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1 Submitted to Applied and Environmental Microbiology 1 2 Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile 3 derived from Geobacillus kaustophilus HTA426 4 5 Jyumpei Kobayashi, 1,2 Misaki Tanabiki, 1 Shohei Doi, 1 Akihiko Kondo, 3 Takashi Ohshiro, 1 6 and Hirokazu Suzuki 1,2,# 7 8 1 Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori 9 University, Tottori 680-8552, Japan 10 2 Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu 11 University, Fukuoka 812-8581, Japan 12 3 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe 13 University, Kobe, Hyogo 657-8501, Japan 14 15 Present address: Jyumpei Kobayashi, Organization of Advanced Science and Technology, Kobe 16 University, Hyogo 657-8501, Japan 17 18 19 20 Running title: pUC replicon mutagenesis in G. kaustophilus 21 22 # Correspondence to: Hirokazu Suzuki; Department of Chemistry and Biotechnology, Graduate 23 School of Engineering, Tottori University, Tottori 680-8552, Japan 24 E-mail: [email protected]; Tel.: +81 857 31 5907; Fax: +81 857 31 5907 25 AEM Accepted Manuscript Posted Online 28 August 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01574-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved. on February 15, 2018 by guest http://aem.asm.org/ Downloaded from

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Submitted to Applied and Environmental Microbiology 1

2

Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile 3

derived from Geobacillus kaustophilus HTA426 4

5

Jyumpei Kobayashi,1,2 Misaki Tanabiki,1 Shohei Doi,1 Akihiko Kondo,3 Takashi Ohshiro,1 6

and Hirokazu Suzuki1,2,# 7

8 1Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori 9

University, Tottori 680-8552, Japan 10 2Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu 11

University, Fukuoka 812-8581, Japan 12 3Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe 13

University, Kobe, Hyogo 657-8501, Japan 14

15

Present address: Jyumpei Kobayashi, Organization of Advanced Science and Technology, Kobe 16

University, Hyogo 657-8501, Japan 17

18

19

20

Running title: pUC replicon mutagenesis in G. kaustophilus 21

22 #Correspondence to: Hirokazu Suzuki; Department of Chemistry and Biotechnology, Graduate 23

School of Engineering, Tottori University, Tottori 680-8552, Japan 24

E-mail: [email protected]; Tel.: +81 857 31 5907; Fax: +81 857 31 5907 25

AEM Accepted Manuscript Posted Online 28 August 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01574-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 26

The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding 27

thermostable chloramphenicol acetyltransferase (CATA138T), serves as an Escherichia 28

coli–Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance 29

on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed 30

mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid 31

pGKE75αβ-catA138T responsible for substantial chloramphenicol resistance at 65°C. 32

pGKE75αβ-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC 33

replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical 34

characterization suggested that the efficient chloramphenicol resistance conferred by 35

pGKE75αβ-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A 36

following a decrease in incomplete forms of pGKE75αβ-catA138T. The decrease in incomplete 37

plasmids may be due to optimization of plasmid replication by RNA species transcribed from the 38

mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. 39

kaustophilus was transformed with pGKE75αβ-catA138T using chloramphenicol selection at 60°C. 40

In addition, a pUC18 derivative with the two mutations propagated in E. coli with high copy 41

number independent of culture temperature and high plasmid stability. Since these properties 42

have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. 43

kaustophilus and E. coli. 44

45

Keywords: Geobacillus kaustophilus, chloramphenicol resistance, pUC replicon, 46

thermoadaptation-directed evolution, error-prone thermophile 47

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Introduction 48

ColE1-type plasmids, such as pBR322 and pUC, replicate in Escherichia coli autonomously with 49

substantial copy numbers and are extensively utilized in microbial genetic engineering. As 50

shown by pBR322 (Fig. 1), the replicon of ColE1-type plasmids generally contains genes for a 51

precursor of RNA primer (RNA II), a replication regulatory RNA (RNA I), and a replication 52

regulatory protein (Rom). The precursor RNA II is transcribed from 555 bp upstream of the 53

replication origin and adopts a stem-loop structure that forms a persistent hybrid at the origin. 54

This structure is subsequently cleaved by RNase H to serve as a primer for plasmid replication 55

(1). RNA I consists of 108 nucleotides transcribed from 445 bp upstream of the origin to near the 56

initiation site for RNA II synthesis (2–4). Because RNA I synthesis proceeds in the direction 57

opposite to that of RNA II synthesis, RNA I can hybridize to RNA II and trigger conformational 58

changes in RNA II. This event, which prevents RNA II from hybridization at the replication 59

origin, inhibits plasmid replication and decreases plasmid copy number. Plasmid replication is 60

also negatively regulated by the Rom protein (2, 5–8), which facilitates the initial interaction 61

between RNA I and RNA II. Thus, the copy number of ColE1-type plasmids is under the 62

tripartite control of RNA I, RNA II, and Rom protein. 63

Genetic alterations in or near the replicon are known to affect the copy number of 64

