1
Journal section: Invertebrate microbiology 1
2
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
14
Running title: Cell wall synthesis gene in insect-bacterium symbiosis 15
16
#Address correspondence to Bok Luel Lee, [email protected] or 17
Takema Fukatsu, [email protected] 18
19
Key words: insect-bacterium symbiosis, bacterial cell wall, undecaprenyl pyrophosphate 20
phosphatase (UppP) 21
22
23
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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