrations of antibiotics to inhibit the growth of the test strain. E. coli TOP10 strain 136

without pEMB1 was used as a negative control. 137

Construction of knock-out plasmids of orf5 and orf6 and cloning/expression of orf6. Orf5 138

and orf6, which were tentatively annotated as putative antitoxin (parD) and toxin (parE) genes in 139

plasmid pEMB1, respectively, were functionally characterized. To construct knock-out plasmids 140

of orf5 and orf6, pEMB1parD and pEMB1parE, plasmid pEMB1 was separately digested 141

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with MfeI and NcoI, single cutting enzymes that cleave orf5 and orf6 genes, respectively and 142

subsequently treated with mung bean nuclease (New England Biolabs, UK) to generate blunt 143

ends. Each blunt-ended plasmid DNA was self-ligated using T4 DNA ligase and transformed 144

into competent E. coli TOP10 cells by electroporation. The construction of pEMB1parD and 145

pEMB1parE with 4-bp deletion in orf5 and orf6, respectively, in transformed E. coli TOP10 146

cells was confirmed via PCR using the primer pair OparD-F and OparE-R (Table 1), and 147

sequencing. 148

To characterize the functional properties of orf6 as a putative toxin gene (parE), orf6 was cloned 149

into expression vector pET21b (Novagen, USA) as described previously (35). Briefly, orf6 was 150

PCR-amplified from plasmid pEMB1 using pfu DNA polymerase (Solgent, Korea) and the 151

primer pair ParE-F and ParE-R, which contained NdeI and BamHI restriction sites, respectively 152

(Table 1). The amplified PCR product was excised with NdeI and BamHI and ligated into the 153

NdeI and BamHI sites of pET21b. The resulting recombinant plasmid, designated as 154

pET21bparE, was transformed into E. coli BL21 (DE3) and the cloning of orf6 in transformed E. 155

coli BL21 (DE3) cells was confirmed using PCR and sequencing. The function of orf6 as a toxin 156

protein (ParE)-encoded gene was evaluated by the growth of E. coli BL21 (DE3) cells containing 157

pET21bparE on LB agar with or without induction using IPTG (isopropyl-β-D-158

thiogalactopyranoside; 1 mM) at 37°C after 2 days. Additionally, the growth of E. coli BL21 159

(DE3) harboring pET21bparE was also tested in LB broth with and without IPTG. One ml of 160

overnight cultures of E. coli BL21 (DE3) harboring pET21bparE in LB broth containing 161

ampicillin (50 μg/ml) at 37°C was inoculated into two flasks containing 100 ml of LB broth with 162

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ampicillin, and the flasks were incubated with shaking at 37°C. When the optical density (660 163

nm) of the cultures reached approximately 0.6, IPTG (1 mM) was added to one flask and the 164

cells were continually cultivated. The growth of E. coli BL21 (DE3) without pET21bparE was 165

tested in parallel in LB broth without ampicillin. The growth and viability of E. coli cells were 166

monitored by measuring optical density (660 nm) and colony forming unit (CFU). 167

Construction of a pEMB1 plasmid with orf5 and orf6 deleted and plasmid stability assays. 168

For the construction of pEMB1with the putative antitoxin (orf5) and toxin (orf6) genes deleted, 169

plasmid pEMB1 was digested with BsrGI and PvuII cutting upstream and downstream of orf5 170

and orf6, and the plasmid DNA ends were repaired using the Klenow treatment to generate blunt 171

ends. The blunt-ended plasmid DNA was ligated using T4 DNA ligase and transformed into E. 172

coli TOP10 by electroporation, resulting in pEMB1parDE. The deletion of orf5 and orf6 in 173

plasmid pEMB1 in transformed E. coli TOP10 cells was verified by PCR using the outer primer 174

pair OparD-F/OparE-R, and by sequencing (Table 1). 175

The stability of pEMB1 and pEMB1parDE was investigated in triplicate as previously 176

described, with some modifications (24). E. coli TOP10 cells containing pEMB1 and 177

pEMB1parDE were cultivated in LB broth containing ampicillin (50 µg/ml) overnight at 37°C 178

and 50 µl of the overnight-incubated cultures were used to inoculate 5 ml of fresh LB broth with 179

or without ampicillin. After 8 h-cultivation at 37°C, 1% of the cultures were used to inoculate 5 180

ml of fresh LB broth with or without ampicillin. This process was repeated until approximately 181