ColE1-type plasmids (9–12). Although the pUC plasmids are pBR322 derivatives, their replicons 65

lack rom gene and have a point mutation in the RNA II gene (Fig. 1). Consequently, pUC 66

plasmids are present in higher copy numbers than pBR322 (9). Both of the alterations are 67

essential for pUC plasmids to achieve high copy numbers; pBR322 derivatives that have either 68

the rom deletion or the point mutation show copy numbers comparable to that of pBR322 (9). It 69

is known that the copy numbers of pUC plasmids change depending on the culture temperature 70

of the host E. coli cells. This appears to arise from a temperature-dependent secondary structure 71

within RNA II (9). 72

Geobacillus kaustophilus HTA426 is an aerobic, Gram-positive, Bacillus-related 73

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thermophile that had been isolated from the deep-sea sediments of the Mariana Trench (13, 14). 74

We have studied this strain as a model for Geobacillus spp., because basal genetic tools (15–18) 75

and a whole genome sequence (19) are available for this strain. In a previous study (20), we 76

constructed G. kaustophilus MK480 from the HTA426 strain by inactivating DNA repair genes 77

and demonstrated that MK480, an error-prone thermophile that exhibits frequent mutations in 78

vivo, can generate mutant genes encoding more thermostable variants than the parent enzymes 79

during periods of cell growth at high temperatures. This approach, thermoadaptation-directed 80

evolution, was further employed on the plasmid pGKE75 carrying the cat gene (pGKE75-cat; 81

Fig. 2A) (21). The cat gene encodes the chloramphenicol acetyltransferase from Staphylococcus 82

aureus (CAT), which confers chloramphenicol (Cm) resistance on host bacteria (22). pGKE75 is 83

an E. coli–G. kaustophilus shuttle plasmid that includes the pUC18 plasmid (Fig. 2B). The 84

thermoadaptation-directed evolution was performed by successive propagation of pGKE75-cat 85

in MK480 cells under growth inhibition by Cm pressure, resulting in the generation of a mutant 86

plasmid, pGKE75-catA138T, which encodes a CAT variant (CATA138T) having enhanced 87

thermostability due to an A138T amino acid replacement (21). 88

Antibiotic resistance genes are commonly used as selectable markers during complex 89

genetic modifications of microbes. However, a single kanamycin resistance gene has been the 90

only antibiotic resistance marker used in G. kaustophilus HTA426 (15–18, 20). Although cat and 91

catA138T genes may serve as selectable markers, neither pGKE75-cat nor pGKE75-catA138T 92

conferred Cm resistance on G. kaustophilus at temperatures higher than 65°C (21). Therefore, 93

this study was designed to generate catA138T mutants that function at higher temperatures via the 94

further thermoadaptation-directed evolution of pGKE75-catA138T. Here we report a new plasmid 95

that confers Cm resistance at 65°C, which was generated via unexpected mutations in the pUC 96

replicon of pGKE75-catA138T. 97

98

Materials and methods 99

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Bacterial strains and media 100

G. kaustophilus strains MK242 and MK480 were previously constructed (20). Strain MK480 101

was used as an error-prone thermophile for thermoadaptation-directed evolution, and MK242 102

was used for genetic characterization because of its genetic stability. These strains were 103

essentially grown at 60°C in Luria–Bertani (LB) medium, but G. kaustophilus [pGKE75 104

derivative], where square brackets denote the plasmid-carrier state, was grown in LB medium 105

supplemented with 5 mg l−1 of kanamycin (LK5). E. coli DH5α (Takara Bio, Otsu, Japan) was 106

used for DNA manipulation. E. coli DH5α [pUC18 or pGKE75-cat derivative] and E. coli DH5α 107

[pUB307] were grown at 37°C in LB medium supplemented with ampicillin (50 mg l−1) and 108

kanamycin (25 mg l−1), respectively. 109

110

Plasmids and primers 111

Table 1 lists plasmids used, and Fig. 2 shows their structures. Primers are summarized in Table 2. 112

pGKE75-cat and pGKE75-catA138T were previously constructed (21). pUC18αβ was constructed 113

from pUC18 (Takara Bio) by site-directed mutagenesis as follows. The pUC18 replicon was 114

amplified using primers colEF1 and colER2. PCR was performed using PrimeSTAR HS DNA 115

polymerase (Takara Bio) in accordance with the manufacturer’s protocol. Using the two strands 116

of the resulting fragment (458 bp) as mutagenesis primers, pUC18 was replicated in vitro in a 117

mixture (25 µl) that contained the fragment (0.2 µg), pUC18 (0.1 µg), dNTPs (0.4 mM each), 118

1×buffer, and PrimeSTAR HS DNA polymerase (1.2 units). The reaction was performed by 17 119

cycles of 98°C for 10 s, 55°C for 10 s, and 72°C for 8.5 min. The template pUC18 was digested 120

with DpnI. Subsequently, the resulting DNA was propagated in E. coli to obtain pUC18αβ. This 121

approach was also used to construct the plasmids pUC18α, pUC18β, pUC18γ, pGKE75α-catA138T, 122

and pGKE75β-catA138T. pUC18α, pUC18β, and pUC18γ were generated from pUC18 using 123

mutagenesis primers colEF1 and colER1, colEF2 and colER2, and colEF3 and colER3, 124

respectively. pGKE75α-catA138T and pGKE75β-catA138T were generated from pGKE75-catA138T 125

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using primers colEF1 and colER1, and colEF2 and colER2, respectively. 126

