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Journal section: Invertebrate microbiology 1

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The bacterial cell wall synthesis gene uppP is required for Burkhoderia colonization of 3

the stinkbug gut 4

5

Jiyeun Kate Kima, Ho Jin Leea, Yoshitomo Kikuchib, Wataru Kitagawab, Naruo Nikohc, 6

Takema Fukatsud#, Bok Luel Leea# 7

8

Global Research Laboratory, College of Pharmacy, Pusan National University, Pusan 609-735, 9

Koreaa; National Institute of Advanced Industrial Science and Technology (AIST), Hokkaido 10

Center, Sapporo 062-8517, Japanb; Department of Liberal Arts, The Open University of Japan, 11

Chiba 261-8586, Japanc; Institute for Biological Resources and Functions, National Institute 12

of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japand 13

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Running title: Cell wall synthesis gene in insect-bacterium symbiosis 15

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#Address correspondence to Bok Luel Lee, brlee@pusan.ac.kr or 17

Takema Fukatsu, t-fukatsu@aist.go.jp 18

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Key words: insect-bacterium symbiosis, bacterial cell wall, undecaprenyl pyrophosphate 20

phosphatase (UppP) 21

22

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01269-13 AEM Accepts, published online ahead of print on 7 June 2013

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ABSTRACT 24

To establish a host-bacterium symbiotic association, a number of factors involved in 25

symbiosis must operate in a coordinated manner. In insects, bacterial factors for symbiosis 26

have been poorly characterized at the molecular and biochemical levels, since many 27

symbionts have not yet been cultured, or are as yet genetically intractable. Recently, the 28

symbiotic association between a stinkbug, Riptortus pedestris, and its beneficial gut 29

bacterium, Burkholderia sp., has emerged as a promising experimental model system, 30

providing opportunities to study insect symbiosis using genetically manipulated symbiotic 31

bacteria. Here, in search of bacterial symbiotic factors, we targeted cell wall components of 32

the Burkholderia symbiont by disruption of uppP gene, which encodes undecaprenyl 33

pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall 34

components. Under culture conditions, the ΔuppP mutant showed higher susceptibility to 35

lysozyme than the wildtype strain, indicating impaired integrity of peptidoglycan of the 36

mutant. When administered to the host insect, the ΔuppP mutant failed to establish normal 37

symbiotic association: the bacterial cells reached to the symbiotic midgut but neither 38

proliferated nor persisted there. Transformation of the ΔuppP mutant with uppP-encoding 39

plasmid complemented these phenotypic defects: lysozyme susceptibility in vitro was 40

restored, and normal infection and proliferation in the midgut symbiotic organ were observed 41

in vivo. The ΔuppP mutant also exhibited susceptibility to hypotonic, hypertonic and 42

centrifugal stresses. These results suggest that peptidoglycan cell wall integrity is a stress 43

resistance factor relevant to the successful colonization of the stinkbug midgut by 44

Burkholderia symbiont. 45

46

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INTRODUCTION 48

Many insects are in intimate symbiotic associations with bacteria. Such symbiotic bacteria 49

exist in the gut lumen, body cavity or inside cells. To establish a successful host-symbiont 50

association, a number of molecular factors from the symbiont side, and also from the host 51

side, must work in a coordinated manner. To understand the mechanisms of these intricate 52

host-symbiont interactions, several model symbiotic systems have been used to identify novel 53

symbiotic factors and to determine their molecular functions (1). For example, the legume-54

Rhizobium nitrogen-fixing symbiosis and the squid-Vibrio luminescent symbiosis have been 55

studied in depth. In both systems, the symbiotic bacteria are easily cultivable and genetically 56

manipulatable, and thus suitable for elucidating the molecular properties of their symbiotic 57

factors (2-8). 58

59

However, among insect-microbe symbiotic systems, molecular factors relevant to 60

symbiosis have been poorly characterized except for inferences from genomic information 61

(9-11). The paucity of molecular and biochemical studies is attributed to the difficulty in 62

isolating and culturing symbiotic bacteria outside insect hosts. Consequently, powerful 63

mutant-based molecular genetic approaches have not been effectively applied to insect-64

microbe symbiotic systems in general. Obligate insect symbionts, such as Buchnera in aphids 65

and Wigglesworthia in tsetse flies, have been associated with their hosts over evolutionary 66

time and are incapable of independent living and thus are uncultivable (9, 12). As for 67

facultative insect symbionts, such as Wolbachia in various insects and Sodalis in tsetse flies, 68

which are transmitted through host generations not only vertically but also horizontally, at 69

least some of them are cultivable outside their host insects and thus potentially genetically 70

manipulable (13-15). However, culturing these symbionts is generally not easy because it 71

