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Development of a double-crossover markerless gene deletion system in 1

Bifidobacterium longum: Functional analysis of the α-galactosidase 2

gene for raffinose assimilation 3

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Running title: Markerless gene deletion in B. longum 5

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Yosuke Hirayama,1 Mikiyasu Sakanaka,1 Hidenori Fukuma,2 Hiroki Murayama,2 7

Yasunobu Kano,2 Satoru Fukiya,1* and Atsushi Yokota1 8

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Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido 10

University, Kita 9 Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan1; Department 11

of Molecular Genetics, Kyoto Pharmaceutical University, 5, Nakauchi-cho, Misasagi, 12

Yamashina-ku, Kyoto 607-8414, Japan2 13

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*Corresponding author. Mailing address: Laboratory of Microbial Physiology, 15

Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, 16

Sapporo, Hokkaido 060-8589, Japan. Phone/Fax: +81-11-706-2501. E-mail: 17

[email protected]. 18

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00588-12 AEM Accepts, published online ahead of print on 11 May 2012

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ABSTRACT 20

Functional analysis of Bifidobacterium genes is essential for understanding 21

host-Bifidobacterium interactions with beneficial effects on human health; however, 22

lack of an effective targeted gene inactivation system in bifidobacteria has prevented 23

the development of functional genomics in this bacterium. Here, we report the 24

development of a markerless gene deletion system involving a double crossover in 25

Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second 26

crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the 27

plasmid replication gene repA, was integrated into the target gene by a first crossover 28

event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was 29

introduced into this integrant in order to facilitate the second crossover step and 30

subsequent elimination of the excised conditional replication vector from the cells by 31

the plasmid incompatibility. The proposed system was confirmed to work as expected 32

in B. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. 33

Then, markerless gene deletion was tested using the aga gene, which encodes 34

α-galactosidase, whose substrates include raffinose. Almost all the 35

pTBR101-CM-transformed strains became double-crossover recombinants after 36

subculture, and four out of the 270 double-crossover recombinants had lost the ability 37

to assimilate raffinose. Genotype analysis of these strains revealed markerless gene 38

deletion of aga. Carbohydrate assimilation analysis and α-galactosidase activity 39

measurement were conducted using both the representative mutant and a 40

plasmid-based aga-complemented strain. These functional analyses revealed that aga 41

is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A. 42

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INTRODUCTION 44

Bifidobacteria are generally known as beneficial microbes that produce 45

health-promoting effects by interacting with their hosts (20, 23). Some bifidobacteria 46

are commercially used as probiotics in yogurt products and as pharmaceutical agents. 47

To better understand bifidobacteria, the genome sequences of several probiotic 48

bifidobacterial species—Bifidobacterium longum NCC2705 (46), B. longum DJO10A 49

(26), B. longum subsp. infantis ATCC15697 (47), Bifidobacterium breve UCC2003 50

(34), and Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04 (4)—have 51

been analyzed. Although knowledge of the genome sequences enables the prediction of 52

bifidobacterial gene function, clarification of the detailed mechanisms of interactions is 53

not possible with genomic information alone. The most effective approach for directly 54

analyzing gene function is targeted gene mutagenesis. However, this approach is still 55

not easily applicable to studies of bifidobacteria (14), despite recent progress (15, 56

32-34, 41). 57

Currently, Escherichia coli-Bifidobacterium shuttle vectors based on cryptic 58

plasmids have been developed, but they have relatively low transformation frequencies 59

(14). As for gene mutagenesis systems, the construction of vector-insertion mutants by 60

single crossover and gene disruption mutants by double crossover through allelic 61

exchange between drug resistance genes and target genes have been reported (15, 62

32-34, 41). Markerless gene deletion, which leaves no exogenous DNA in mutated 63

alleles, has not yet been achieved in bifidobacteria. Markerless gene deletion has 64

several advantages: i) it is stable; ii) it avoids polar effects on the expression of 65

adjacent genes; and iii) it enables multiple gene deletions in a genome. 66

Markerless gene deletion using the double-crossover method has been achieved in 67

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various bacteria (19, 28, 31, 35, 38). Generally, a low frequency of the second 68

crossover event is an obstacle to successful gene deletion, and strategies for efficiently 69

selecting second-crossover recombinants are required. Conditional replication vectors 70

such as thermosensitive replication vectors (7, 17, 35, 45) and counter-selectable 71

marker genes, e.g., sacB (31, 35, 38) and pheS encoding phenylalanyl-tRNA synthase 72

α-subunit (5, 50), have been used to overcome this problem in other bacteria. A similar 73

system should be applicable in bifidobacteria, but reliable conditional replication 74

vectors and suitable counter-selectable marker genes are currently unavailable. 75

To achieve markerless gene deletion in the bifidobacterial genome, a strategy based 76

on conditional plasmid replication was conceived (Fig. 1). The plasmid for the first 77

crossover functions as a conditional replication vector as a result of deletion of the 78

gene encoding the replication protein RepA. This plasmid can only replicate in 79

bifidobacterial cells when RepA is provided in trans. When this plasmid is integrated 80

into the target gene, supplying RepA is expected to induce the conditional replication 81

of the integrated plasmid. This will result in the severe growth defects (17), 82

presumably due to the interference with chromosome replication as replication starts 83

from not only the chromosome’s origin of replication but also the origin in the 84

integrated plasmid. Under these conditions, the only cells in which a second crossover 85

has occurred, excising the integrated plasmid from the chromosome and leaving only 86

the chromosomal replication origin, can survive. To supply RepA, a second plasmid 87

harboring the repA gene is introduced into first-crossover integrants. Second-crossover 88

recombinants that lost the first plasmid can be efficiently isolated through selection 89

with appropriate antibiotics, as the second plasmid is incompatible with the conditional 90

replication vector. In this study, the proposed strategy was tested to introduce 91

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markerless gene deletion in B. longum 105-A chromosome using aga gene, which 92

encodes α-galactosidase (α-Gal), as a target. 93

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MATERIALS AND METHODS 95

Bacterial strains, plasmids and culture conditions. The bacterial strains and 96

plasmids used in this study are listed in Table 1. All chemicals were of analytical grade 97

and were commercially available. Cultures of B. longum 105-A (29) were routinely 98

grown in half-strength (1/2) MRS broth containing glucose as the fermentable 99

carbohydrate source (Becton, Dickinson and Co., Franklin Lakes, NJ) (22) and 100

supplemented with 0.02% (w/v) L-cysteine and 0.34% (w/v) sodium ascorbate (1/2 101

MRSCS). Carbohydrate assimilation by B. longum was examined in semi-synthetic 102

medium (SSM) containing raffinose, melibiose, or glucose as the sole carbohydrate 103

source (20 g/L) (39). The SSM, which did not contain L-tryptophan and L-asparagine, 104

was autoclaved at 110°C for 10 min, and then its pH was adjusted to pH 6.8 with 105