127

Plasmid introduction into G. kaustophilus 128

pGKE75-cat derivatives were introduced into G. kaustophilus using conjugative DNA transfer 129

from E. coli DH5α (16). G. kaustophilus was used as the DNA recipient. E. coli [pGKE75-cat 130

derivative] and E. coli [pUB307] were used as the DNA donor and conjugation helper, 131

respectively. These strains were cultured until the optical density at 600 nm (OD600) reached 0.3. 132

Cultures were then mixed and spotted on LB plates, followed by incubation at 37°C to achieve 133

conjugation. In this process, E. coli [pGKE75-cat derivative] transfers the plasmid to G. 134

kaustophilus with the mediation of E. coli [pUB307]. Subsequently, cell mixture was recovered 135

from LB plates and incubated at 60°C on LK5 plates to isolate G. kaustophilus transformants. 136

137

Thermoadaptation-directed evolution of pGKE75-catA138T 138

LK5 plates containing Cm (10 mg l−1) were used as solid media throughout this section. G. 139

kaustophilus MK480 [pGKE75-catA138T] was cultured on solid media at 60°C for 24 h. The 140

resulting cells were collected and incubated for 1 h at 30°C in the presence of 20 mM hydrogen 141

peroxide to induce mutations (cell survival rate, 1%). The surviving cells (104 cells) were 142

regrown on solid media at 60°C. This process was repeated three more times. Subsequently, cells 143

were cultivated on solid media at 60°C followed by cell collection. This process was 144

successively performed at 65°C and 70°C. Plasmid mixtures were then extracted from the 145

resultant cells and reintroduced into G. kaustophilus MK242. Transformants were incubated on 146

solid media at 65°C to obtain clones obviously resistant to Cm. pGKE75-catA138T derivatives 147

were isolated from these clones and subjected to DNA sequence analysis. 148

149

Cm resistance assay 150

G. kaustophilus MK242 [pGKE75-cat derivative] was precultured in liquid LK5. Equal volume 151

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aliquots of culture were incubated at various temperatures for 24 h on LK5 plates with or without 152

Cm (5 mg l−1). The resulting colonies were counted to calculate the Cm resistance efficiency, 153

which is the ratio of Cm-resistant colonies (grown on LK5 with Cm) to the total number of 154

colonies (grown on LK5 without Cm). The efficiency was determined from four independent 155

experiments and is presented as the mean ± standard deviation (SD). 156

157

CAT activity assay 158

G. kaustophilus MK242 [pGKE75-cat derivative] was cultured at 65°C for 18 h on LK5 plates. 159

Cells were collected and suspended in buffer (50 mM potassium phosphate, pH 7.0). The 160

suspension was homogenized by sonication and then centrifuged to remove cell debris. The 161

supernatant (cell extract) was used for further analysis. The protein concentration in cell extracts 162

was analyzed using the Bradford method. CAT activity was assayed at 60°C according to an 163

established method (21). One unit was defined as the amount of enzyme that produces 1 µmol of 164

coenzyme A (CoA) per min. 165

166

Acetyl-CoA assay 167

The concentration of acetyl-CoA in cell extracts (see above) was determined using the PicoProbe 168

Acetyl-CoA Fluorometric Assay Kit (BioVision Inc., CA, USA). Data were normalized by the 169

protein concentration of cell extracts. 170

171

Assay of plasmid copy number 172

G. kaustophilus MK242 [pGKE75-cat derivative] was cultured on LK5 plates at 65°C for 18 h. E. 173

coli DH5α [pUC18 derivative] was cultured in liquid LB at 20–42°C until OD600 reached 1.0. 174

Total DNA was extracted from cells using the method of Wu and Welker (23). Plasmid copy 175

numbers of pGKE75-cat and pUC18 derivatives in total DNA were analyzed using the 176

quantitative competitive-PCR (QC-PCR) (24). The plasmid concentration was determined using 177

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primers blaF and blaR, which amplify a portion of the bla gene (positions in the open reading 178

frame, 99–612; 514 bp). Artificial short fragments that consisted of positions 99–251 and 179

459–612 of the bla gene were used as competitor DNA. The chromosome concentration was 180

determined using primers to amplify a portion of the rpoA gene. Primers GKrpoAF and 181

GKrpoAR were used to amplify G. kaustophilus rpoA (positions 101–620; 520 bp). E. coli rpoA 182

(positions 87–613; 527 bp) was amplified using ECrpoAF and ECrpoA. Competitor DNA 183

consisted of positions 101–240 and 440–620 of G. kaustophilus rpoA and positions 87–249 and 184

445–613 of E. coli rpoA. QC-PCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, 185

Japan) in the presence of competitor DNA (1.0 pM–54 nM) by 30 cycles of 94°C for 30 s, 55°C 186

for 30 s, and 68°C for 1 min. The products were separated by agarose gel electrophoresis and 187

visualized using ethidium bromide. DNA bands were quantified using the ImageJ program 188

(http://rsb.info.nih.gov/ij). These data were used to calculate the ratio of bla to rpoA copies in 189

total DNA, which was defined as the plasmid copy number. Data are presented as the mean ± SD 190