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requires complex culture media containing either mammalian sera or live insect cells, and the 72

symbionts grow very slowly, are prone to contamination, and are reluctant to form colonies 73

on agar plates (16). Therefore, previous studies on bacterial symbiotic factors using 74

genetically manipulated symbionts have been limited (16-21). 75

76

The bean bug, Riptortus pedestris, belongs to the stinkbug family Alydidae in the insect 77

order Hemiptera. In contrast to previously known insect-bacterium symbiotic systems, 78

nymphal R. pedestris acquires a β-proteobacterial symbiont of the genus Burkholderia not 79

vertically but from the soil environment every generation (22). A posterior region of the 80

insect midgut bears numerous crypts whose lumens are filled with bacterial cells of the 81

symbiotic Burkholderia (23). Reflecting its free-living origin in the environment, the 82

symbiotic Burkholderia is easily cultivable on standard microbiological media and can be 83

experimentally re-infected into the host insect by oral administration (24, 25). Comparisons 84

between symbiotic and asymbiotic insects showed beneficial fitness consequences of 85

Burkholderia infection to the host insect (22, 26). These features of the Riptortus-86

Burkholderia gut symbiotic system provide unprecedented opportunities to study insect 87

symbiosis at molecular and biochemical levels. 88

89

The cell wall of Gram-negative bacteria is the front-line of interacting with the 90

surrounding environment. It consists of an inner membrane, an outer membrane in which 91

lipopolysaccharide (LPS) forms the outer leaflet, and a periplasmic region where the 92

peptidoglycan layer resides (27). Bacterial cell wall components such as LPS and 93

peptidoglycan are essential for maintaining the structural integrity of bacterial cells and 94

generally required for viability (27, 28). In addition, these cell wall components most likely 95

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play a role in bacterial association with host and hence, may function as symbiotic factors. 96

Biosynthesis of bacterial cell wall components, such as LPS and peptidoglycan, requires a 97

key lipid carrier, undecaprenyl phosphate (C55-P), which is generated from dephosphorylation 98

of undecaprenyl pyrophosphate (C55-PP) (29-34). C55-P is a precursor of various cell wall 99

components that are synthesized in the cytoplasm and transported to the periplasm, where 100

further polymerization occurs. After release from the cell wall component precursors, the 101

lipid carrier is in a pyrophosphate form (C55-PP) and requires another dephosphorylation step 102

before being reused as a lipid carrier (35). This dephosphorylation step is catalyzed by C55-PP 103

phosphatase enzymes. Four C55-PP phosphatases have been identified in Escherichia coli: 104

UppP (also called BacA), YbjG, YeiU and PgpB, of which UppP is regarded as the major 105

phosphatase (36, 37). 106

107

To identify bacterial symbiotic factors in the Riptortus-Burkholderia symbiosis, we 108

targeted the bacterial cell wall-related uppP gene. We generated an uppP-deficient mutant 109

(ΔuppP) of the Burkholderia symbiont by allelic exchange and homologous recombination. 110

Because ΔuppP mutant shows 75% reduction of C55-PP phosphatase activity in E. coli (36), 111

we hypothesized that the decrease of C55-PP phosphatase activity affects the cell wall 112

component synthesis, resulting in defected cell wall. Since the actual effects on the cell wall 113

by the uppP mutation are not well-characterized, we first examined cell wall components of 114

ΔuppP Burkholderia. Furthermore, the growth phenotypes in vitro and symbiotic phenotypes 115

in vivo of the ΔuppP mutant were compared with those of the wildtype Burkholderia 116

symbiont and an ΔuppP/uppP-complemented mutant transfected with a plasmid encoding a 117

functional uppP gene. 118

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MATERIALS AND METHODS 120

Bacterial plasmids and culture media. Bacterial strains and plasmids used in this study 121

are listed in Table 1. Escherichia coli cells were cultured at 37°C in LB medium (1% [w/v] 122

tryptone, 0.5% [w/v] yeast extract, and 0.5% [w/v] NaCl). Cells of Burkholderia symbiont 123

strain RPE161, a spontaneous chloramphenicol resistant mutant derived from RPE64 (24), 124

were cultured at 30°C in YG medium (0.5% [w/v] yeast extract, 0.4% [w/v] glucose, and 125

0.1% [w/v] NaCl). Antibiotics were used at the following concentrations unless otherwise 126

described: kanamycin at 30 μg/ml and chloramphenicol at 10μg/ml. 127

128

Generation of ΔuppP mutant. Deletion of the chromosomal uppP gene from the 129

Burkholderia symbiont was accomplished by allelic exchange and homologous 130

recombination using a suicide vector pK18mobsacB containing the 5’ (uppP-L) and 3’ (uppP-131

R) regions of uppP gene. The wildtype Burkholderia symbiont strain RPE161 was subjected 132

to PCR using the primers uppP-L-P1 (5’- TTT AAG CTT GAG TTC GAC TTC GAG CGT 133

GT-3’) and uppP-L-P2 (5’- TTT GGA TCC AAG ACT GCT GAC CGG AAA AA-3’) for 134

the uppP-L region, and the primers uppP-R-P1 (5’- TTT GGA TCC TTC TTC TTC GGC 135

TGG TTC AT-3’) and uppP-R-P2 (5’- TTT GAA TTC GCA CTG GAA AAC CTC AGC A-136

3’) for the uppP-R region. PCR products and the pK18mobsacB vector were digested with 137

proper restriction enzymes, ligated and transformed into E. coli DH5α cells. The transformed 138