NaOH. Bacterial growth was monitored by measuring the optical density at 660 nm 106

(OD660) using a Mini Photo 518R (Taitec, Saitama, Japan) or UV-1800 (Shimadzu, 107

Kyoto, Japan) spectrophotometer. When necessary, the following antibiotics were 108

added to both the broth and the agar medium: ampicillin (100 µg/ml), chloramphenicol 109

[Cm (2.5 µg/ml)], rifampicin [Rif (0.1 µg/ml)], and spectinomycin [Spc (75 µg/ml)]. 110

Bifidobacterial strains were cultured at 37°C under anaerobic conditions in an 111

anaerobic chamber (80:10:10, N2:CO2:H2; Coy Laboratory Products, Inc., Grass Lake, 112

MI). Cultures of E. coli were grown aerobically in Luria-Bertani (LB) medium or SOC 113

medium as described previously (44). 114

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DNA manipulations. The oligonucleotide primers used in this study are listed in 116

Supplemental Table S1. Reagents used in molecular biological procedures were 117

purchased from Takara Bio, Inc. (Otsu, Japan) unless otherwise indicated. 118

Chromosomal DNA was purified from cultured bifidobacterial cells using a previously 119

described method (13) with one modification: prior to the extraction of chromosomal 120

DNA using ISOPLANT II (Nippon Gene Co., Ltd., Tokyo, Japan), the cells were 121

treated with 30 mg/ml lysozyme dissolved in a 25% (w/v) solution of sucrose, at 37°C 122

for 1 h. Molecular cloning in E. coli was conducted as described elsewhere (44). 123

Plasmid DNA from E. coli was prepared using a QIAprep Miniprep kit (Qiagen, 124

Hilden, Germany). Plasmids were purified from B. longum cells as described 125

previously (27). PCR was performed using PrimeSTAR HS DNA polymerase with GC 126

Buffer (Takara Bio) according to the manufacturer’s instructions. Amplified DNA 127

fragments were purified using a MinElute PCR purification kit (Qiagen) or a MinElute 128

gel extraction kit (Qiagen). Agarose gel electrophoresis was conducted as described 129

previously (13). Nucleotide sequences were determined by the dideoxy method at 130

Takara Bio and Operon Biotechnologies, Inc. (Tokyo, Japan). BLAST programs were 131

used for sequence similarity analysis (1). Southern hybridization analysis was 132

conducted as described previously (13). BamHI-digested chromosomal DNA (4 µg) 133

from each strain was analyzed. The 3′ region of aga (1.1 kbp) was amplified from 134

pBS423-ΔrepAΔaga with the primer pair aga13f/aga8r and used as a probe. 135

136

Electro-transformation of B. longum strains. Electro-transformation of B. 137

longum was performed using a previously described method with some modifications 138

(3). In brief, B. longum 105-A and first-crossover integrants were cultured 139

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anaerobically until the OD660 reached 0.5 to 0.6 in 1/2 MRSCS medium and 1/2 140

MRSCS-Spc medium, respectively, both of which were supplemented with 50 mM 141

sucrose. Cells were then harvested by centrifugation at 32,300 × g at 4°C for 15 min 142

and washed twice with ice-cold buffered sucrose (1 mM ammonium citrate buffer 143

containing 50 mM sucrose, pH 6.0) and resuspended in the buffered sucrose. 144

Electroporation was conducted using Gene Pulser (Bio-Rad Laboratories, Hercules, 145

CA) in the settings of 12 kV/cm, 25 μF and 200 Ω. After an electric pulse, the cells 146

were cultured anaerobically in 1/2 MRSCS supplemented with 50 mM sucrose for 3 h 147

and spread onto 1/2 MRSCS agar medium (without sucrose supplementation) 148

containing antibiotics corresponding to the plasmid used. The plates were incubated 149

anaerobically at 37°C for 48 h. Sucrose in the 1/2 MRSCS medium and the buffered 150

sucrose has been reported to improve the transformation efficiency in bifidobacteria 151

(3). 152

153

Construction of incompatible plasmids for markerless gene deletion. Two 154

incompatible plasmids, pBS423-ΔrepA and pTBR101-CM, were constructed for 155

markerless gene deletion. All of the plasmids named below were constructed by the 156

introduction of ligated products into E. coli HMS174 (8) and subsequent selection on 157

LB agar medium containing appropriate antibiotics. To construct pBS423-ΔrepA, a 158

conditional replication vector for the first crossover event, the Spc resistance (Spcr) 159

gene [Spc adenyltransferase AAD(9)] from pBRASTA101 (48) was first ligated with a 160

pMB1 ori-containing fragment from pUC18. The resulting plasmid was named 161

pTANS19. The cryptic plasmid pTB4 from B. longum BK25 (13) was digested with 162

BamHI and ligated into pTANS19. The resulting plasmid, E. coli-Bifidobacterium 163

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shuttle vector pBS423 (Fig. 2A), was digested with HincII to remove repA. The 164

HincII-digested pBS423 was separated into 4.4-kbp and 0.5-kbp fragments (the latter 165

contained a part of repA), and the 4.4-kbp fragment was self-ligated. The resulting 166

plasmid was named pBS423-ΔrepA [RepA-, pTB4 ori+, Spcr (Fig. 2B)] and was 167

shown not to replicate in B. longum 105-A. 168

To construct pTBR101-CM, a repA-positive plasmid for facilitation of the second 169

crossover step, bifidobacterial plasmid pTB4 was purified from B. longum BK25, 170

digested with BamHI, and ligated into BamHI-digested pBR322. The resulting plasmid, 171

E. coli-Bifidobacterium shuttle vector pTBR101 (Fig. 2C), was digested with HindIII. 172

The HindIII linker fragment was ligated to the MspI-Sau3AI-digested DNA fragment 173

from pC194 containing the Cm resistance (Cmr) gene (18). The HindIII-digested 174

fragment containing the Cmr gene was ligated into HindIII-digested pTBR101, and the 175

resulting plasmid was named pTBR101-CM [RepA+, pTB4 ori+, Cmr (Fig. 2D)]. 176

Nucleotide sequences of the plasmids constructed were deposited into the 177

DDBJ/EMBL/GenBank databases (Accession no. AB687999 for pBS423 including the 178

information of deleted region in pBS423-ΔrepA and AB688000 for pTBR101-CM). 179

180

Construction of vectors for the first crossover. Two first-crossover vectors were 181

constructed for different objectives: pBS423-ΔrepAβgal for validation of the 182

integration and efficient excision/exclusion of the vector at the βgal locus and 183

pBS423-ΔrepAΔaga for introduction of the markerless gene deletion in aga locus. 184