500 generations of growth were achieved. Periodically, culture broths were serially diluted in 0.9% 182

saline and plated onto LB agar without ampicillin for viable cell counts. Viable-count plates 183

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containing approximately 100 colonies were replica-plated onto LB agar plates with ampicillin. 184

The proportion of E. coli cells containing pEMB1 or pEMB1parDE plasmids was calculated 185

by comparing E. coli cell numbers grown on LB agar with and without ampicillin. The 186

maintenance or loss of pEMB1 or pEMB1parDE in E. coli after the stability assays was 187

confirmed by plasmid extraction from selected colonies. 188

PCR-based monitoring of pEMB1-like plasmids in additional habitats. A nested touchdown-189

PCR approach using PCR primer pairs targeting plasmid pEMB1 was used to monitor the 190

presence of pEMB1-like plasmids in samples aseptically gathered from various environments 191

described below. Two nested PCR primer pairs, BlaParD-OF/BlaParD-OR and BlaParD-192

IF/BlaParD-IR, were designed to target orf3 (bla), encoding β-lactamase, and orf5, encoding a 193

putative ParD in plasmid pEMB1 (Table 2). Besides the Tancheon WWTP sludge and Mt. 194

Jeombong soil samples, twenty additional environmental samples were obtained from various 195

habitats (four WWTPs, ten remote mountain soils, three tidal-flats, and three rice paddies) and 196

total DNA was extracted using a FastDNA Spin kit (MPbio, USA) according to the 197

manufacturer’s instructions. The first-round of touchdown-PCR was carried out using the outer 198

primer pair (BlaParD-OF/BlaParD-OR) with a cycling regime of 94°C for 5 min (1 cycle); 94°C 199

for 45 s, 60°C for 45 s, and 72°C for 2 min 30 s, followed by 20 cycles at decreasing annealing 200

temperature in decrements of 0.5°C per cycle, then 94°C for 45 s, 50°C for 45 s, and 72°C for 2 201

min 30 s (15 cycles) and 72°C for 10 min (1 cycle). The second-round of touchdown-PCR was 202

performed using the inner primer pair (BlaParD-IF/BlaParD-IR) and 1 µl of first-round PCR 203

products as with the first-round of touchdown-PCR. The PCR products were verified by direct 204

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sequencing of the second-round PCR products. 205

Nucleotide sequence accession number. The plasmid DNA sequences of pEMB1, pEMB2, and 206

pEMB3 have been deposited in GenBank under accession nos. KJ631729, KJ631730, and 207

KJ631731, respectively. 208

209

RESULTS 210

Isolation of plasmids harboring ampicillin resistance genes. To find environmental plasmids 211

conferring ampicillin resistance, total DNA extracted from activated sludge (Tancheon WWTP) 212

and remote mountain soil (Mt. Jeombong) samples was directly transformed into E. coli TOP10 213

cells. Restriction fragment length polymorphism (RFLP) analysis of plasmids recovered from 214

transformed ampicillin-resistant E. coli colonies revealed that only a single plasmid type was 215

obtained from the remote mountain soil sample (Fig. 1A), while plasmids with three different 216

RFLP patterns were obtained from the sludge sample (Fig 1B; plasmid #1, lanes 4 and 6; 217

plasmid #2, lanes 1, 2, 5, 8; and plasmid #3, lanes 3 and 7). Interestingly, the RFLP pattern of the 218

plasmids from the remote mountain soil was identical to that of plasmid #1 derived from the 219

activated sludge sample, and partial plasmid sequencing of approximately 2 kb revealed no 220

sequence differences between representatives of each (data not shown). We selected one plasmid 221

representative of plasmid RFLP patterns #1, #2, and #3 (Fig. 1) and designated the three 222

identified plasmids as pEMB1, pEMB2, and pEMB3, respectively. 223

Plasmid sequencing and sequence analysis. After sequencing, plasmids pEMB1, pEMB2, and 224

