Bacterial Cell Wall Synthesis Gene uppP Is Required for BurkholderiaColonization of the Stinkbug Gut

Jiyeun Kate Kim,a Ho Jin Lee,a Yoshitomo Kikuchi,b Wataru Kitagawa,b Naruo Nikoh,c Takema Fukatsu,d Bok Luel Leea

Global Research Laboratory, College of Pharmacy, Pusan National University, Pusan, South Koreaa; National Institute of Advanced Industrial Science and Technology,Hokkaido Center, Sapporo, Japanb; Department of Liberal Arts, The Open University of Japan, Chiba, Japanc; Institute for Biological Resources and Functions, NationalInstitute of Advanced Industrial Science and Technology, Tsukuba, Japand

To establish a host-bacterium symbiotic association, a number of factors involved in symbiosis must operate in a coordinatedmanner. In insects, bacterial factors for symbiosis have been poorly characterized at the molecular and biochemical levels, sincemany symbionts have not yet been cultured or are as yet genetically intractable. Recently, the symbiotic association between astinkbug, Riptortus pedestris, and its beneficial gut bacterium, Burkholderia sp., has emerged as a promising experimental modelsystem, providing opportunities to study insect symbiosis using genetically manipulated symbiotic bacteria. Here, in search ofbacterial symbiotic factors, we targeted cell wall components of the Burkholderia symbiont by disruption of uppP gene, whichencodes undecaprenyl pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall components. Underculture conditions, the �uppP mutant showed higher susceptibility to lysozyme than the wild-type strain, indicating impairedintegrity of peptidoglycan of the mutant. When administered to the host insect, the �uppP mutant failed to establish normalsymbiotic association: the bacterial cells reached to the symbiotic midgut but neither proliferated nor persisted there. Transfor-mation of the �uppP mutant with uppP-encoding plasmid complemented these phenotypic defects: lysozyme susceptibility invitro was restored, and normal infection and proliferation in the midgut symbiotic organ were observed in vivo. The �uppP mu-tant also exhibited susceptibility to hypotonic, hypertonic, and centrifugal stresses. These results suggest that peptidoglycan cellwall integrity is a stress resistance factor relevant to the successful colonization of the stinkbug midgut by Burkholderiasymbiont.

Many insects are in intimate symbiotic associations with bac-teria. Such symbiotic bacteria exist in the gut lumen, body

cavity, or inside cells. To establish a successful host-symbiont as-sociation, a number of molecular factors from the symbiont side,and also from the host side, must work in a coordinated manner.To understand the mechanisms of these intricate host-symbiontinteractions, several model symbiotic systems have been used toidentify novel symbiotic factors and to determine their molecularfunctions (1). For example, the legume-Rhizobium nitrogen-fix-ing symbiosis and the squid-Vibrio luminescent symbiosis havebeen studied in depth. In both systems, the symbiotic bacteria areeasily cultivable and genetically manipulatable and are thus suit-able for elucidating the molecular properties of their symbioticfactors (2–8).

However, among insect-microbe symbiotic systems, molecu-lar factors relevant to symbiosis have been poorly characterizedexcept for inferences from genomic information (9–11). The pau-city of molecular and biochemical studies is attributed to the dif-ficulty in isolating and culturing symbiotic bacteria outside insecthosts. Consequently, powerful mutant-based molecular geneticapproaches have not been effectively applied to insect-microbesymbiotic systems in general. Obligate insect symbionts, such asBuchnera in aphids and Wigglesworthia in tsetse flies, have beenassociated with their hosts over evolutionary time and are incapa-ble of independent living and thus are uncultivable (9, 12). As forfacultative insect symbionts, such as Wolbachia in various insectsand Sodalis in tsetse flies, which are transmitted through host gen-erations not only vertically but also horizontally, at least some ofthem are cultivable outside their host insects and thus potentiallygenetically manipulable (13–15). However, culturing these sym-bionts is generally not easy because it requires complex culture

media containing either mammalian sera or live insect cells, andthe symbionts grow very slowly, are prone to contamination, andare reluctant to form colonies on agar plates (16). Therefore, pre-vious studies on bacterial symbiotic factors using genetically ma-nipulated symbionts have been limited (16–21).

The bean bug Riptortus pedestris belongs to the stinkbug familyAlydidae in the insect order Hemiptera. In contrast to previouslyknown insect-bacterium symbiotic systems, nymphal R. pedestrisacquires a betaproteobacterial symbiont of the genus Burkholderianot vertically but from the soil environment every generation(22). A posterior region of the insect midgut bears numerouscrypts whose lumens are filled with bacterial cells of the symbioticBurkholderia (23). Reflecting its free-living origin in the environ-ment, the symbiotic Burkholderia is easily cultivable on standardmicrobiological media and can be experimentally reinfected intothe host insect by oral administration (24, 25). Comparisons be-tween symbiotic and asymbiotic insects showed beneficial fitnessconsequences of Burkholderia infection to the host insect (22, 26).These features of the Riptortus-Burkholderia gut symbiotic systemprovide unprecedented opportunities to study insect symbiosis atmolecular and biochemical levels.