For the construction of pBS423-ΔrepAβgal, the full-length βgal of B. longum 185

105-A, which shows 99% nucleotide identity to BLLJ_1486 in B. longum subsp. 186

longum JCM 1217T (15), was used as the target gene. A 4.6-kbp fragment containing 187

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full-length βgal was amplified by PCR, using B. longum 105-A chromosomal DNA as 188

a template and the lacZ Fw/lacZ Rv primer pair. The amplified product was digested 189

with PstI and ligated into PstI-digested pBS423-ΔrepA. Ligated products were 190

introduced into E. coli JM109 by electroporation. Transformants were selected on LB 191

agar containing Spc and supplemented with X-Gal 192

(5-bromo-4-chloro-3-indolyl-β-D-galactoside; 40 µg/ml) and IPTG 193

(isopropyl-1-thio-β-D-galactopyranoside; 0.1 mM). Colonies that showed blue color, 194

indicating successful cloning of βgal, were isolated. 195

For the construction of pBS423-ΔrepAΔaga, the 5′ and 3′ regions of aga, but 196

lacking the internal region of aga containing the predicted α-Gal catalytic site (Fig. 197

3A), were used for the construction. A 4.9-kbp fragment containing the full-length aga 198

gene was amplified by PCR using B. longum 105-A chromosomal DNA as the template 199

and the aga1f/aga2r oligonucleotide primer pair. The amplified fragment was purified 200

and cloned into TA-cloning vector pGEM-T Easy (Promega Corp., Madison, WI). The 201

resulting plasmid was named pGEM-T-aga. The 5′ and 3′ regions of the cloned 202

fragment (0.9 and 1.1 kbp, respectively) were separately amplified by PCR using 203

pGEM-T-aga as the template and aga11f/aga12r and aga13f/aga8r as the respective 204

primer pairs. These fragments were fused together by performing a second 205

amplification, covering the 25-bp overlapping region inserted at the 5′ terminus of the 206

primer aga13f, with the aga11f/aga8r primer pair. The amplified 2.0-kbp fragment was 207

digested with PstI and ligated into PstI-digested pBS423-ΔrepA. The ligated products 208

were introduced into E. coli JM109 and transformants were selected on LB-Spc agar 209

medium. 210

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Construction of the first-crossover integrants. The first-crossover integrants 212

were constructed by the introduction of the first-crossover vectors, either 213

pBS423-ΔrepAβgal or pBS423-ΔrepAΔaga, to B. longum 105-A by electroporation. 214

Transformants were selected on 1/2 MRSCS-Spc agar medium. Integration of the 215

first-crossover vector into objective gene locus was verified by genomic PCR using 216

following primers: pBS423-ΔrepAβgal integrants, SpR1/SpR3 and 217

bgal1/UC18pBS423 (Fig. S1A) for the detection of the Spcr gene and 218

pBS423-ΔrepAβgal integration sequence, respectively; pBS423-ΔrepAΔaga integrants, 219

aga11f/aga8r, aga11f/TB4-1 and aga8r/SpR4 (Fig. 3A) for the detection of the 220

pBS423-ΔrepAΔaga integration sequence. The validated strains were used as the 221

first-crossover integrant of each vector. 222

223

Triggering of the second crossover by the introduction of pTBR101-CM. 224

pTBR101-CM was introduced into the first-crossover integrant, either 105-A 225

βgal::pBS423-ΔrepAβgal or 105-A aga::pBS423-ΔrepAΔaga, by electroporation to 226

facilitate the second crossover step through excision of integrated 227

pBS423-ΔrepA-derived first-crossover vectors. Transformants were selected on 1/2 228

MRSCS-Cm agar medium as the Cm-resistant colonies, and they were anaerobically 229

subcultured overnight in 1/2 MRSCS-Cm medium. Subcultures were conducted twice. 230

Excised plasmid was expected to be eliminated from the cells due to plasmid 231

incompatibility during the subcultures. Second-crossover recombinants were selected 232

by using Spc sensitive (Spcs) phenotype as an indicator of the elimination of the 233

excised plasmid. This was tested by a replica-plating method using 1/2 MRSCS-Cm 234

and 1/2 MRSCS-CmSpc agar media. Excision of pBS423-ΔrepA-derived first 235

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crossover vectors was verified by genomic PCR using the following primer pairs: for 236

pBS423-ΔrepAβgal, SpR1/SpR3 and bgal1/UC18pBS423; for pBS423-ΔrepAΔaga, 237

aga11f/aga8r, aga11f/TB4-1 and aga8r/SpR4. In principle, the second crossover event 238

in 105-A βgal::pBS423-ΔrepAβgal yields original wild-type 105-A strain with respect 239

to βgal allele and that in 105-A aga::pBS423-ΔrepAΔaga yields either wild-type 240

revertant or mutant with respect to aga allele. In order to check if the recombination 241

rates actually change when pTBR101-CM is introduced into the cell, control 242

experiments were conducted in both the first-crossover integrants without adding 243

pTBR101-CM in the reaction mixture for electroporation step (mock transformation 244

experiment) for comparison. In this case Cm was not added to the culture media. 245

246

Construction of the markerless aga-defective mutants. The second-crossover 247

recombinants from 105-A aga::pBS423-ΔrepAΔaga was cultured in SSM 248

supplemented with raffinose as the sole carbohydrate source using 96-well microtiter 249

plates. Strains that showed no growth were selected as candidates for aga deletion. The 250

genotypes of the candidates were checked by aga-specific PCR, conducted using 251

chromosomal DNA as the template and the aga11f/aga8r primer pair. Subsequently, 252

one candidate was cultured anaerobically overnight in 1/2 MRSCS-Rif broth at 37°C to 253

cure pTBR101-CM, which was propagated in the cells. The culture was streaked onto 254

1/2 MRSCS agar medium. Plasmid-cured strains were selected as Cm-sensitive (Cms) 255

colonies by replica plating using 1/2 MRSCS and 1/2 MRSCS-Cm agar media. After 256

curing, detailed genotype analysis of the candidate aga-deletion strain was conducted 257

by Southern hybridization and sequencing of the aga-deletion region. The aga-deletion 258

mutant was named 105-A Δaga. 259

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260

Construction of the aga-complemented strain. The upstream region of aga 261

containing the putative promoter was determined from the draft genome sequence of B. 262

longum 105-A (Yoshikawa et al., unpublished result) and a previous report (21). A 263

2.5-kbp DNA fragment containing aga and an upstream region (135 bp) was amplified 264

by PCR using pGEM-T-aga as the template and the aga20f/aga21r primer pair. The 265

products were digested with SalI and ligated into SalI-digested pKKT427 (51) (Spcr). 266