E. coli cells were selected on LB-agar plates containing 100 μg/ml of kanamycin. Positive 139

colonies carrying a vector with the correct insert were further selected by colony PCR using 140

the primer uppP-L-P1 and the vector primer aphII (5’-ATC CAT CTT GTT CAA TCA TGC 141

G-3’). Donor E. coli cells carrying the pK18mobsacB containing uppP-L and uppP-R were 142

mixed with recipient Burkholderia RPE161 cells and also E. coli CC118λpir cells carrying a 143

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helper plasmid pEVS104 to transfer the cloned vector to the RPE161 cells. After allowing a 144

single crossover by culturing cell mixtures of triparental conjugation on YG-agar, RPE161 145

cells with the first crossover were selected on YG-agar containing chloramphenicol (30 146

μg/ml) and kanamycin. Positive colonies with the genomic integration of vector DNA were 147

confirmed by PCR using the chromosomal primer uppP-up (5’- GAG GCA ATG AAA CGT 148

ATC GAC-3’) and the vector primer aphII. The second crossover was allowed by culturing 149

cells with the single crossover in YG media and Burkholderia cells with a double crossover 150

were selected on YG-agar containing chloramphenicol and sucrose (200 μg/ml). The mutant 151

strain with deletion of the uppP gene (BBL005) was identified by PCR using the primers 152

uppP-up and uppP-down (5’-CCA GCA TCT GCT CTT TGT CA-3’) and sequencing of the 153

PCR product. 154

155

Generation of ΔuppP/uppP-complemented mutant. A DNA fragment containing the 156

open reading frame of uppP gene was amplified from RPE161 using the primers uppP-com-157

P1 (5’- GCA CGG CAA TTT TTC TCT TC-3’) and uppP-com-P2 (5’- CGA CTC GAA CGT 158

GTG ACC TA-3’). The amplified DNA fragment was cloned into the DraI site of pBBR122 159

to generate the plasmid pBL5. The cloned plasmid was introduced into E. coli DH5α cells to 160

generate donor cells. By tri-parental conjugation with the BBL005 recipient cells and E. coli 161

CC118λpir helper cells, the pBL5 plasmid carried by the donor E. coli DH5α cells was 162

transferred to the recipient Burkholderia BBL005 cells, yielding the complemented 163

Burkholderia BBL105 cells. The complemented mutant strain was selected on YG-agar with 164

chloramphenicol (30 μg/ml) and kanamycin. 165

166

Measurement of bacterial growth in liquid media. Growth curves of the Burkholderia 167

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symbiont strains were examined either in YG medium or in minimal medium (0.6% 168

Na2HPO4·2H2O, 0.3% KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 0.0003% CaCl2, 1 mM MgSO4, 169

0.2% glucose). The starting cell solutions were prepared by adjusting OD600 to 0.05 in either 170

YG medium or minimal medium using primary culture grown in YG medium at 30°C for 18 171

h. The cell solutions were incubated on a rotator shaker at 180 rpm at 30°C for 36 h, whose 172

OD600 was monitored every 2 h using a spectrophotometer (Mecasys, Korea). 173

174

Protein analysis of bacterial lysates. Burkholderia symbiont cells were harvested at 175

OD600 = 1 after culturing in YG medium. The cells were washed with PBS (137 mM NaCl, 176

2.7 mM KCl, 8 mM NaH2PO4, and 3 mM KH2PO4 at pH 7) and resuspended at 2 x 107 177

cells/μl in PBS. An aliquot of this solution was saved for the whole lysate (WL) fraction. The 178

cell suspension was then sonicated and further diluted to 107 cells/μl equivalent in PBS 179

containing 10 mM EDTA, 100 ug/ml egg white lysozyme (BioShop Canada Inc., Canada) 180

and protease inhibitors (Roche, Germany). After adding one-fourth volume of 10% Triton X-181

114 (final 2%), the cell solution was agitated for 1 h at 4°C. The sample was centrifuged 182

(15,000 x g for 20 min at 4°C), and the pellet was saved for the insoluble fraction (IS), while 183

the supernatant was transferred to a new tube. The IS fraction was washed with PBS and 184

resuspended in 1x Laemmli sample buffer. The liquid was incubated at 37°C for 10 min and 185

centrifuged (10,000 x g for 10 min at 25°C). Following a 10 min incubation at room 186

temperature, the separated aqueous fraction (AQ) was transferred to a fresh tube and 187

supplemented with Triton X-114 solution to a final concentration of 2% for additional phase 188

partitioning before collecting the final AQ fraction. The Triton X-114 fractions (TX) from 189

both partitionings were combined and an equal volume of TBSE solution (20 mM Tris/HCl, 190

pH 8, 130 mM NaCl and 5 mM EDTA) was added. After agitation for 10 min at 4°C, the 191

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samples were centrifuged (10,000 x g for 10 min at 25°C) and separated into upper and lower 192

phases at room temperature. The upper layer was then discarded, TBSE was added, and the 193

procedure was repeated. Final TX fractions were precipitated with cold ethanol, and dried 194

precipitates were resuspended in 1x Laemmli sample buffer. Proteins from different phase 195

fractions were separated by 15% SDS-PAGE and visualized by staining with Coomassie 196