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pEMB3 were determined to be 8744 bp, 9042 bp, and 9344 bp in size, respectively. Sequence 225

analysis of the plasmids showed that all three plasmids contained a putative β-lactamase gene 226

that might mediate ampicillin resistance. Plasmids pEMB1 and pEMB3 were novel, but plasmid 227

pEMB2 was very similar to plasmid pQ7 of E. coli TB7 isolated from the urine sample of a 228

Swiss patient, with only 7 bp differences (36). Interestingly, the analysis of potential protein-229

coding sequences using BlastX searches showed that plasmid pEMB1, identified from both 230

wastewater sludge and remote mountain soil samples, harbored a putative TA system with the β-231

lactamase gene (Table 2). Therefore, in this study, we focused only on the characterization of 232

plasmid pEMB1. 233

The average G + C content of pEMB1 was 55.41%. Analysis of the plasmid nucleotide 234

sequences revealed six putative open reading frames (ORFs), all more than 78 amino acid 235

residues in length (Table 2). A putative oriC region with five possible E. coli DnaA box motifs 236

was identified in the pEMB1 plasmid, using the Ori-Finder program (Fig. 2). Orf1, orf2, and orf3 237

in plasmid pEMB1were predicted to encode transposase (tnpA), DNA invertase (tnpR), and β-238

lactamase (bla), respectively, which are three components of the Tn3 transposon. Moreover, a 239

Tn3 transposase DDE domain (Asp689-Asp765-Glu895; pfam01526) was found in the predicted 240

protein product of orf1 (tnpA), suggesting that plasmid pEMB1 might be a chimeric plasmid 241

derived from the easily transferable Tn3 transposon. The predicted protein product of orf3 (bla) 242

shared 82% sequence identity with the class A β-lactamase from Pseudomonas putida HB3267 243

(YP_007232230.1). The protein product of orf3 (bla) contained four specific elements of class A 244

β-lactamases, an STHK tetrad (positions 78 to 81), an SDN triad (positions 139 to 141), an 245

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EPELN pentad (positions 175 to 179), and a KTG triad (positions 243 to 245) (37), similar to 246

other reported class A β-lactamases with carbenicillin-hydrolyzing activity (Supplementary Fig. 247

1). The translated orf4 encoded an 80-amino acid protein that was most closely related to the 248

plasmid replication proteins (RepB) from Burkholderia caribensis MBA4 (ETY75818.1) and 249

Aeromonas salmonicida (WP_005321398.1), with 100% and 96% amino acid sequence identities, 250

respectively (Table 2). The predicted protein product of orf4 was therefore designated as RepB. 251

The predicted protein product of orf5 showed 92% and 79% identities to a hypothetical protein 252

PAAH01 (predicted as a transcriptional regulator) from Aeromonas hydrophila 253

(YP_005230994.1) and to the CopG family transcriptional regulator from Shewanella 254

putrefaciens 200 (4 YP_006010550.1), respectively. The translated orf5 encoded a 79-amino acid 255

protein that included a ribbon-helix-helix (RHH) structure from the RHH family of proteins, 256

which includes an antitoxin ParD and transcriptional repressors CopG, Arc, and Mnt (38), 257

although their sequence identities were very low (Fig. 3). The ORF5 protein also contained 258

specifically positioned hydrophobic residues found in the hydrophobic cores of folding motifs 259

and a highly conserved turn with a typical GXT/S pattern between two helices, as seen in the 260

RHH family of proteins. 261

The predicted protein of orf5 had a C-terminal tail region, which is a distinct feature of ParD and 262

Mnt among the RHH family proteins (Fig. 3) (39, 40). The predicted protein product of orf6 was 263

most closely related to the plasmid stabilization system protein (ParE) of Shewanella 264

putrefaciens 200 and Pectobacterium wasabiae WPP163, although their amino acid sequences 265

were evolutionarily distant from known plasmid stabilization system proteins (70% and 62% 266