The ligated products were introduced into E. coli DH5α and transformants were 267

selected on LB-Spc agar medium. The resulting aga-complementation plasmid was 268

named pAga135. Purified pAga135 was subsequently introduced into 105-A Δaga by 269

electroporation, and a Spc-resistant strain was isolated on 1/2 MRSCS-Spc agar 270

medium. Propagation of pAga135 was confirmed by agarose gel electrophoresis of the 271

purified plasmid. The strain, named 105-A Δaga/pAga135, was used as a 272

plasmid-based aga-complemented strain. An empty vector-introduced strain, 105-A 273

Δaga/pKKT427, was similarly constructed through introduction of the shuttle vector 274

pKKT427 into 105-A Δaga. 275

276

Measurement of α-Gal activity. Strains 105-A, 105-A Δaga, 105-A 277

Δaga/pAga135 and 105-A Δaga/pKKT427 were cultured overnight in 40 ml of 1/2 278

MRSCS (Spc was added when necessary). Cells were harvested by centrifugation at 279

19,000 × g at 4°C for 15 min and washed with saline. The cells were resuspended in 280

SSM containing 20 g/L raffinose and incubated for 6 h under anaerobic conditions at 281

37°C to induce α-Gal expression. The cells were harvested by centrifugation, washed 282

twice with ice-cold 50 mM potassium phosphate buffer (pH 6.0) containing 0.015% 283

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(w/v) dithiothreitol, and disrupted three times using a French pressure cell (Ohtake 284

Works, Tokyo, Japan) at 125 MPa. Cell debris was removed by centrifugation. The 285

resulting supernatant was used as a crude extract. Protein concentration was 286

determined by the Bradford method using a Bio-Rad protein assay kit (Bio-Rad 287

Laboratories, Hercules, CA). Measurement of α-Gal activity was performed according 288

to a previously described method (36) in a 300 µl of reaction mixture containing 50 289

mM potassium phosphate buffer (pH 6.0), 5 mM p-nitrophenyl-α-D-galactopyranoside 290

(25026-81; Nacalai Tesque, Inc., Kyoto, Japan) (as a substrate), and 60 µl of crude 291

extract, diluted appropriately in 50 mM potassium phosphate buffer (pH 6.0) 292

containing 0.5 mg/ml bovine serum albumin. The enzyme reaction was started by 293

adding the crude extract to the reaction mixture followed by incubation at 35°C for 5 294

or 10 min. The reaction was stopped by adding 600 µl of 1M Na2CO3. A blank reaction 295

mixture was prepared by adding 600 µl of 1M Na2CO3 prior to the addition of the 296

crude extract. The release of p-nitrophenol was monitored in a 1-cm-wide cuvette by 297

measuring absorbance at 400 nm with a DU800 spectrophotometer (Beckman Coulter 298

Inc., Fullerton, CA). The absorbance value for the blank was subtracted from that for 299

the samples. A molar extinction coefficient of 5,560 M-1 cm-1 for p-nitrophenol was 300

used in the calculation. The specific activity was expressed as µmol of p-nitrophenol 301

per min per mg protein. 302

303

RESULTS 304

Validation of the proposed system at β-galactosidase gene locus in B. longum 305

105-A. In the proposed system, the plasmid pBS423-ΔrepA (RepA-, pTB4 ori+, Spcr), 306

which lacks the gene encoding the replication protein RepA, was used as the 307

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conditional replication vector. A second plasmid, pTBR101-CM (RepA+, pTB4 ori+, 308

Cmr), which was incompatible with pBS423-ΔrepA, was used to supply RepA. 309

Uncertainty as to the presence of sufficient recombination activity in B. longum 105-A 310

led us to first examine whether integration and excision events readily occur, using 311

full-length βgal as the target. 312

Spcr transformants carrying the pBS423-ΔrepAβgal were obtained with an 313

efficiency of 50 cfu/μg DNA. Genomic PCR analysis of the isolated transformants 314

indicated that pBS423-ΔrepAβgal was successfully integrated into the chromosomal 315

βgal locus as a result of the first crossover (Supplemental Fig. S1B and C). 316

Introduction of pTBR101-CM into the first-crossover pBS423-ΔrepAβgal integrants 317

yielded transformants (7.5 × 102 cfu/μg DNA) on 1/2 MRSCS-Cm agar. After 318

subsequent culture of pTBR101-CM transformants in 1/2 MRSCS-Cm medium, 100% 319

of the colonies tested showed Spcs (Table 2). In contrast, only 0.125% of the mock 320

transformants without using pTBR101-CM showed Spcs phenotype after the culture in 321

1/2 MRSCS medium. Therefore, it was found that the introduction of pTBR101-CM 322

dramatically increased the frequency of appearance of the second-crossover 323

recombinants. 324

No amplification in genomic PCR analysis of selected Spcs strains (Supplemental 325

Fig. S1D and E) indicated successful excision of the pBS423-ΔrepAβgal from the 326

chromosome by the second crossover. To confirm elimination of the excised 327

pBS423-ΔrepA by using incompatibility, plasmids in the second-crossover 328

recombinants were extracted and introduced into E. coli DH5α. Many transformants 329

derived from pTBR101-CM appeared in the LB-Cm agar. In contrast, none of the 330

transformants derived from pBS423-ΔrepA were detected in LB-Spc agar. These 331

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results indicate that the excised pBS423-ΔrepA was eliminated from cells because of 332

plasmid incompatibility. Taken together, the proposed system was verified to function 333

as expected. 334

335

Markerless deletion of aga in B. longum 105-A. This system was next applied for 336

markerless gene deletion into the aga gene, which putatively encodes α-Gal (Fig. 3A). 337

The α-Gal-encoding gene was selected as the target because α-Gal hydrolyzes raffinose, 338

which is known to increase bifidobacterial populations in the intestinal tract of rats and 339

humans (9, 10). As shown in Table 3, three genes annotated as α-Gal were found in the 340

draft genome sequence of B. longum 105-A (Yoshikawa et al., unpublished data). The 341

contribution of each gene to the α-Gal activity in B. longum 105-A cells was not 342

known. Among these genes, aga and galA3 are conserved in the genomes of B. longum 343

NCC2705 and B. longum DJO10A (Table 3). Furthermore, the molecular weight of the 344

deduced aga polypeptide was similar to those of purified α-Gals from other 345

bifidobacteria (approximately 80 kDa as a monomer) (16, 24, 49). Therefore, aga was 346

inferred to contribute to α-Gal activity and raffinose assimilation in B. longum 105-A. 347

Fragments of both the 5′ and 3′ regions of aga (each approximately 1 kbp in 348

length), which lacked the sequence for the putative active site of the protein, were used 349

as the homologous regions to construct the conditional replication vector 350

pBS423-ΔrepAΔaga. Outline of experimental procedures is illustrated in Fig. 3A. 351

When pBS423-ΔrepAΔaga was introduced into B. longum 105-A by electroporation, 352