Brilliant Blue (CBB)-R250. The loading quantity for each fraction was 7 x 107 cells 197

equivalent for WL, 7 x 107 cells equivalent for AQ, 6 x 108 cells equivalent for TX, and 3 x 198

108 cells equivalent for IS. 199

200

Carbohydrate analysis. The whole lysate (WL) samples prepared for protein analysis by 201

SDS-PAGE were used for the analysis of bacterial carbohydrates. The WL sample was boiled 202

in 1x Laemmli sample buffer, de-proteinated by incubating with 400 μg/ml proteinase K at 203

60°C for 1 h, and re-boiled prior to SDS-PAGE. Loading amount was 1 x 108 cells equivalent 204

per lane for 12% Laemmli SDS-PAGE gels and 2 x 108 cells equivalent per lane for 12% 205

Tris/Tricine SDS-PAGE gels. Bacterial carbohydrates separated in the gels were visualized 206

using the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (Invitrogen). Briefly, the gels 207

were fixed with 5% acetic acid and 50% methanol, washed three times with 3% acetic acid, 208

incubated with oxidizing solution containing periodic acid for 30 min, washed three times 209

again with 3% acetic acid, and stained with Pro-Q® Emerald 300 Staining Solution for 2 h. 210

After washing twice with 3% acetic acid, the gels were observed with the gel documentation 211

system GDS-200. 212

213

Lysozyme susceptibility assay. Frozen mid-log phase Burkholderia cells were thawed 214

and resuspended in PB (10 mM sodium phosphate, pH 7). After washing with PB, 0.9 ml of 215

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the Burkholderia cell suspension was prepared at OD600 = 0.77-0.78 in PB and transferred to 216

a cuvette for spectrophotometry. Following an addition of 0.08 ml of lysis solution (500 217

μg/ml egg white lysozyme in PB with 100 mM EDTA), the OD600 of the cell suspension was 218

measured every 2 min up to 28 min and then every 5 min until 73 min. As a control, 0.08 ml 219

of PB containing 100 mM EDTA was added to the cell suspension. 220

221

Insect rearing and symbiont inoculation. The bean bugs R. pedestris were reared in our 222

insect laboratory at 28°C under a long day regime of 16 h light and 8 h dark as described (38). 223

Nymphal insects were reared in clean plastic containers (34 cm x 19.5 cm wide and 27.5 cm 224

high) supplied with soybean seeds and DWA (distilled water containing 0.05% ascorbic acid). 225

Upon reaching adulthood, the insects were transferred to a bigger container (35 cm x 35 cm 226

wide and 40 cm high) in which soybean plant pots were placed for food and cotton pads were 227

attached to the walls as substrate for egg laying. Eggs were collected daily and transferred to 228

new cages for hatching. Newly molted second instar nymphs were provided with wet cotton 229

balls soaked with a symbiont inoculum solution consisting of mid-log phase Burkholderia 230

cells suspended in DWA at a concentration of 107 cells/ml. 231

The care and treatment of Burkholderia cells and insects in all procedures strictly 232

followed the guidelines of Pusan National University (PNU)-Institutional Animals care and 233

Use Committee (IACUC) and Living Modified Organ (LMO) Committee. 234

235

Diagnostic PCR. Insects were surface-sterilized briefly with 70 % ethanol, and dissected 236

in PBS in a glass Petri dish using fine scissors and forceps under a dissection microscope. 237

Dissected samples of the posterior midgut M4 region were individually subjected to DNA 238

extraction using the QIAamp DNA Mini Kit (Qiagen). Diagnostic PCR was conducted using 239

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GoTaq Green Master Mix (Promega) with the supplied buffer system under a temperature 240

profile of 95°C for 10 min followed by 30 cycles of 95°C for 30 sec, 58°C for 30 sec and 241

72°C for 1 min, and finally 72 °C for 2 min using the primers Burk16SF (5’-TTT TGG ACA 242

ATG GGG GCA AC-3’) and Burk16SR (5’-GCT CTT GCG TAG CAA CTA AG-3’), which 243

specifically target 16S rRNA gene of the Burkholderia symbiont (38). PCR products were 244

analyzed by 1 % agarose gel electrophoresis and a 100 bp DNA ladder was used to estimate 245

product size. 246

247

CFU assay. Each of the M4 midgut regions dissected from second instar Riptortus 248

nymphs was collected in 50 μl of PB and homogenized by a pestle mortar. The homogenized 249

sample was diluted if necessary and spread on YG-agar plates containing chloramphenicol. 250

Following two days incubation at 30°C, colonies on the plates were counted and the number 251

of symbiont cells in the sample was calculated by CFU x dilution factor. 252

253

Quantitative PCR. Quantitative PCR for estimating titers of the Burkholderia symbiont 254

was performed as described (38). Dissected midgut samples (either M3 or M4) were 255

individually subjected to DNA extraction by QIAamp DNA mini kit (Qiagen). DNA samples 256

were mixed with a master PCR solution containing 2 x qPCR premix of QuantiMix SYBR 257