(n = 3–4). 191

192

Transcriptional analysis of the mutant pUC replicon 193

G. kaustophilus MK242 [pGKE75αβ-catA138T] was cultured on LK5 plates at 65°C for 18 h. Total 194

RNA was prepared from resulting cells using an RNeasy Mini Kit with RNAprotect Bacteria 195

Reagent (Qiagen, Venlo, Netherlands). The RNA obtained from this procedure was treated with 196

gDNA Eraser (Takara Bio) to eliminate genomic DNA and then used for two-step reverse 197

transcription-PCR (RT-PCR) to detect transcripts from the mutant pUC replicon. Reverse 198

transcription was performed using the PrimeScript RT reagent Kit (Takara Bio) with primer 199

repF1 or repR2. PCR was performed using Quick Taq HS DyeMix by 30 cycles of 94°C for 30 s, 200

55°C for 30 s, and 68°C for 1 min. Primers repF1 and repR1 were used to detect transcripts from 201

between −559 and +1), and repF1 and repR2 were used for those between −559 and +205. 202

203

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Plasmid stability assay of pUC18 derivatives 204

E. coli [pUC18 derivative] was precultured in liquid LB supplemented with ampicillin. An 205

aliquot of culture (103 cells) were then cultured at 20–42°C in liquid LB without antibiotics until 206

the culture reached stationary phase. This culture was successively repeated for two more times. 207

The resulting cells were grown at 37°C on LB plates and then 100 colonies were screened for 208

ampicillin resistance to calculate the plasmid retention rate. Data are presented as the mean ± SD 209

(n = 3). 210

211

Plasmid transformation of G. kaustophilus using Cm selection 212

pGKE75-cat derivatives were introduced into G. kaustophilus by conjugative DNA transfer (see 213

above). E. coli donor and conjugation helper were cultured in 10 ml of LB medium; G. 214

kaustophilus MK242 was cultured in 100 ml. These cultures were mixed and incubated on LB 215

plates at 37°C for 18 h, followed by incubation at 60°C for 1 h. The resulting cells were collected 216

in LB medium, and equal volume aliquots (G. kaustophilus cells, 107–108) were incubated at 217

60°C for 24 h on LB plates supplemented with Cm (10 mg l−1) and LK5 plates. Colonies were 218

counted to calculate the selection efficiency, which is the ratio of the number of colonies grown 219

using Cm selection (grown on LB plates with Cm) to the number of colonies grown using 220

kanamycin selection (grown on LK5 plates). Data are presented as the mean ± SD (n = 5). 221

222

Results 223

Generation of pGKE75αβ-catA138T 224

We first examined thermoadaptation-directed evolution of pGKE75-catA138T by simple successive 225

cultures of G. kaustophilus MK480 [pGKE75-catA138T] under Cm selection pressure. While the 226

strain could intrinsically grow at 65°C with tiny colonies only when spread at high cell density, 227

this approach provided no colonies that were more Cm resistant than the parent cells (population 228

rate, <0.01%). The result led us to treat cells with hydrogen peroxide between successive cultures 229

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for more frequent mutations. This approach produced many colonies that showed substantial Cm 230

resistance at 70°C following 7 successive cultures (population rate, approximately 1%). 231

pGKE75-catA138T was extracted from these colonies in a mixture and reintroduced into G. 232

kaustophilus MK242 to eliminate false positives (21, 25, 26). Using this process, we identified 233

four clones that showed obvious Cm resistance at 65°C in dozens of clones. The 234

pGKE75-catA138T derivatives were isolated from these clones, and their sequences in the 235

Pgk704-catA138T region were determined. Since none of the four sequences contained mutations, we 236

further analyzed the entire sequence of the plasmid that conferred most efficient Cm resistance 237

on G. kaustophilus. The mutant plasmid, designated as pGKE75αβ-catA138T, contained two 238

mutations in the pUC replicon: a C·G→A·T transversion and a C·G→T·A transition (Fig. 2). 239

The C·G→T·A transition was presumably due to a spontaneous mutation in MK480 because this 240

type of mutation is intrinsically frequent in G. kaustophilus (20). Meanwhile, the C·G→A·T 241

transversion was attributable to hydrogen peroxide because it facilitates generation of the 242

aberrant base 7,8-dihydro-8-oxoguanine (27), which causes C·G→A·T transversions (28, 29). 243

244

Cm resistance conferred by pGKE75-cat derivatives 245

G. kaustophilus MK242 [pGKE75-cat] showed efficient Cm resistance at 50°C and MK242 246

[pGKE75-catA138T] showed at 55°C; however, MK242 [pGKE75αβ-catA138T] showed substantial 247

Cm resistance at 60–65°C (Table 3). The pGKE75-catA138T derivatives with either of the two 248

mutations, pGKE75α-catA138T and pGKE75β-catA138T, conferred moderate Cm resistance at 65°C 249

(Fig. 3A). The observation suggests that both mutations in the mutant pUC replicon participate in 250

the process by which pGKE75αβ-catA138T confers efficient Cm resistance. 251

252

CAT activity in G. kaustophilus 253

To examine the reasons for efficient Cm resistance conferred by pGKE75αβ-catA138T, we analyzed 254

the intracellular CAT activity in G. kaustophilus MK242 [pGKE75-cat derivative] (Fig. 3B). 255