Spcr transformants were formed with an efficiency of 2.2 × 102 cfu/μg DNA. After 353

single colony isolation, strains harboring pBS423-ΔrepAΔaga integrated into the 354

chromosomal aga locus were selected by aga-specific PCR (primer pair aga11f/aga8r). 355

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The amplicons of both wild-type (3.4 kbp) and mutant (2.0 kbp) aga alleles were 356

identified in Spcr transformants (Fig. 3B). In order to identify integration patterns, PCR 357

was conducted using different primer pairs. Amplicons of 3.8 kbp (primer pair 358

aga11f/TB4-1, Fig. 3C) and 2.2 kbp (primer pair aga8r/SpR4, Fig. 3D) were detected 359

in the same transformants. These results demonstrated that the selected strains were 360

first-crossover integrants of pBS423-ΔrepAΔaga (105-A aga::pBS423-ΔrepAΔaga) in 361

which recombination had taken place in the 3′ region of the aga locus (region B, Fig. 362

3A). 363

To construct the markerless aga-deletion mutants, pTBR101-CM was introduced 364

into the 105-A aga::pBS423-ΔrepAΔaga, and Cmr transformants were formed with an 365

average efficiency of 3.9 × 102 cfu/μg DNA. Randomly selected Cmr transformants 366

(288 colonies) were subcultured in 1/2 MRSCS-Cm broth to select for 367

second-crossover recombinants. The subcultures were checked for Spcs and the 368

inability to assimilate raffinose by the replica method. The results indicated a high 369

frequency of appearance of the second-crossover recombinants, as 270 of the 288 370

subcultured strains were Spcs (94%). In this case, four of the 270 Spcs strains (1.5%) 371

lacked raffinose assimilation ability and were thus considered candidate aga-deletion 372

mutants. Genotype analysis of all of these candidates by aga-specific PCR supported 373

their inability to grow on raffinose (data not shown). In addition, same aga-specific 374

PCR analysis of raffinose-assimilating Spcs strains indicated that these strains were the 375

revertants to wild-type aga allele (data not shown). In a separate experiment, 376

usefulness of pTBR101-CM was also confirmed at the aga locus. As shown in Table 2 377

when pTBR101-CM was not introduced into the integrant, the ratio of Spcs colonies 378

among the tested colonies was found to be dramatically decreased, indicating the 379

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facilitation of the second crossover step by the plasmid. One of the four candidate 380

strains was selected and used to cure pTBR101-CM, which remained in the cells. Rif 381

was employed as a curing agent because it was previously used to cure plasmids in E. 382

coli (6, 30). After Rif treatment, 84% of the colonies grown on 1/2 MRSCS agar plates 383

showed Cms. Curing of the plasmid was also confirmed by the introduction of the 384

extracted plasmid fraction from a Cms strain into E. coli, which yielded no Cmr E. coli 385

transformants. These results indicated that pTBR101-CM was successfully removed 386

from the candidate strain by Rif treatment. Deletion of aga from the 387

pTBR101-CM-cured candidate strain was further confirmed by Southern hybridization 388

(Fig. 3E) and sequence analysis. These results demonstrated successful markerless aga 389

gene deletion in B. longum 105-A. This mutant was named 105-A Δaga. 390

391

Functional analyses of aga using B. longum 105-A Δaga. To clarify the function 392

of aga, complementation of aga in 105-A Δaga was conducted using the plasmid 393

pAga135, which harbors the aga ORF and a 135-bp upstream region containing the 394

putative aga promoter. A transformant, 105-A Δaga/pAga135, was used in the 395

functional analysis, together with the wild-type 105-A strain, 105-A Δaga, and the 396

empty vector-introduced derivative 105-A Δaga/pKKT427. 397

Carbohydrate assimilation analysis using SSM revealed that 105-A Δaga was 398

unable to grow after cultivation for 24 h on either melibiose or raffinose, two substrates 399

for α-Gal (Fig. 4). In contrast, Δaga/pAga135 showed comparable growth levels to 400

105-A on all tested carbohydrates. These results indicated that aga is the gene 401

responsible for the assimilation of melibiose and raffinose. 402

Next, α-Gal activity was measured in cells incubated for 6 h in SSM containing 403

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raffinose as the sole carbohydrate. As expected, α-Gal activity was not detected in 404

105-A Δaga (Table 4). α-Gal activity was successfully restored in Δaga/pAga135 but 405

not in 105-A Δaga/pKKT427 (Table 4). Taken together, these results clearly identify 406

aga as the only functional α-Gal gene necessary for the assimilation of α-galactosides 407

such as melibiose and raffinose in B. longum 105-A. These results also suggest that the 408

other annotated α-Gal genes in the B. longum 105-A genome, galA2 and galA3 (Table 409

3), make little contribution, if any, to α-Gal activity under the conditions used in this 410

study. 411

412

DISCUSSION 413

In this study, a markerless gene deletion system was successfully developed using a 414

double-crossover method in B. longum 105-A. Despite the emerging importance of 415

bifidobacteria in human health, genetic manipulation systems for improving our 416

understanding of these bacteria, especially efficient methods for targeted gene 417

mutagenesis, have not yet been established. Several B. breve UCC2003 gene-insertion 418

mutants (32-34, 41), in which a non-replicating vector containing internal DNA 419

fragments of the target gene was integrated into the target gene by a single crossover 420

method, were recently reported. A mutant of B. longum NCC2705 has also been 421

generated (15). In this mutant, gene disruption was achieved by a double-crossover 422

method in which a region homologous to the target gene along with an inserted 423

antibiotic resistance gene was cloned into a general E. coli vector, and allelic exchange 424

was performed. These methods represent important progress in the function analysis of 425

bifidobacterial genes, although exogenous sequences (vector sequences and/or 426

selection marker genes) were left in the mutated allele with both methods. In contrast, 427

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the proposed method is a markerless deletion method that does not leave any 428

exogenous sequences in the mutated allele. 429

The following factors are considered to be the characteristics of the developed 430

system. (i) Facilitation of the second crossover step. A low-frequency second crossover 431

event is generally the bottleneck in gene mutagenesis based on the double-crossover 432

system. In this study, a conditional replication vector lacking repA [pBS423-ΔrepA 433

(Spcr)] for integration into the chromosome (first crossover event) and a second 434

plasmid to supply RepA (pTBR101-CM) were used to facilitate the second crossover 435

step. As shown in Table 2, pTBR101-CM apparently increased the second crossover 436

frequency. Still one can also consider the possibility that the introduced pTBR101-CM 437

served simply as a strong selective pressure to the first-crossover integrants allowing 438

the preferential growth of pre-existing spontaneous second-crossover recombinants 439

after the electro-transformation step. However, this may not be the case as there 440

seemed to be no contamination of the spontaneous second-crossover recombinants in 441

the competent cells (Spc was included in the medium for preparation of the competent 442

cells). Though not shown in the results, colony PCR analysis of the pTBR101-CM 443

transformants clearly indicated that the excision of the integrated plasmid from the 444

chromosome had already occurred in the transformed colonies before subcultures. 445