Kit (PhileKorea) and the primers BSdnaA-F (5’-AGC GCG AGA TCA GAC GGT CGT CGA 258

T-3’) and BSdnaA-R (5’-TCC GGC AAG TCG CGC ACG CA-3’), which target a 0.15 kb 259

region of dnaA gene of the Burkholderia symbiont. The PCR temperature profile was 40 260

cycles of 95°C for 10 s, 60°C for 15 s and 72°C for 15 s. A standard curve for dnaA gene 261

copies was generated using a series of extracted DNA samples containing known numbers of 262

Burkholderia cells. 263

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CFU assay after stress treatments. For each test, the starting CFUs were compared to 265

CFUs after the following treatments. (i) M4 lysate treatment: Midgut M4 regions dissected 266

from fifth instar Riptortus nymphs were homogenized and heat-treated at 55°C for 5 min to 267

kill intrinsic symbiont cells prior to the assay. Different concentrations of the M4 lysate, 268

ranging from 0.0 to 0.4 mg/ml, were incubated with cultured Burkholderia cells at mid-log 269

phase for 1 h at room temperature. After incubation, the samples were diluted, spread on YG-270

agar plates, cultured for two days, and subjected to colony counting. (ii) Hypotonic test: Mid-271

log phase Burkholderia cells in YG medium were washed with 10 mM PB and adjusted to 272

approximately 107 cells/ml in PB. The cell suspensions were incubated at 30°C for 24 h and 273

subjected to CFU assay. (iii) Hypertonic test: Mid-log phase Burkholderia cells were adjusted 274

to OD600 = 0.5-0.7 in YG medium. The cell suspension was combined with an equal volume 275

of 2 M glucose solution, incubated at 30°C for 24 h, and subjected to CFU assay. (iv) 276

Centrifugal pressure test: Mid-log phase Burkholderia cells cultured in YG medium were 277

adjusted to 104 cells/ml, placed in 1.5 ml microcentrifuge tubes, centrifuged at 15,000 rpm 278

(20,000 x g) for 30 min, and subjected to CFU assay. 279

280

RESULTS 281

282

Growth rates of wildtype and mutant Burkholderia symbiont strains. We disrupted 283

the uppP gene of the wildtype Burkholderia symbiont strain RPE161, thereby establishing a 284

ΔuppP mutant Burkholderia symbiont strain BBL005. By transforming the ΔuppP mutant 285

strain with a plasmid encoding a functional uppP gene, we also generated a ΔuppP/uppP-286

complemented mutant Burkholderia symbiont strain BBL105. Growth curves of these 287

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Burkholderia strains in nutritionally rich yeast-glucose (YG) medium revealed that the 288

wildtype strain and the ΔuppP mutant exhibited similar growth rates, while the ΔuppP/uppP-289

complemented mutant grew a little slower (Fig. 1A). Growth curves in nutritionally limited 290

minimal medium exhibited similar patterns, although growth rates overall were much slower 291

in minimal medium than in YG medium (Fig. 1B). These results indicate that deletion of the 292

uppP gene does not affect growth of the Burkholderia symbiont under in vitro culture 293

conditions. The slower growth of the ΔuppP/uppP-complemented mutant may be due to a 294

cost of harboring the plasmid. 295

296

Susceptibility of the ΔuppP mutant to lysozyme. Previous studies have shown that the 297

product of the UppP-mediated enzymatic reaction, C55-P, is involved in biosynthesis of 298

various cell wall components including peptidoglycan, LPS, colanic acid and teichoic acid 299

(30-34, 39). Hence, we compared protein composition, carbohydrate expression and 300

lysozyme susceptibility of the wildtype Burkholderia symbiont strain and the ΔuppP mutant 301

strain. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins 302

extracted from the cultured Burkholderia cells, the whole lysates (WL) were partitioned into 303

aqueous soluble (AQ), Triton X-114 soluble (TX) and insoluble (IS) fractions. No notable 304

differences in protein profiles were detected between the wildtype strain and ΔuppP mutant 305

(Fig. 2A). Carbohydrates extracted from proteinase K-treated bacterial lysates were separated 306

by SDS-PAGE and subjected to periodic acid oxidation and fluorescent staining. Ladder 307

patterns representing repeating units of LPS O-antigen and high molecular weight bacterial 308

carbohydrates were commonly detected in the wildtype strain and ΔuppP mutant, and the 309

profiles exhibited no apparent differences between them (Fig. 2B). On the other hand, when 310

lysozyme was added to bacterial cell suspensions, the ΔuppP mutant exhibited a much 311

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greater reduction in turbidity than the wildtype strain, and the reduction in turbidity was 312

restored in the ΔuppP/uppP-complemented mutant to the level of the wildtype strain (Fig. 313

2C). These results indicate that cell wall integrity of the ΔuppP mutant is impaired by 314

disruption of the uppP gene. 315

316

Atrophied host symbiotic organ and symbiosis defect in the Riptortus host infected 317

with the ΔuppP mutant. To examine the symbiotic properties of the ΔuppP mutant, wildtype, 318