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identities, respectively) (Table 2). Moreover, web-based conserved domain analysis based on the 267

Conserved Domain Database showed that the translated protein of orf6 contained a conserved 268

domain of ParE (COG3668), suggesting that orf6 in pEMB1 might be a parE toxin gene. It is 269

known that toxin genes of bacterial TA systems are expressed with an antitoxin gene in a single 270

gene cluster. It was predicted that a transcription promoter and a termination sequence were 271

present upstream and downstream of the orf5-orf6 genes, respectively, suggesting that the orf5 272

and orf6 genes in pEMB1 might be transcribed as a single operon (data not shown). These gene 273

analyses therefore suggested that orf5 that formed a single gene cluster with a putative toxin 274

gene (parE) may be an antitoxin gene (parD), rather than a CopG family transcriptional 275

regulator (Table 2). 276

Determination of MIC. MICs of five representative β-lactam antibiotics belonging to four β-277

lactam classes, penicillins (ampicillin and penicillin G), cephalosporins (cefotaxime), 278

carbapenems (meropenem), and monobactams (aztreonam), against E. coli TOP10 cells 279

harboring pEMB1 were determined to investigate the antibiotic resistance range of β-lactamase 280

in pEMB1. As shown in Table 3, MICs of ampicillin and penicillin G belonging to the penicillin 281

class were >512 and >1,024 μg/ml, respectively (Table 3), meaning that β-lactamase in pEMB1 282

can readily hydrolyze the β-lactam rings of ampicillin and penicillin G. However, the MIC test 283

showed that E. coli harboring pEMB1 was very sensitive to cefotaxime, meropenem, and 284

aztreonam (<1 μg/ml), suggesting that the β-lactam rings of antibiotics belonging to other β-285

lactam classes besides penicillins are resistant to the hydrolysis by β-lactamase in pEMB1. 286

Characterization of orf5 and orf6 as a TA system. To verify the function of orf5 and orf6 as 287

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antitoxin and toxin genes in plasmid pEMB1, two knock-out plasmids of orf5 and orf6, 288

pEMB1parD and pEMB1parE, were constructed, respectively. pEMB1parE was 289

successfully transformed into competent E. coli TOP10 cells by electroporation, while no 290

colonies were obtained in spite of repeated electroporation of pEMB1parD, which is 291

consistent with the hypothesis that these constitute a TA system (orf6 may be lethal in the 292

absence of orf5). In search of confirmation that orf6 functions as a toxin gene, the putative toxin 293

(orf6) gene was cloned into pET21b (pET21bparE). When the growth of recombinant E. coli 294

BL21 (DE3) cells harboring pET21bparE was tested on LB agar with or without IPTG, they 295

grew well on LB agar without IPTG, while they did not grow on LB agar with IPTG (data not 296

shown). The growth of E. coli cells harboring pET21bparE was also tested in LB broth (Fig. 4). 297

Recombinant E. coli BL21 (DE3) cells harboring pET21bparE grew well in LB broth without 298

IPTG as host E. coli BL21 (DE3) cells did, but their cell counts decreased rapidly by the addition 299

of IPTG (Fig. 4B). These results indicated that E. coli cells were killed by the expression of orf6 300

gene and is fully consistent with its role as a toxin gene in a TA system. 301

Stability assay of plasmids pEMB1 and pEMB1parDE. To investigate the stability 302

enhancement of plasmid pEMB1 by orf5 and orf6 as a TA system, a pEMB1 plasmid with orf5 303

and orf6 deleted (pEMB1parDE) was constructed and the stabilities of plasmids pEMB1 and 304

pEMB1parDE were compared by repeated cultivation of E. coli cells harboring pEMB1 and 305

pEMB1parDE in LB with or without ampicillin. Plasmid pEMB1 in E. coli was found to be 306

very stable even in the absence of ampicillin, surviving for more than 500 bacterial generations 307