The cell wall of Gram-negative bacteria is the front-line of in-

Received 22 April 2013 Accepted 4 June 2013

Published ahead of print 7 June 2013

Address correspondence to Bok Luel Lee, brlee@pusan.ac.kr, or Takema Fukatsu,t-fukatsu@aist.go.jp.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01269-13

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teracting with the surrounding environment. It consists of an in-ner membrane, an outer membrane in which lipopolysaccharide(LPS) forms the outer leaflet, and a periplasmic region where thepeptidoglycan layer resides (27). Bacterial cell wall componentssuch as LPS and peptidoglycan are essential for maintaining thestructural integrity of bacterial cells and generally required forviability (27, 28). In addition, these cell wall components mostlikely play a role in bacterial association with host and hence, mayfunction as symbiotic factors. Biosynthesis of bacterial cell wallcomponents, such as LPS and peptidoglycan, requires a key lipidcarrier, undecaprenyl phosphate (C55-P), which is generated fromdephosphorylation of undecaprenyl pyrophosphate (C55-PP)(29–34). C55-P is a precursor of various cell wall components thatare synthesized in the cytoplasm and transported to the periplasm,where further polymerization occurs. After release from the cellwall component precursors, the lipid carrier is in a pyrophosphateform (C55-PP) and requires another dephosphorylation step be-fore being reused as a lipid carrier (35). This dephosphorylationstep is catalyzed by C55-PP phosphatase enzymes. Four C55-PPphosphatases have been identified in Escherichia coli: UppP (alsocalled BacA), YbjG, YeiU and PgpB, of which UppP is regarded asthe major phosphatase (36, 37).

To identify bacterial symbiotic factors in the Riptortus-Burk-holderia symbiosis, we targeted the bacterial cell wall-related uppPgene. We generated an uppP-deficient mutant (�uppP) of theBurkholderia symbiont by allelic exchange and homologous re-combination. Because the �uppP mutant shows 75% reduction ofC55-PP phosphatase activity in E. coli (36), we hypothesized thatthe decrease of C55-PP phosphatase activity affects the cell wallcomponent synthesis, resulting in defected cell wall. Since the ac-tual effects on the cell wall by the uppP mutation are not wellcharacterized, we first examined cell wall components of a �uppPBurkholderia strain. Furthermore, the growth phenotypes in vitroand symbiotic phenotypes in vivo of the �uppP mutant were com-pared to those of the wild-type Burkholderia symbiont and an�uppP/uppP-complemented mutant transfected with a plasmidencoding a functional uppP gene.

MATERIALS AND METHODS

Bacteria, plasmids, and culture media. Bacterial strains and plasmidsused in the present study are listed in Table 1. E. coli cells were cultured at37°C in LB medium (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract,and 0.5% [wt/vol] NaCl). Cells of Burkholderia symbiont strain RPE161, aspontaneous chloramphenicol-resistant mutant derived from RPE64(24), were cultured at 30°C in YG medium (0.5% [wt/vol] yeast extract,0.4% [wt/vol] glucose, and 0.1% [wt/vol] NaCl). Antibiotics were used atthe following concentrations unless otherwise described: kanamycin at 30�g/ml and chloramphenicol at 10 �g/ml.

Generation of �uppP mutant. Deletion of the chromosomal uppPgene from the Burkholderia symbiont was accomplished by allelic ex-change and homologous recombination using a suicide vectorpK18mobsacB containing the 5= (uppP-L) and 3= (uppP-R) regions ofuppP gene. The wild-type Burkholderia symbiont strain RPE161 was sub-jected to PCR using the primers uppP-L-P1 (5=-TTT AAG CTT GAG TTCGAC TTC GAG CGT GT-3=) and uppP-L-P2 (5=-TTT GGA TCC AAGACT GCT GAC CGG AAA AA-3=) for the uppP-L region, and the primersuppP-R-P1 (5=-TTT GGA TCC TTC TTC TTC GGC TGG TTC AT-3=)and uppP-R-P2 (5=-TTT GAA TTC GCA CTG GAA AAC CTC AGC A-3=)for the uppP-R region. PCR products and the pK18mobsacB vector weredigested with proper restriction enzymes, ligated, and transformed into E.coli DH5� cells. The transformed E. coli cells were selected on LB-agarplates containing 100 �g of kanamycin/ml. Positive colonies carrying avector with the correct insert were further selected by colony PCR usingthe primer uppP-L-P1 and the vector primer aphII (5=-ATC CAT CTTGTT CAA TCA TGC G-3=). Donor E. coli cells carrying the pK18mobsacBcontaining uppP-L and uppP-R were mixed with recipient BurkholderiaRPE161 cells and also E. coli CC118�pir cells carrying a helper plasmidpEVS104 to transfer the cloned vector to the RPE161 cells. After allowinga single crossover by culturing cell mixtures of triparental conjugation onYG-agar, RPE161 cells with the first crossover were selected on YG-agarcontaining chloramphenicol (30 �g/ml) and kanamycin. Positive colo-nies with the genomic integration of vector DNA were confirmed by PCRusing the chromosomal primer uppP-up (5=-GAG GCA ATG AAA CGTATC GAC-3=) and the vector primer aphII. The second crossover wasallowed by culturing cells with the single crossover in YG media andBurkholderia cells with a double crossover were selected on YG-agarcontaining chloramphenicol and sucrose (200 �g/ml). The mutantstrain with deletion of the uppP gene (BBL005) was identified by PCR