ΔuppP or ΔuppP/uppP Burkholderia cells were orally administered to early second instar 319

Riptortus nymphs. The insects were reared to the fourth instar, and their midgut symbiotic 320

organs were dissected and inspected morphologically. In wildtype-infected insects, the 321

symbiotic organs were well developed and hazy in color, which was indicative of bacterial 322

cells filling the midgut crypts (Fig. 3A). In ΔuppP-infected insects, by contrast, the symbiotic 323

organs were atrophied and translucent in color (Fig. 3B), which was reminiscent of the 324

symbiotic organs of uninfected control insects (Fig. 3C). In ΔuppP/uppP-infected insects, the 325

well-developed hazy symbiotic organs were restored (Fig. 3D). Diagnostic PCR of the 326

dissected symbiotic organs confirmed the absence of symbiont infection in the ΔuppP-327

infected insects (Fig. 3E). These results indicate that the ΔuppP mutant strain is deficient in 328

symbiosis and that disruption of the uppP gene is responsible for this phenotype. 329

330

Initial infection but no proliferation of the ΔuppP mutant in the host symbiotic 331

organ. To compare the initial infection processes of the wildtype strain and the ΔuppP mutant, 332

second instar Riptortus nymphs were orally administered with the cultured symbiont strains 333

and maintained for 10, 15, 20 or 25 h after inoculation. Subsequently, their midguts were 334

dissected, individually subjected to DNA extraction, and analyzed by quantitative PCR 335

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targeting dnaA gene of the Burkholderia symbiont strain (Fig. 4A). The wildtype strain was 336

already detectable in both the M3 region and the M4 symbiotic region of the host midgut at 337

10 h after inoculation, and the symbiont population steadily increased at 15, 20 and 25 h after 338

inoculation. In contrast, the ΔuppP mutant was also detected in both the M3 region and the 339

M4 symbiotic region of the host midgut at 10 h after inoculation, but the symbiont population 340

exhibited no increase at 15, 20 or 25 h after inoculation (Fig. 4A). We also performed a 341

colony forming unit (CFU) assay for the wildtype strain, ΔuppP mutant and ΔuppP/uppP-342

complemented mutant on dissected midgut samples from second instar Riptortus nymphs at 343

36 and 63 h after inoculation (Fig. 4B). At these later stages, the infection titers of the ΔuppP 344

mutant (~102 per insect) were drastically lower than those of the wildtype strain (104~105 per 345

insect). Notably, infection titers of the ΔuppP/uppP-complemented mutant exhibited 346

significant restoration to 103~104 per insect (Fig. 4B). These results indicate that the ΔuppP 347

mutant is certainly incorporated into the host midgut, but cannot proliferate and survive in the 348

symbiotic organ, thereby failing to establish the symbiotic association with the Riptortus host. 349

350

Effect of symbiotic organ lysate on the ΔuppP mutant. Considering the lysozyme 351

susceptibility of the ΔuppP mutant (Fig. 2C) and its incapability of survival in the host 352

symbiotic organ (Figs. 3 and 4), we hypothesized that the host symbiotic organ may possess 353

bactericidal activities to which the wildtype strain is resistant but the ΔuppP mutant is 354

susceptible. To explore this possibility, we dissected fifth instar Riptortus nymphs and 355

collected their midguts. The dissected symbiotic organs were homogenized and heat-treated 356

to inactivate intrinsic Burkholderia cells, and the lysates at different concentrations were 357

applied to cultured wildtype Burkholderia cells and ΔuppP mutant cells. No significant effect 358

of the midgut lysate was observed on either the wildtype strain or the ΔuppP mutant (Fig. 5A). 359

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360

Survival of the ΔuppP mutant under environmental stress conditions. The wildtype 361

strain, the ΔuppP mutant and the ΔuppP/uppP-complemented mutant of the Burkholderia 362

symbiont were exposed to several stressful conditions in vitro, and their survival was 363

evaluated by CFU assay. Under a hypotonic condition in 10 mM phosphate buffer for 24 h, 364

the ΔuppP mutant exhibited a significantly lower survival rate than the wildtype strain and 365

the ΔuppP/uppP-complemented mutant (Fig. 5B). Under a hypertonic condition in 1 M 366

glucose for 24 h, the ΔuppP mutant also showed a significantly lower survival rate than the 367

wildtype strain and the ΔuppP/uppP-complemented mutant (Fig. 5C). When centrifugal 368

pressure at 20,000 x g for 30 min was applied to the cultured Burkholderia cells, the ΔuppP 369

mutant again showed a significantly lower survival rate than the wildtype strain and the 370