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MK242 [pGKE75αβ-catA138T] had higher CAT activity than MK242 [pGKE75-cat or 256

pGKE75-catA138T]. pGKE75α-catA138T and pGKE75β-catA138T caused activity between that of 257

pGKE75αβ-catA138T and those of pGKE75-cat and pGKE75-catA138T. Because the specific 258

activities of the CAT and CATA138T proteins were comparable (21), it is likely that these data 259

reflect the intracellular concentration of active CAT protein. Thus, there was a correlation 260

between Cm resistance efficiency and CAT concentration in MK242 [pGKE75-cat derivative]. 261

262

Plasmid copy numbers of pGKE75-cat derivatives in G. kaustophilus 263

Plasmid copy numbers often affect gene expression from plasmids. In general, a higher number 264

of copies leads to increased gene expression (30). Consequently, we analyzed the copy numbers 265

of pGKE75-cat derivatives in G. kaustophilus MK242 using QC-PCR (Fig. 3C). The copy 266

number of pGKE75αβ-catA138T was 17 ± 2 copies per chromosome, which was lower than those of 267

pGKE75-cat and pGKE75-catA138T, contrary to our expectation. pGKE75α-catA138T and 268

pGKE75β-catA138T showed copy numbers between that of pGKE75αβ-catA138T and those of 269

pGKE75-cat and pGKE75-catA138T. Thus, there was a negative correlation between the Cm 270

resistance efficiencies conferred by pGKE75-cat derivatives and their copy numbers in G. 271

kaustophilus. 272

273

Acetyl-CoA concentration in G. kaustophilus 274

Because CAT inactivates Cm using acetyl-CoA as substrate, we analyzed the acetyl-CoA 275

concentration in G. kaustophilus MK242 [pGKE75-cat derivative] (Fig. 3D). MK242 276

[pGKE75αβ-catA138T] had more abundant acetyl-CoA than MK242 [pGKE75-cat or 277

pGKE75-catA138T]. MK242 [pGKE75α-catA138T or pGKE75β-catA138T] showed acetyl-CoA 278

concentrations higher than those of MK242 [pGKE75-cat or pGKE75-catA138T] but less than that 279

of MK242 [pGKE75αβ-catA138T]. These data indicate a positive correlation between Cm 280

resistance efficiencies and acetyl-CoA concentrations in MK242 [pGKE75-cat derivative]. 281

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282

Transcription from the mutant pUC replicon 283

RNA transcribed from the mutant pUC replicon in G. kaustophilus MK242 [pGKE75αβ-catA138T] 284

was analyzed using RT-PCR (Fig. S1). The analysis detected a transcript from −559 to +1 in the 285

mutant pUC replicon (RNA−559/+1) and its complementary transcript (RNA+1/−559). RT-PCR also 286

detected a longer transcript from −559 to +205 (RNA−559/+205), which could contain the two 287

mutations, although not its complement. The observation suggests that the mutant pUC replicon 288

is actually transcribed in G. kaustophilus, even though it arises from incidental promoter 289

sequences. 290

291

G. kaustophilus transformation using Cm selection 292

To examine whether G. kaustophilus can be transformed with pGKE75αβ-catA138T using Cm 293

selection, the plasmid was introduced into G. kaustophilus MK242 by conjugative DNA transfer. 294

A cell mixture containing donor, recipient, and conjugation helper was incubated at 37°C for 295

conjugation and subsequently preincubated at 60°C. An aliquot of cells was then incubated at 296

60°C on LB plates supplemented with Cm, providing 53 transformants (conjugation efficiency, 297

10−5–10−6 recipient−1). An equal aliquot provided 122 transformants on LK5 plates. Five repeated 298

analyses indicated that transformants obtained using Cm selection accounted for 36 ± 13% of 299

those obtained using kanamycin selection. Preincubation was essential for transformant growth 300

probably because of sufficient production of CATA138T prior to Cm exposure. Almost 301

transformants (>95%) obtained using Cm selection were resistant to kanamycin, confirming that 302

the number of false positives was negligible. pGKE75-cat and pGKE75-catA138T provided few 303

transformants using Cm selection. Thus, pGKE75αβ-catA138T is a unique plasmid for G. 304

kaustophilus; its introduction can be selected using Cm resistance at 60°C. 305

306

Plasmid copy numbers of pUC18 derivatives in E. coli 307

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The plasmid pUC18αβ, which contains the mutant pUC replicon of pGKE75αβ-catA138T, was 308

constructed from pUC18 and analyzed for plasmid copy numbers (Fig. 4). QC-PCR analysis 309

indicated that pUC18 propagates with 310 ± 12 copies at 37°C. The copy number increased with 310

increasing culture temperature in agreement with previous observations (9). However, pUC18αβ 311

exhibited a substantial but temperature-independent copy number (120–210 copies at 25–42°C). 312

pUC18α and pUC18β showed temperature-dependent and independent profiles, respectively, but 313

both had lower copy numbers than pUC18αβ. Because a pBR322 derivative lacking rom gene is 314

also known to exhibit temperature-independent copy number, we analyzed the copy number of 315

pUC18γ, which had the replicon of pBR322 lacking the rom gene. Although pUC18γ exhibited a 316

temperature-independent profile, like pUC18αβ, its copy number was much lower at all 317