In contrast, plasmid pEMB1parDE in E. coli in the absence of ampicillin was rapidly lost after 308

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250 bacterial generations (Fig. 5). 309

Monitoring of pEMB1-like plasmids in the additional habitats. Besides Mt. Jeombong soil 310

and Tancheon wastewater treatment plant (WWTP) sludge, the presence of pEMB1-like 311

plasmids was investigated in samples from twenty other environments. Among these, pEMB1-312

like plasmids harboring a β-lactamase gene and a toxin (ParD)-antitoxin (ParE) system were 313

identified in two other human-influenced sites (sludge samples) and two other remote mountain 314

soil samples (Supplementary Fig. S2). 315

316

DISCUSSION 317

The global dissemination of antibiotic resistance genes is related to the selective pressure 318

generated by the use of antibiotics in clinical and veterinary practices, and in animal feeds. This 319

is supported by many studies showing correlations between the dissemination and concentrations 320

of antibiotic resistance genes and the use of antibiotics (2, 8, 9). It is generally accepted that 321

bacteria readily lose their antibiotic resistance genes in the absence of selective pressures 322

because, in most cases, antibiotic resistance is associated with reduced bacterial fitness due to a 323

decrease in growth rate (41, 42). However, some studies have shown that antibiotic resistance 324

genes do not disappear from bacterial populations even after the antibiotic is no longer used (3, 325

43–46). To date, it is not known why antibiotic resistance genes in bacteria are so stable in the 326

absence of antibiotics (47). It has been proposed that bacteria harboring antibiotic resistance 327

genes may be more competitive in microbial communities because bacteria can be exposed to 328

antibiotics intermittently in nature, or antibiotic resistance genes may play roles other than in 329

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antibiotic resistance in natural ecosystems (3, 41, 48). This suggests that bacteria may harbor 330

maintenance mechanisms for antibiotic resistance genes in the absence of antibiotic selective 331

pressure. 332

In this study, a nonculture-based strategy was used to recover a variety of plasmids 333

harboring ampicillin resistance genes in two quite distinct habitats, Tancheon WWTP and Mt. 334

Jeombong. Tancheon WWTP receives sewage from private households, hospitals, and animal 335

facilities, and previous research detected antibiotic residues at relatively high concentrations (49), 336

which suggests that antibiotic resistance genes may be abundant in the Tancheon WWTP sludge. 337

Congruent with this, three different plasmids harboring ampicillin resistance genes were 338

identified from the Tancheon WWTP sludge sample (Fig. 1B), whereas only one kind of plasmid 339

showing ampicillin resistance was identified from the remote Mt. Jeombong soil, where medical 340

antibiotic exposure has been likely absent or minimal. Interestingly, the plasmid (pEMB1) from 341

the remote Mt. Jeombong soil was indistinguishable from one of the three plasmids retrieved 342

from the Tancheon WWTP sludge sample; pEMB1 harbored an ampicillin-resistance gene as 343

well as a TA system. Typically, TA systems are identified by the primary sequence of toxin genes 344

because toxin genes are relatively well conserved, whereas antitoxin genes are phylogenetically 345

diverse (50). In plasmid pEMB1, the predicted protein product of orf6 was annotated as a 346

plasmid stabilization system protein (ParE), while the translated protein of orf5 was annotated as 347

a putative CopG family transcriptional regulator from its sequence (Table 2). Based on the 348

annotation of orf6 as a toxin gene, we hypothesized that orf5 may function as an antitoxin (ParD) 349

gene within a TA system. The impact of orf5 (Fig. 3) on plasmid retention was experimentally 350

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characterized (Fig. 5). It is known that ParE inhibits DNA gyrase and thereby blocks 351

chromosomal DNA replication, whereas ParD exhibits inhibition or the reversal of the toxic 352

activity of ParE and regulation of the parD-parE cluster (51, 52). In the future, genetic and 353

biochemical investigations must be completed to verify that the protein products of orf5 and orf6 354

on pEMB1function as a ParD-ParE TA system. 355

ParD-ParE toxin-antitoxin systems are commonly found in bacterial plasmids and 356

chromosomes of Gammaproteobacteria and Alphaproteobacteria (53–55) and plasmids 357

harboring both TA systems and antibiotic resistance genes are widely distributed in enterococci 358