ΔuppP/uppP-complemented mutant (Fig. 5D). These results strongly suggest that the ΔuppP 371

mutant is susceptible to environmental stresses, which is likely attributable to the impaired 372

cell wall integrity caused by the disruption of uppP gene. 373

374

DISCUSSION 375

In this study, we show that the ΔuppP mutant of the Burkholderia symbiont fails to 376

establish symbiosis in the host midgut M4 region while the ΔuppP/uppP-complemented 377

mutant restores normal association with the host midgut (Figs. 3 and 4). These results 378

indicate that uppP gene of the Burkholderia symbiont is essential for establishing normal gut 379

symbiotic association with the Riptortus host. 380

381

In E. coli and other bacteria, uppP gene is involved in the biosynthesis of various cell 382

wall components, including peptideglycan, LPS and others (29-34). The ΔuppP mutant of the 383

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Burkholderia symbiont exhibits higher susceptibility to lysozyme than the wildtype strain, 384

while the ΔuppP/uppP-complemented mutant shows a restored level of lysozyme 385

susceptibility comparable to that of the wildtype strain (Fig. 2C). These results indicate that 386

disruption of the uppP gene impairs integrity of the symbiont cell wall, and suggest that the 387

cell wall defect is likely relevant to the symbiosis defect of the ΔuppP mutant. 388

389

Why the ΔuppP mutant cannot establish infection in the host’s symbiotic organ is 390

currently elusive. Considering the lysozyme susceptibility and the impaired cell wall integrity 391

of the ΔuppP mutant (Fig. 2C), a hypothesis is that the symbiotic midgut is producing 392

bactericidal factors such as lysozymes or antimicrobial peptides, to which the wildtype 393

symbiont is resistant but the ΔuppP mutant (and possibly also non-symbiotic bacteria) is 394

susceptible. Our results that lysates of the midgut M4 region of fifth instar Riptortus nymphs 395

affected neither the wildtype symbiont nor the ΔuppP mutant (Fig. 5A) do not support this 396

hypothesis, but it should be noted that the lysate was heat-treated to kill intrinsic 397

Burkholderia cells, and thus heat-sensitive bactericidal factors may have been inactivated by 398

the treatment. Although the midgut lysate was prepared from fifth instar nymphs due to 399

difficulty in collecting sufficient amount of the sample from younger nymphs, it should also 400

be noted that the Burkholderia infection initially establishes in the host midgut at the second 401

instar, not the fifth. Interestingly, a recent transcriptomic analysis of the midgut regions of 402

Riptortus nymphs revealed that host antimicrobial genes, such as a c-type lysozyme gene and 403

a defensin-like gene, are highly expressed in asymbiotic insects but scarcely expressed in 404

symbiotic insects (40). In the bacteriocytes of the grain weevils, an antimicrobial peptide, 405

coleoptericin A, regulates the population and proliferation of the Sodalis-allied endosymbiont 406

(41). In the bacteriocytes of the pea aphid, two i-type lysozyme genes are specifically 407

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expressed and represent the most abundant transcripts in the symbiotic cells, presumably 408

regulating the population and proliferation of the Buchnera endosymbiont (42). Hence, the 409

possibility cannot be ruled out that such bactericidal gene products are preferentially 410

expressed in the Riptortus midgut, act on the symbiont cell wall, and result in the infection 411

failure of the ΔuppP mutant of the Burkholderia symbiont. 412

413

Considering the susceptibility of the ΔuppP mutant to environmental stresses, such as 414

low osmolality, high osmolality and high centrifugal pressure (Fig. 5B-D), an alternative 415

hypothesis is that the symbiotic conditions within the host midgut entail some environmental 416

stresses, to which the wildtype symbiont is resistant but the ΔuppP mutant is susceptible. 417

While the nature of the “symbiotic stress” is unknown, it may be osmotic, anoxic, nutritional, 418

immunological or a combination of these. In this context, a recent study demonstrated a 419

crucial involvement of bacterial stress-related genes in the Riptortus-Burkholderia symbiosis: 420

disruption of symbiont genes for synthesizing an endocellular storage polyester, 421

polyhydroxyalkanoate (PHA), which confers bacterial resistance to nutritional depletion and 422

other environmental stresses, resulted in failure of normal symbiotic association, while 423

complementation of the PHA synthesis genes rescued the symbiosis defect (54). It should be 424

noted that the “bactericidal factor hypothesis” and the “symbiotic stress hypothesis” may not 425

necessarily be mutually exclusive, on the ground that the bactericidal factors could be 426

regarded as comprising host-derived immunological stresses. 427

428

The cell wall is located on the outer surface of bacterial cells as a front line of host-429

symbiont interactions. Therefore, considerable attention has been paid to the possible 430

relevance of the symbiont cell wall to symbiosis, particularly to interactions with host’s 431

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innate immunity. For example, some endosymbiotic bacteria, such as Spiroplasma and 432