temperatures examined. Thus, pUC18αβ had a unique copy number profile different from known 318

pUC plasmids. 319

320

Plasmid stability of pUC18 derivatives in E. coli 321

The plasmid stability of pUC18 derivatives was assessed on the basis of plasmid retention rates 322

following 3 successive cultures of E. coli [pUC18 derivative]. pUC18 and pUC18α were notably 323

unstable at 42°C and 20–30°C, respectively (Table 4). However, pUC18αβ, pUC18β, pUC18γ 324

showed excellent stability at 20–42°C. pUC18αβ, pUC18β, and pUC18γ were completely retained 325

even following 7 successive cultures at 37°C, whereas pUC18 and pUC18α were retained at the 326

rate of 79 ± 7% and 55 ± 8%, respectively. 327

328

Discussion 329

A new plasmid responsible for Cm resistance at high temperatures, pGKE75αβ-catA138T, was 330

generated from pGKE75-catA138T through a directed evolution process. The mutations in 331

pGKE75αβ-catA138T were found in the pUC replicon and caused efficient Cm resistance at high 332

temperatures, although the pUC replicon has no role, in theory, in G. kaustophilus. An immediate 333

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reason for this is that the mutation increased the intracellular concentration of active CATA138T 334

protein. An increased concentration of intracellular acetyl-CoA could also contribute to the Cm 335

resistance because acetyl-CoA is the substrate for CAT reaction. This idea is supported by the 336

fact that some E. coli strains exhibit Cm sensitivity despite sufficient cat expression because of a 337

low intracellular concentration of acetyl-CoA (31, 32). The increased concentrations of CATA138T 338

and acetyl-CoA can be attributed to the decreased copy number of pGKE75αβ-catA138T. It is 339

known that plasmid maintenance places a metabolic burden on bacteria and/or inhibits cell 340

growth (30, 33–35), presumably because of the considerable bioenergy required for replication. 341

In addition, acetyl-CoA is often used during the synthesis of metabolites that store redundant 342

bioenergy in bacteria, such as polyhydroxybutyrate (36) and fatty acids. Therefore, it is possible 343

that the decrease in plasmid copy number leads to redundant bioenergy and facilitates the 344

biosynthesis of both CATA138T and acetyl-CoA. It is unlikely that the decreased copy number 345

resulted in much slower CATA138T production, thereby increasing soluble and active CATA138T, 346

because CATA138T seemed to be more abundantly produced from pGKE75αβ than pGKE75 as 347

soluble forms while being equally produced as insoluble forms, when analyzed by Western 348

blotting (Fig. S2). 349

We note that the actual copy numbers of the intact pGKE75-cat derivatives could be much 350

lower than the values determined in this study. We previously determined the copy number of 351

pUCG18T, which is a parent plasmid of pGKE75-cat (16, 21), and another plasmid pSTE33T 352

using the Wu and Welker method (23). This method, using agarose gel electrophoresis, clearly 353

detected the pSTE33T band in agarose gel and determined its copy number as 16 copies. 354

Meanwhile, the copy number of pSTE33T was determined as 7 ± 3 (n = 4) using QC-PCR, 355

confirming that QC-PCR can determine plasmid copy number as well as the Wu and Welker 356

method. However, the Wu and Welker method could not detect pUCG18T, indicating a lower 357

abundance of the plasmid (17), although QC-PCR determined the copy number as 13 ± 9 (n = 4). 358

QC-PCR also determined that there were >17 copies of each pGKE75-cat derivative, whereas 359

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none of pGKE75-cat derivatives were detected by agarose gel electrophoresis. These 360

observations imply the presence of incomplete forms of pUCG18T and pGKE75-cat derivatives, 361

suggesting that the decrease in pGKE75αβ-catA138T copies, which was indicated by QC-PCR 362

analysis, may reflect the decrease in incomplete forms. Taken together, efficient Cm resistance 363

conferred by pGKE75αβ-catA138T can be explained by an increase in intracellular concentrations 364

of CATA138T and acetyl-CoA following a decrease in the number of incomplete plasmids. 365

We speculate that the incomplete forms are single-stranded DNA rather than 366

randomly-truncated DNA, because these were undetectable by agarose gel electrophoresis and 367

PCR analysis exclusively detected intact cat gene, but not partial fragments, in G. kaustophilus 368

MK242 [pGKE75-cat derivative]. It is unclear why the mutations in pUC replicon suppress 369

production of single-stranded plasmids. However, the pBST1 replicon in pGKE75-cat 370

derivatives shows sequence similarity to the replicon of a plasmid from B. megaterium, pBM300, 371

which is thought to replicate via a theta-type mechanism, as do ColE1-type plasmids (37–39). In 372

theta-type mechanisms, plasmids replicate continuously on leading strand but discontinuously on 373

lagging strand. Therefore, single-stranded DNA may accumulate when the plasmid is replicated 374

efficiently on leading strand but not on lagging strand. This hypothesis allows us to speculate 375

that RNA species from the mutant pUC replicon, which were actually produced in G. 376

kaustophilus [pGKE75αβ-catA138T], may form thermostable secondary structures and hybridize 377

with leading strand at the pUC replication origin, as do in E. coli, under high-temperature 378

conditions and that this event may inhibit DNA elongation on leading strand to reduce 379

incomplete plasmids. 380

A kanamycin resistance gene is currently in use as an efficient selectable marker for 381