Taken together, the supply of RepA was judged to increase the second crossover 446

frequency, which led to the facilitation of the second crossover step. 447

(ii) Exclusion of excised vector by plasmid incompatibility. Plasmid incompatibility 448

has been used as a method to eliminate unnecessary plasmids from cells during 449

bacterial gene mutagenesis by double-crossover recombination (11, 25, 37, 43). This 450

system was applied to markerless gene deletion. The use of two incompatible vectors, 451

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pBS423-ΔrepA and pTBR101-CM, enabled the excised vector pBS423-ΔrepA 452

(containing a wild-type or mutated allele) to be effectively excluded from cells. In 453

principle, either plasmid could be excluded. However, in this case, retaining 454

pTBR101-CM was the only option because pBS423-ΔrepA cannot replicate on its own. 455

The Spcs plus Cmr combination ensured acquisition of the desired recombinants. 456

(iii) Successful curing of pTBR101-CM by Rif. The remaining pTBR101-CM in 457

cells was easily cured by Rif treatment, as has been reported in E. coli (6, 30). 458

Successful curing of the replicative vector in the markerless gene deletion mutant 459

means that multiple gene deletions are possible using the developed system. Multiple 460

gene mutations have not been introduced into bifidobacteria and are theoretically 461

limited with mutagenesis methods that leave antibiotic resistance markers such as Spc 462

(29), tetracycline (2), and erythromycin (42) in the mutated allele, owing to the limited 463

availability of these makers in bifidobacteria. 464

Temperature-sensitive vectors harboring temperature-sensitive rep gene have been 465

commonly used for targeted gene mutagenesis in other bacteria (7, 17, 35, 45). 466

However, no reliable temperature-sensitive vector is currently available for use in 467

bifidobacteria. In addition, mutagenic effects of heat shock stress on the genome 468

sequence are concerned, because heat shock-induced GroEL/GroES chaperone is 469

known to protect error-prone DNA polymerases, Pol IV and Pol V, from degradation in 470

E. coli (12). Homologs of the chaperone and the DNA polymerases are also found in 471

the genome sequences of B. longum 105-A and other bifidobacteria. 472

Theoretically, after the introduction of gene mutations by the double-crossover 473

method, the frequencies of the appearance of the wild-type and the mutant genotypes 474

should be the same. However, it was found that the number of aga-deletion mutants 475

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was far lower than that of revertants (4 vs. 266). The reason for the low frequency of 476

gene deletion mutants is unclear, but it is possible that there was a difference in 477

recombination efficiency between the homologous regions A and B used in aga 478

deletion (Fig. 3A). Therefore, the recombination frequency was checked using 479

plasmids consisting of homologous region A or B in pBS423-ΔrepA. These plasmids 480

were introduced into B. longum 105-A, and the appearance of first-crossover integrants 481

was examined. However, no significant difference between homologous regions A and 482

B was observed (data not shown). These results suggest that a difference in 483

recombination frequency between these homologous regions was not a cause of the 484

low occurrence of gene deletion mutants. An unexpected selective disadvantage 485

associated with the aga deletion might be involved in this tendency. Further 486

comparisons using other genes are needed to address this point. 487

The aga was shown to be the sole responsible gene for α-Gal activity in B. longum 488

105-A. Successful complementation in both the growth on melibiose and the α-Gal 489

enzyme activity by introduction of pAga135 into α-Gal-deficient E. coli JW4080 490

(melA-) also supported the observation (data not shown). Although gene deletion 491

mutants for the other annotated α-Gal genes (galA2 and galA3) were not constructed, it 492

seemed that these genes are either not expressed or do not encode proteins with α-Gal 493

activity. The nucleotide similarity between aga and galA2 or galA3 was calculated to 494

be 47% or 48%, respectively. The molecular weights of the predicted polypeptides 495

encoded by these genes were lower than that of aga (Table 3) and those of purified 496

α-Gals in other bifidobacteria (approximately 80 kDa) (16, 24, 49), suggesting that 497

these genes are unlikely to encode proteins with α-Gal activity. 498

The applicability of the developed system to other bifidobacterial species is unclear. 499

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It may depend on the host range of the plasmids pBS423-ΔrepA and pTBR101-CM, 500

both of which were derived from a cryptic plasmid pTB4 harbored by B. longum BK25 501

(13). Another factor will be the transformation efficiency of a given bifidobacterial 502

strain. The average transformation efficiency of an E. coli-Bifidobacterium shuttle 503

vector pBS423 into B. longum 105-A was 1.1 × 105 cfu/μg DNA, whereas in the case 504

of aga deletion, transformation efficiency of pBS423-ΔrepAΔaga for the construction 505

of the first crossover integrants decreased to 2.2 × 102 cfu/μg DNA. The average 506

transformation efficiency of the replicative pTBR101-CM vector into the 507

first-crossover integrants was 3.9 × 102 cfu/μg DNA. According to our calculation, 508

from previously reported data on host/vector systems, the median transformation 509

efficiency in bifidobacteria was up to 103 cfu/μg DNA (14). It therefore appears that 510

application of the developed system to other bifidobacteria will be limited to the strains 511

that have relatively high transformation efficiency at least over 104 cfu/μg DNA with 512

the replicative vector pBS423. In fact, preliminary trials for the deletion of aga in B. 513

longum subsp. longum JCM 1217T, B. longum subsp. infantis JCM 1222T and B. breve 514

JCM 1192T were not successful probably due to the low-transformation efficiencies as 515

well as appearance of false-positive transformants. Optimization of transformation 516

conditions and antibiotic concentrations for transformants selection will allow the 517

markerless gene deletion in other strains. Methylation of the vectors using 518

bifidobacterial modification enzymes is one way to increase the transformation 519

efficiency by avoiding cleavage of introduced DNA by restriction system in 520

bifidobacteria (33, 51). 521

The markerless gene deletion method reported here can be used for functional gene 522

analysis as well as for other applications. For example, this method may have 523

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advantages in the construction of food-grade host strains, as the use of antibiotic 524

resistance genes has not been approved for the construction of food-grade hosts (40, 525

45). Moreover, the method may be used to insert exogenous genes into the 526

bifidobacterial chromosome. It is anticipated that therapeutic proteins and hormones 527

for medical treatments could be stably expressed in genetically engineered 528

bifidobacteria (14). 529

In conclusion, a rational system for markerless gene deletion was successfully 530

developed in B. longum 105-A. Although the applicability of this system to other 531

bifidobacteria remains to be demonstrated, and there is much room for improvement, 532

we believe that this technique will accelerate functional gene analysis in bifidobacteria. 533

534

535

ACKNOWLEDGMENTS 536

We are grateful to Masayuki Okuyama (Research Faculty of Agriculture, Hokkaido 537