(24, 56). However, to the best of our knowledge, pEMB1 is the first plasmid harboring a ParD-359

ParE TA system and an ampicillin resistance gene identified from human-influenced (wastewater 360

sludge) and pristine (remote mountain soil) environments. Moreover, PCR-based monitoring 361

showed that pEMB1-like plasmids harboring a ParD-ParE TA system occurred more broadly in 362

human-influenced, as well as remote, environmental samples (Supplementary Figure S2). As 363

shown in Fig. 5, even in the absence of ampicillin, there was no detectable plasmid loss from 364

transformed E. coli cells over 500 generations of bacterial growth, which suggests that the ParD-365

ParE TA system may play an important role in maintaining plasmids stably in hosts in habitats 366

without antibiotic selective pressures. This study, therefore, adds to understanding of the 367

development of antibiotic-resistant bacteria, and the dissemination and persistence of antibiotic-368

resistance genes in the environment. 369

370

ACKNOWLEDGEMENTS 371

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These efforts were supported by the "National Research Foundation of Korea (No. 372

2013R1A2A2A07068946)" grant funded by the Korean Government (MEST), Republic of 373

Korea. ELM was supported by NSF grant #DEB-0841999. 374

375

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528

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Figure legends 529

FIG. 1. RFLP analysis of plasmids from environmental samples conferring antibiotic resistance 530

to E. coli. Plasmids derived from remote mountain soil (Mt. Jeombong, A) and sludge (Tancheon 531

WWTP, B) samples were digested with Sau3AI. The RFLP analysis showed that the fragment 532

patterns of plasmids in panel A were identical with those of plasmids B4 and B6. Plasmids A1, 533

B1, and B3 were designated as pEMB1, pEMB2, and pEMB3, respectively. M, 100-bp ladder 534

(Bioneer, Korea). 535

536

FIG. 2. Genetic map of plasmid pEMB1 showing open reading frames (ORFs), putative 537

functions (in parentheses), and oriC as described in the text. Restriction enzyme sites used for 538

genetic manipulation of this study are also indicated on the map. 539

540

Fig. 3. Multiple alignment of the predicted protein product of orf5 in pEMB1 with other ribbon-541

helix-helix proteins. Conserved hydrophobic residues are shaded in light grey and the highly 542

conserved turns between helices A and B are shaded in dark grey. ParD, Pseudomonas 543

aeruginosa (YP_758694.1); CopG, Streptococcus agalactiae (2CPG_A); Arc, Enterobacteria 544

phage P22 (1PAR_A); Mnt, Enterobacteria phage P22 (1MNT_A). 545

546

Fig. 4. Overexpression of the toxin (parE) gene of pEMB1 in E. coli. Growth and viability of 547

recombinant E. coli BL21 (DE3) harboring pET21bparE in LB broth with or without IPTG were 548

monitored by optical density every 40 min (A) and colony forming unit (CFU) every 80 min (B). 549

Host E. coli BL21 (DE3) without pET21bparE was also tested in parallel. Arrows indicate the 550

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time with the addition of IPTG (1 mM). 551

552

Fig. 5. Plasmid stability assay. Plasmid stability was determined by replica plating onto LB agar 553

with or without ampicillin (amp) after repeated cultivation of E. coli cells harboring pEMB1 and 554

pEMB1parDE in LB broth with or without ampicillin. Error bars represent standard deviations 555

from the mean values. 556

557

558

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Table 1. Bacterial strains, plasmids, and PCR primers used in this study* 559

Names of strains, plasmids, and primers

Description or sequence Source or reference(s)