Wolbachia, exhibit remarkable degeneration in their cell wall, thereby eliciting no or little 433

innate immune responses of their host insects (43-46). Transcriptomic comparisons between 434

symbiotic and asymbiotic host insects have revealed that a variety of immunity-related genes, 435

including lysozyme genes and antimicrobial peptide genes, are up-regulated in symbiosis-436

associated patterns (40, 42, 47-50) . To our knowledge, apart from general studies of bacterial 437

cell wall changes and host immune responses, this study is the first to unequivocally identify 438

that a specific cell wall biosynthesis-related symbiont gene is required for an insect-bacterium 439

symbiotic association. 440

441

On the basis of previous studies in squid-Vibrio, nematode-Photorhabdus/Xenorhabdus 442

and other model symbiotic systems, Ruby (2008) classified symbiosis-deficient bacterial 443

mutants into (i) initiation mutants, which are unable to establish infection in the host, (ii) 444

accommodation mutants, which can establish infection but fail to reach the usual infection 445

density, and (iii) persistence mutants, which at first establish infection normally but are 446

unable to maintain the normal infection level (1). Under these criteria, the ΔuppP mutant can 447

be regarded as a mutant between an initiation mutant and accommodation mutant, because it 448

is able to infect initially but fails to establish colonization in the Riptortus host. The cell wall 449

deficiency of the ΔuppP mutant most likely affects the initial host-symbiont association, 450

which highlights a previously under-explored aspect of insect-bacterium symbiotic 451

associations. 452

453

454

455

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ACKNOWLEDGMENTS. This work was supported by the Global Research Laboratory 456

Grant of the National Research Foundation of Korea (grant number 2011-0021535) to B.L.L. 457

and T.F. We thank Joerg Graf (University of Connecticut) for providing plasmids. 458

459

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611

Figure legends 612

FIG 1 Growth curves of the wildtype Burkholderia symbiont strain (RPE161), the ΔuppP 613

mutant strain (BBL005) and the ΔuppP/uppP-complemented mutant strain (BBL105) in YG 614

medium (A) and in minimal medium (B). 615

FIG 2 In vitro characterization of the ΔuppP mutant strain BBL005. (A) Protein analysis of 616

the Burkholderia symbiont strains by SDS-PAGE. WL, whole lysate; AQ, aqueous soluble 617

fraction; TX, Triton X-114 soluble fraction; IS, insoluble fraction. (B) Carbohydrate analysis 618

by SDS-PAGE. (C) Lysozyme susceptibility assay of the Burkholderia strains. Error bars 619

indicate standard deviations. 620

FIG 3 (A-C) Morphology of host symbiotic midgut inoculated with Burkholderia symbiont 621

strains: wildtype strain RPE161(A), ΔuppP mutant strain BBL005(B), uninfected control (C), 622

and ΔuppP/uppP-complemented mutant strain BBL105 (D). Insects were orally administered 623

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with the Burkholderia cells at the second instar and dissected for inspection of the midgut at 624

the fourth instar. (E) Diagnostic PCR detection of the Burkholderia infection in midgut 625

dissected from third, fourth and fifth instar nymphs. 626

FIG 4 Quantitative analyses of the Burkholderia symbiont strains in the host symbiotic 627

organs of second instar Riptortus nymphs. (A) Quantitative PCR analysis of infection 628

densities of the wildtype strain RPE161 and the ΔuppP mutant BBL005 at 10, 15, 20 and 25 629

h after inoculation. (B) CFU quantification of infection densities of the wildtype strain 630

RPE161, the ΔuppP mutant BBL005, and the ΔuppP/uppP-complemented mutant BBL105 at 631

36 and 63 h after inoculation. Different letters (a, b) indicate statistically significant 632

differences (unpaired t-test; *, P<0.05; **, P< 0.01; ****, P<0.0001). 633

FIG 5 (A) Survival of the wildtype Burkholderia symbiont strain RPE161 and the ΔuppP 634

mutant strain BBL005 when symbiotic midgut lysates from fifth instar Riptortus nymphs 635

were added to the cultured bacteria. (B-D) Survival of the wildtype strain RPE161, the ΔuppP 636

mutant BBL005, and the ΔuppP/uppP-complemented mutant BBL105 under environmental 637

stress conditions. (B) Under a hypotonic condition in 10 mM phosphate buffer for 24 h. (C) 638

Under a hypertonic condition in 1 M glucose for 24 h. (D) Under a high gravity condition of 639

centrifugation at 20,000 x g for 30 min. Different letters (a, b) indicate statistically significant 640

differences (unpaired t-test with Bonferroni correction; P < 0.05). 641

642

643

644

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TABLE 1 Bacteria strains and plasmids used this study Strain or plasmid Relevant characteristics Reference

Burkholderia symbiont

RPE161 Burkholderia symbiont (RPE64); CmR

(24)

BBL005 RPE161 ΔuppP; CmR This study

BBL105 BBL005 / pBL5, complementation of uppP; CmR, KmR

This study

Escherichia coli

DH5α

deoR, endA1, gyrA96, hsdR17(rk-,mk+), phoA, recA1, relA1, supE44, thi-1,Δ(lacZYA-argF)U169, φ80dlacZΔM15, F-, λ-

TOYOBO

CC118λpir Carrying helper plasmid pEVS104; RifR, KmR

(51)

Plasmid

pEVS104 oriR6K helper plasmid containing conjugal tra and trb; KmR

(51)

pK18mobsacB pMB1ori allelic exchange vector containing oriT; KmR

(52)

pBBR122 Broad host vector; CmR, KmR (53)

pBL5 pBBR122 derivative containing uppP; KmR

This study

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