Geobacillus spp. at temperatures above 65°C (17, 38, 40, 41). Although cat can be used as a 382

selectable marker (23, 42), neither cat nor catA138T conferred Cm resistance on G. kaustophilus at 383

temperatures above 65°C (21). Therefore, it is noteworthy that pGKE75αβ-catA138T conferred Cm 384

resistance at 65°C. Moreover, G. kaustophilus was efficiently transformed with 385

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pGKE75αβ-catA138T using Cm selection at 60°C. The selection was performed by incubation for 386

only 1 day without producing false positives. These observations suggest this new plasmid is 387

useful for genetic modifications of G. kaustophilus and maybe other Geobacillus spp. 388

In addition to pGKE75αβ-catA138T, this study provided a unique plasmid for E. coli, pUC18αβ, 389

which shows high plasmid stability and temperature-independent high copy number. pUC18αβ 390

has two mutations, compared with pUC18. Based on the properties of pUC18α and pUC18β, 391

which have either of the two mutations, both mutations obviously participate in the process that 392

results in high plasmid stability and high copy number of pUC18αβ. Mutant RNA species from 393

the mutant pUC replicon are probably responsible for the unique profile of pUC18αβ. One 394

mutation in the RNA II sequence can affect secondary structures within RNA II, thereby 395

affecting plasmid replication. Although the other mutation is outside the RNA II sequence, the 396

mutation can affect the secondary structure of extended RNAs that are partially transcribed 397

beyond the origin during plasmid replication (1). The temperature-independent plasmid copy 398

number of pUC18αβ may arise from thermostable secondary structures of mutant RNA species, in 399

contrast to temperature-sensitive RNAs from pUC18. 400

In summary, this study generated two plasmids: pGKE75αβ-catA138T and pUC18αβ. Because 401

of their unique properties, these plasmids extend the genetic toolboxes for G. kaustophilus and E. 402

coli. The results also suggest that thermoadaptation-directed evolution using an error-prone 403

thermophile, G. kaustophilus MK480, can generate not only mutant genes encoding thermostable 404

enzyme variants, but also plasmids with unique properties via unexpected mutations. 405

406

Acknowledgments 407

We wish to thank Dr. Jun Ishii of Kobe University for advice on Western blotting. This work was 408

supported by the following organizations: Programme for Promotion of Basic and Applied 409

Researches for Innovations in Bio-oriented Industry, Japan; the Science and Technology 410

Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Japan; JSPS 411

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KAKENHI (Grant Number 25450105); and the Institute for Fermentation, Osaka, Japan. 412

413

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526

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Figure legends 527

528

Fig. 1 pBR322 and pUC replicons 529

The replication origin is indicated at position +1. RNA I and RNA II are indicated with their 530

transcription directions. Compared with the pBR322 replicon, pUC replicon lacks a rom (also 531

known as rop) gene and has a point mutation (C·G→T·A transition) at position −444. 532

533

Fig. 2 The structure of pGKE75-cat (A), pUC18 (B), and pUC replicons of their derivatives (C) 534

The replication origin is indicated at position +1. Mutation sites are indicated by hollow circles. 535

pGKE75-cat and pGKE75-catA138T shared the usual pUC replicon with pUC18. 536

pGKE75αβ-catA138T and pUC18αβ carry both a G·C→T·A transversion at +175 and a G·C→A·T 537

transition at −252. pGKE75α-catA138T and pUC18α carry a G·C→T·A transversion at +175. 538

pGKE75β-catA138T and pUC18β carry a G·C→A·T transition at −252. pUC18γ carries a 539

T·A→C·G transition at −444 and thus has a pBR322 replicon lacking the rom gene. 540

Abbreviations: Pgk704, a promoter functional in G. kaustophilus (18); bla, ampicillin resistance 541

gene functional in E. coli; TK101, kanamycin resistance gene functional at high temperatures 542

(40); pUC, pUC replicon functional in E. coli; pBST1, pBST1 replicon functional in Geobacillus 543

spp. (38); and oriT, conjugative transfer origin from pRK2013 (44). 544

545

Fig. 3 Effects of pGKE75-cat derivatives on G. kaustophilus 546

G. kaustophilus MK242 [pGKE75-cat derivative] was grown at 65°C on LK5 plates and 547

analyzed for Cm resistance efficiency (A), intracellular CAT activity (B), plasmid copy number 548

(C), and acetyl-CoA concentration (D). (A) Cells were incubated at 65°C on LK5 plates with and 549

without Cm to determine Cm resistance efficiency. Data are presented as the mean ± SD (n = 4). 550

(B) Cell extract was prepared and analyzed for CAT specific activity. Data are presented as the 551

mean ± SD (n = 5). (C) Total DNA was extracted from cells and used to analyze plasmid copy 552