University) for the instruction of the α-galactosidase activity measurement. This study 538

was supported in part by a Grant-in-Aid for Young Scientists (B) from the Japan 539

Society for the Promotion of Science (No. 23780072 to S.F.), by a Grant-in-Aid for 540

Regional R&D Proposal-Based Program Grant from Northern Advancement Center for 541

Science & Technology of Hokkaido Japan (No. T-3-5 to S.F.), and by a Ph.D. Student 542

Research Support Program Grant from Hokkaido University Clark Memorial 543

Foundation to Y.H. We also thank the Research Center, Nippon Beet Sugar Mfg. Co., 544

Ltd. (Obihiro, Japan) for financial support. 545

546

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A, Rokhsar DS, Lebrilla CB, German JB, Price NP, Richardson PM, Mills 702

DA. 2008. The genome sequence of Bifidobacterium longum subsp. infantis 703

reveals adaptations for milk utilization within the infant microbiome. Proc. Natl. 704

Acad. Sci. U. S. A. 105:18964-18969. 705

48. Tanaka K, Samura K, Kano Y. 2005. Structural and functional analysis of 706

pTB6 from Bifidobacterium longum. Biosci. Biotechnol. Biochem. 69:422-425. 707

49. Xiao M, Tanaka K, Qian XM, Yamamoto K, Kumagai H. 2000. High-yield 708

production and characterization of α-galactosidase from Bifidobacterium breve 709

grown on raffinose. Biotechnol. Lett. 22:747-751. 710

50. Xie Z, Okinaga T, Qi F, Zhang Z, Merritt J. 2011. Cloning-independent and 711

counterselectable markerless mutagenesis system in Streptococcus mutans. 712


51. Yasui K, Kano Y, Tanaka K, Watanabe K, Shimizu-Kadota M, Yoshikawa 714

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H, Suzuki T. 2009. Improvement of bacterial transformation efficiency using 715

plasmid artificial modification. Nucleic Acids Res. 37:e3. 716

717

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Figure legends 718

FIG. 1. Outline of markerless gene deletion system in Bifidobacterium longum 719

105-A. GeneA represents a model target gene for markerless gene deletion. ΔGeneA 720

lacks the internal region of GeneA and is cloned in the conditional replication vector 721

(first plasmid), which lacks the repA gene encoding the plasmid replication initiation 722

protein RepA (indicated as ΔrepA). The region absent in ΔGeneA is identified by a 723

cross. Theoretically two types of GeneA alleles can be generated by the first crossover 724

and ΔGeneA deletion mutants and wild-type revertants can arise in the 725

second-crossover recombinants. Here only the simplified scheme for generation of 726

ΔGeneA deletion mutants was described to represent the concept. 1) First crossover. 727

The first plasmid harboring ΔGeneA was introduced into the host strain B. longum 728

105-A. Here the 5′ region of ΔGeneA is used as a homologous region for the first 729

crossover. The first-crossover integrant harbors both the GeneA and ΔGeneA alleles in 730

its chromosome. The region derived from the first plasmid harboring ΔGeneA in the 731

chromosome of the first-crossover integrants is identified by a dashed line. 2) 732

Transformation of the RepA+ plasmid. Introduction of the repA-harboring second 733

plasmid (RepA+ plasmid) into the first-crossover integrant results in the supply of the 734

plasmid replication protein RepA to the cells. 3) Second crossover and excision. 735

Integrated first plasmid is expected to be excised with GeneA from the chromosome as 736

a result of the second crossover event facilitated by RepA. 4) Exclusion. The excised 737

first plasmid with GeneA has been excluded due to the incompatibility between two 738

plasmids. Objective ΔGeneA deletion mutants can be found among the 739

second-crossover recombinants. Abbreviations: AntAr, resistance gene for antibiotic A; 740

AntBr, resistance gene for antibiotic B. 741

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FIG. 2. Structures of the plasmids used in markerless gene deletion. Genes are 742

identified by open arrows except for the repA gene identified by black arrows. 743

Representative restriction sites in the plasmids are shown. DNA regions in plasmids 744

pBS423 (A), pTBR101 (C), and pTBR101-CM (D) are shown in the surrounding circle 745

(dashed lines) of each plasmid and are divided according to the source of the DNA 746

region. Names of derived genetic materials are shown on the surrounding circle. The E. 747

coli-Bifidobacterium shuttle vector pBS423 (A) was constructed by cloning the cryptic 748

plasmid pTB4 from B. longum BK25 into the BamHI site of pTANS19. The 749

conditional replication vector pBS423-ΔrepA (B) was constructed by 750

self-circularization of the 4.4-kbp HincII-fragment of pBS423. Because pBS423-ΔrepA 751

lacks the repA gene, which encodes the replication initiation protein RepA, it cannot 752

replicate in B. longum 105-A. To construct pTBR101-CM (D), which is incompatible 753

with pBS423-ΔrepA, a chloramphenicol resistance gene (Cmr) from pC194 (cryptic 754

plasmid of Staphylococcus aureus) was cloned into the HindIII site of pTBR101 (C), 755

which consists of pTB4 from B. longum BK25 and pBR322. Spcr, spectinomycin 756

resistance gene; Apr, ampicillin resistance gene. 757

758

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FIG. 3. (A) Schematic of the markerless gene deletion procedures conducted in B. 759

longum 105-A. The first crossover event is shown in the upper panel, and the second 760

crossover event is in the lower panel. The first crossover between the target gene (aga) 761

and the conditional replication vector pBS423-ΔrepAΔaga harboring the Δaga allele, 762

which lacks the internal region of aga (C in the chromosome structure), can take place 763

with homologous region A or B, yielding two types of first-crossover integrants, a) and 764

b). Primers used to check the genotypes of first-crossover integrants are shown as open 765

arrows, with names above the predicted structure of the target locus of each 766

first-crossover integrant. The expected lengths of the amplicons generated using these 767

primers are indicated by dashed lines below the predicted structures. The second 768

crossover event, facilitated by the supply of RepA by the plasmid pTBR101-CM, can 769

take place with homologous region A or B in the first-crossover integrants, a) and b). 770

The second crossover event yielded two types of strains: 1) aga-deletion mutants, and 771

2) wild-type revertants. Predicted restriction sites for BamHI and the predicted lengths 772

of the restriction fragments are indicated in the structure of the aga locus for each 773

strain. (B), (C), and (D) Genotype analyses of the pBS423-ΔrepAΔaga transformants to 774

confirm integration of pBS423-ΔrepAΔaga into the aga locus. Amplified fragments 775

produced using the aga11f/aga8r primer pair (B), the aga11f/TB4-1 primer pair (C), 776

and the SpR4/aga8r primer pair (D), were subjected to agarose gel electrophoresis. A 1 777

kb DNA ladder (New England Biolabs, Inc., Ipswich, MA) was used as a molecular 778

weight marker (lane M). The sizes of representative marker fragments are shown to the 779

left of each panel. Each lane shows amplified DNA generated from a DNA template: 780

lanes 1-8, candidate first-crossover integrants; lane P, pBS423-ΔrepAΔaga (positive 781

control for amplification); lane W, B. longum 105-A (wild-type); lane N, no template 782