E. coli

TOP10 F-mcrAΔ (mrr-hsdRMs-mcrBC) Invitrogen

BL21(DE3) F- dcm ompT hsdS (rB- mB

-) gal λ (DE3) Novagen

Plasmids or vectors

pEMB1 Carries Apr and a putative parD (antitoxin) and parE (toxin) system This study

pEMB1parD

pEMB1parDE

Carries 4 bp deletion of putative parD in pEMB1

pEMB1 with deletion of putative parD and parE by BsrGI-pvuII-cut

This study

This study

pCR2.1-TOPO TA cloning vector; Kmr Apr Invitrogen

pET21b Expression vector; Apr, T7 tag, multi cloning site, His tag Novagen

pET21bparE NdeI-BamHI-cut PCR fragment PCR product of putative parE inserted into pET21b

This study

PCR primers

ParE-F 5′-AAA CAT ATG CAA GTT AAG TGG CTG CG-3′ (7103–7122) This study, NdeI

ParE-R 5′-CTG GGA TCC ATG TCA CCA GTG GCT TGG-3′ (7373–7390) This study, BamHI

OparD-F 5′-CCT GCA GAA GTG ATG TTT GC-3′ (6496–6515) This study

OparE-R 5′-GCA CAT CAA TCT GAC GGG TG-3′ (77–96) This study

BlaParD-OF 5′-CAT CGC AAA AAT TGG CAC TGC-3′ (4699–4719) This study

BlaParD-OR 5′-AAT CAG CCT CGA TCA ATG CC-3′ (7026–7045) This study

BlaParD-IF 5′-AGC TGA CGC AGT GGA TGC T-3′ (4488–4506) This study

BlaParD-IR 5′-CAA CCA TGA TTT GGT TCT GC-3′ (6944–6963) This study

* Nomenclature: O, outer; I, inner; F, forward; R, reverse. Kmr, kanamycin resistance; Apr, ampicillin resistance. Restriction enzyme sites are 560 underlined. Numbers in parentheses indicate the positions of primer sequences in plasmid pEMB1 561

562

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Table 2. Open reading frames found in plasmid EMB1and their putative protein functions 564

Name Position Putative function Protein size (aa)

Identity (aa)

Best BlastX match (species, accession no.)

orf1 1–2976 (-) Transposase (TnpA) 991 91%

91%

Transposase (Comamonas sp., WP_003056098.1)

Transposase Tn3 family (Providencia rettgeri, YP_008691690.1)

orf2 3273–3830 (+) DNA invertase/

resolvase (TnpR)

185 96%

92%

DNA invertase (Klebsiella pneumoniae, WP_017901435.1)

DNA invertase (Delftia acidovorans, NP_904293.1)

orf3 3843–4742 (+) β-lactamase (Bla) 299 82%

77%

Class A β-lactamase (Pseudomonas putida, YP_007232230.1)

Beta-lactamase AER-1 (Aeromonas hydrophila, Q44056.2)

orf4 5731–5973 (+) Plasmid replication

protein (RepB)

80 100%

96%

Plasmid replication protein RepB (Burkholderia caribensis, ETY75818.1)

Plasmid replication protein RepB (Aeromonas salmonicida, WP_005321398.1)

orf5 6872–7111 (+) Antitoxin (ParD) 79 92%

79%

Hypothetical protein (Aeromonas hydrophila, YP_005230994.1)

CopG family transcriptional regulator (Shewanella putrefaciens, YP_006010550.1)

orf6 7101–7385 (+) Toxin (ParE) 94 70%

62%

Plasmid stabilization system (Shewanella putrefaciens, YP_006010549.1)

Plasmid stabilization system (Pectobacterium wasabiae, YP_003258368.1)

565

566

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Table 3. MICs of various β-lactam antibiotics against E. coli TOP10 with and without 568 pEMB1 569

Antibiotics

MIC (μg/ml)

TOP10 TOP10

with pEMB1

Ampicillin <4 > 512

Penicillin G <8 >1,024

Cefotaxime <1 <1

Meropenem <1 <1

Aztreonam <1 <1

570

571

572

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Figure 1 574

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579

Figure 2 580

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584

Figure 3 585

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Figure 4 590

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Figure 5 595

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