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number, which was determined using the ratio of bla (in pGKE75-cat derivatives) to rpoA (in G. 553

kaustophilus chromosome). Data are presented as the mean ± SD (n = 3). (D) Cell extract was 554

prepared and analyzed for acetyl-CoA concentration. Data are normalized by protein 555

concentration and presented as the mean ± SD (n = 4). 556

557

Fig. 4 Plasmid copy number of pUC18 (upper, solid), pUC18αβ (upper, hollow), pUC18α (lower, 558

solid), pUC18β (lower, hollow), and pUC18γ (lower, gray) in E. coli 559

E. coli [pUC18 derivative] was cultured at the indicated temperature. Total DNA was extracted 560

from cells and used to analyze plasmid copy number, which is the ratio of bla (in pUC18 561

derivatives) to rpoA (in E. coli chromosome). Data are presented as the mean ± SD (n = 3). 562 563 564

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Table 1 Plasmids used in this study 1

pUC and pBST1 denote replicons functional in E. coli and Geobacillus spp., respectively. TK101 is a kanamycin resistance gene 2

functional in G. kaustophilus (40). bla, cat, kan, and tet denote ampicillin, chloramphenicol, kanamycin, and tetracycline resistance 3

genes for E. coli, respectively. oriT denotes the conjugative transfer origin from pRK2013 (44). pUB307 encodes tra genes 4

responsible for conjugative DNA transfer. 5

6

Plasmid Relevant description Reference

pGKE75-cat E. coli–Geobacillus shuttle plasmid; pUC, pBST1, TK101, bla, cat, oriT Fig. 2; (21)

pGKE75-catA138T pGKE75-cat derivative carrying catA138T instead of cat Fig. 2; (21)

pGKE75αβ-catA138T pGKE75-catA138T derivative carrying two mutations in pUC replicon Fig. 2

pGKE75α-catA138T pGKE75-catA138T derivative carrying one mutation in pUC replicon Fig. 2

pGKE75β-catA138T pGKE75-catA138T derivative carrying one mutation in pUC replicon Fig. 2

pUC18 E. coli plasmid; pUC replicon, bla, lacZα Fig. 2

pUC18αβ pUC18 derivative carrying two mutations in pUC replicon Fig. 2

pUC18α pUC18 derivative carrying one mutation in pUC replicon Fig. 2

pUC18β pUC18 derivative carrying one mutation in pUC rreplicon Fig. 2

pUC18γ pUC18 derivative carrying pBR322 replicon without rom Fig. 2

pUB307 Derivative of IncP-1 plasmid RP1, tra, oriT, kan, tet (43)

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Table 2 Primers used in this study 1 2

3

Primer Sequence (5′–3′)

blaF TGCTGAAGATCAGTTGGGTG

blaR TTGTTGCCGGGAAGCTAGAG

catF1 GGACGACGATGACAAAATGCAATTTAATAAAATTG

catF2 GCGCATGCTGGACTACAAGGACGACGATGAC

catR GCCGGATCCTTATAAAAGCCAGTCATTAG

colEF1 CGCTCCAAGCTGGGTTGTGTGCACGAACCC

colER1 GGGTTCGTGCACACAACCCAGCTTGGAGCG

colEF2 GTATTGGGCGCTCTTCAGCTTCCTCGCTCACTG

colER2 CAGTGAGCGAGGAAGCTGAAGAGCGCCCAATAC

colEF3 CGGCTACACTAGAAGGACAGTATTTGGTAT

colER3 ATACCAAATACTGTCCTTCTAGTGTAGCCG

ECrpoAF GCCTTTAGAGCGTGGCTTTG

ECrpoAR TTTCGATGACCAGCTTGTCC

GKrpoAF CAACCTTAGGGAACTCCTTG

GKrpoAR TTCGGCCCAATGCTTCCATC

repF1 GCTTGCAAACAAAAAAACCACCGCTACCAG

repR1 GTTTTTCCATAGGCTCCGCCCCCCTGACGAG

repR2 GAGAGGCGGTTTGCGTATTGGGCGCTCTTC

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Table 3 Cm resistance efficiency (%) conferred by pGKE75-cat derivatives 1

G. kaustophilus MK242 [pGKE75-cat derivative] was precultured in liquid LK5 and incubated 2

at the indicated temperature on LK5 plates with and without Cm (5 mg l−1). Cm resistance 3

efficiency was defined as the ratio of Cm resistant colonies to the total number of colonies. Data 4

are presented as the mean ± SD (n = 4). 5

Plasmids Culture temperature

50°C 55°C 60°C 65°C 70°C

pGKE75-cat 66 ± 25 37 ± 8 27 ± 3 <1 <1

pGKE75-catA138T 56 ± 18 77 ± 4 46 ± 6 <1 <1

pGKE75αβ-catA138T 21 ± 4 20 ± 6 71 ± 6 88 ± 12 2 ± 1

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Table 4 Plasmid retention rates (%) of pUC18 derivatives following successive cultures 1

E. coli [pUC18 derivative] was successively cultured in liquid LB for three times. The resulting 2

cells were screened for ampicillin resistance to calculate the plasmid retention rate. Data are 3

presented as the mean ± SD (n = 3). 4

5

Plasmids Culture temperature

20°C 25°C 30°C 37°C 42°C

pUC18 98 ± 0 97 ± 3 98 ± 2 96 ± 5 3 ± 5

pUC18αβ >99 >99 >99 >99 >99

pUC18α <1 <1 <1 94 ± 4 92 ± 11

pUC18β >99 97 ± 5 97 ± 5 >99 >99

pUC18γ >99 >99 >99 >99 >99

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