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DNA (negative control for amplification). The sizes of the amplified fragments are 783

shown to the right of each panel. (E) Southern hybridization analysis of markerless aga 784

deletion. Samples of BamHI-digested chromosomal DNA (4 μg) were electrophoresed 785

in a 0.7% agarose gel. Each lane corresponds to a DNA sample from a Bifidobacterium 786

strain: lane 1, B. longum 105-A; lane 2, 105-A Δaga; lane 3, wild-type revertant (after 787

the second crossover). Electrophoresed total DNA was transferred to a nylon 788

membrane. The 3′ region of aga (region B in FIG. 3A) was used as a probe. 789

Hybridization signals were detected after exposure for 30 min. The sizes of the 790

hybridized fragments are indicated to the left of the panel.791

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FIG. 4. Growth profiles of the B. longum strains 105-A, 105-A Δaga, 105-A 792

Δaga/pAga135 and 105-A Δaga/pKKT427. Bacteria were cultured anaerobically at 793

37°C for 24 h in semi-synthetic medium containing glucose (filled bars), raffinose 794

(gray bars), or melibiose (open bars) as the sole carbohydrate source. Striped bars 795

indicate cultures without addition of carbohydrate source. Data are shown as means ± 796

SE of three independent experiments. 797

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TABLE 1 Bacterial strains and plasmids used in this studyStrain or plasmid Descriptiona ReferenceEscherichia coli strainsDH5 F-,80lacZM15, lacZYA-argF)U169, recA1, endA1, hsdR17rk

-,mk+), phoA, supE44, -, thi-1, Invitrogen

gyrA96, relA1HMS174 F-, -, recA1, INrrnD-rrnE)1, rph-1, rpoB331R), hsdR19 8JM109 recA1, endA1, gyrA96, thi, hsdR17rk

-, mk+), e14 - mcrA -), supE44, relA1, lac-proAB)/F'[traD36, Takara Bio Inc.

proAB+, lacIq, lacZM15]Bifidobacterium longum strains105-A Human fecal isolate, transformation host strain 29105-A gal::pBS423-repAgal First-crossover integrant of pBS423-repAgal into chromosomal - gal) locus This study105-A aga::pBS423-repAaga First-crossover integrant of pBS423-repAaga into chromosomal - aga) locus This study105-A aga aga-deletion mutant of 105-A This study105-A aga/pAga135 Plasmid-based aga-complementation strain of 105-A by introduction of pAga135 This study105-A aga/pKKT427 -introduced derivative of 105-A This studyBK25 Human fecal isolate harboring cryptic plasmid pTB4 13

PlasmidspAga135 6.4 kbp, pKKT427-derivative containing aga and 135-bp upstream region including putative aga promoter, This study

Spcr

pBS423 4.9 kbp, E. coli-Bifidobacterium shuttle vector, pMB1 ori, pTB4 ori, repA+, Spcr This studypBS423-repA 4.4 kbp, pTB4 ori, repA, Spcr This studypBS423-repAaga 6.2 kbp, derivative of pBS423- containing integrated fragment of 5' and 3' regions of aga, Spcr This studypBS423-repAgal 9.0 kbp, derivative of pBS423-repA containing - gal), Spcr This studypBR322 4.4 kbp, E. coli cloning vector, rop, Tcr, Apr Takara Bio Inc.pBRASTA101 5.0 kbp, E. coli-Bifidobacterium shuttle vector, pMB1 ori, pTB6 ori, Spcr 48pC194 2.9 kbp, Small R-plasmid found in Staphylococcus aureus, Cmr 18pGEM-T Easy 3.0 kbp, TA cloning vector, Apr Promega Corp.pGEM-T-aga 7.9 kbp, 4.9 kbp of B. longum 105-A genomic DNA fragment containing aga was cloned in pGEM-T Easy This studypKKT427 3.9 kbp, E. coli-Bifidobacterium shuttle vector, ColE1 ori, pTB6 ori, Spcr 51pTANS19 1.9 kbp, E. coli cloning vector, pMB1 ori, Spcr This studypTB4 2.9 kbp, Cryptic plasmid in B. longum BK25 This studypTBR101 7.3 kbp, E. coli-Bifidobacterium shuttle vector, pMB1 ori, pTB4 ori, repA+, Apr effective only in E. coli This studypTBR101-CM 8.4 kbp, Cmr gene cloned in pTBR101, pMB1 ori, pTB4 ori, repA+, Apr effective only in E. coli This study

a Antibiotic resistance genes in plasmids were abbreviated as follows: Apr, Ampicillin resistance; Cmr, Chloramphenicol resistance; Spcr, Spectinomycin resistance; Tcr, Tetracycline resistance.

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TABLE 2 Frequency of detection of Spcs colonies during the culture of first-crossover integrants

First-crossover integrant

Addition of pTBR101-CMto reaction mixture for

electroporation

Number of tested

Number of Spcs

second-crossover

Percentage 100)

105-A gal::pBS423-repAgal

- 2396 3 0.125+ 3568 3568 100

105-A aga::pBS423-repAaga

- 963 1 0.104+ 1645 1643 99.9

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TABLE 3 Annotated -galactosidase genes in B. longum 105-A draft genome and their distribution in other B. longum strains, NCC2705 and DJO10AB. longum strain

105-A NCC2705 DJO10A

Annotated -Gala

gene in 105-ALength of

Calculated MWb

Nucleotide sequence identityc

Annotated -Gala

geneNucleotide sequence

identityc Annotated -Gala

geneNucleotide sequence

identityc

aga galA1) 2,307 83.3 100 aga) 98.1 galA1) 99.0galA2 1,863 68.1 100 NDd NDd galA2) 99.8galA3 1,410 52.4 100 BL_0177 98.7 galA3) 99.5

a -Gal, -galactosidase. b Molecular weight of deduced polypeptide was calculated.c !"#!$ % &"'((&&"!-Gal gene in B. longum 105-A.d ND, not determined because galA2 homolog was absent in B. longum NCC2705.

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TABLE 4 -Galactosidase activities of B. longum 105-A and its derivativesStrain Specific activitya

105-A 4.44 0.36105-A aga Not detected105-A aga/pAga135 1.30 0.09105-A aga/pKKT427 Not detecteda The activities were measured after induction by raffinose as described in -galactosidase specific activity [molmin-1 protein)-1] with standard error as determined in duplicate on three biological replicates.

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