TABLE 1 Bacterial strains and plasmids used this study

Strain or plasmid Relevant characteristicsa Source or reference

StrainsBurkholderia symbionts

RPE161 Burkholderia symbiont (RPE64); Cmr 24BBL005 RPE161 �uppP; Cmr This studyBBL105 BBL005/pBL5, complementation of uppP; Cmr Kmr This study

Escherichia coliDH5� deoR endA1 gyrA96 hsdR17(rK

� mK�) phoA recA1 relA1 supE44

thi-1 �(lacZYA-argF)U169 �80dlacZ�M15 F� ��

Toyobo

CC118�pir Carrying helper plasmid pEVS104; Rifr Kmr 51

PlasmidspEVS104 oriR6K helper plasmid containing conjugal tra and trb; Kmr 51pK18mobsacB pMB1ori allelic exchange vector containing oriT; Kmr 52pBBR122 Broad host vector; Cmr Kmr 53pBL5 pBBR122 derivative containing uppP; Kmr This study

a Cmr, chloramphenicol resistance; Rifr, rifampin resistance; Kmr, kanamycin resistance.

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using the primers uppP-up and uppP-down (5=-CCA GCA TCT GCTCTT TGT CA-3=) and sequencing of the PCR product.

Generation of �uppP/uppP-complemented mutant. A DNA frag-ment containing the open reading frame of uppP gene was amplified fromRPE161 using the primers uppP-com-P1 (5=-GCA CGG CAA TTT TTCTCT TC-3=) and uppP-com-P2 (5=-CGA CTC GAA CGT GTG ACC TA-3=). The amplified DNA fragment was cloned into the DraI site ofpBBR122 to generate the plasmid pBL5. The cloned plasmid was intro-duced into E. coli DH5� cells to generate donor cells. By triparental con-jugation with the BBL005 recipient cells and E. coli CC118�pir helper cells,the pBL5 plasmid carried by the donor E. coli DH5� cells was transferredto the recipient Burkholderia BBL005 cells, yielding the complementedBurkholderia BBL105 cells. The complemented mutant strain was selectedon YG-agar with chloramphenicol (30 �g/ml) and kanamycin.

Measurement of bacterial growth in liquid media. Growth curves ofthe Burkholderia symbiont strains were examined either in YG medium orin minimal medium (0.6% Na2HPO4·2H2O, 0.3% KH2PO4, 0.1% NH4Cl,0.05% NaCl, 0.0003% CaCl2, 1 mM MgSO4, 0.2% glucose). The startingcell solutions were prepared by adjusting the optical density at 600 nm[OD600] to 0.05 in either YG medium or minimal medium using primaryculture grown in YG medium at 30°C for 18 h. The cell solutions wereincubated on a rotator shaker at 180 rpm at 30°C for 36 h, whose OD600

was monitored every 2 h using a spectrophotometer (Mecasys, SouthKorea).

Protein analysis of bacterial lysates. Burkholderia symbiont cells wereharvested at an OD600 of 1 after culturing in YG medium. The cells werewashed with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM NaH2PO4, and 3mM KH2PO4 at pH 7) and resuspended at 2 � 107 cells/�l in PBS. Analiquot of this solution was saved for the whole-lysate fraction (WL). Thecell suspension was then sonicated and further diluted to 107 cells/�lequivalent in PBS containing 10 mM EDTA, 100 �g of egg white lysozyme(BioShop Canada, Inc., Canada)/ml, and protease inhibitors (Roche, Ger-many). After adding one-fourth volume of 10% Triton X-114 (final, 2%),the cell solution was agitated for 1 h at 4°C. The sample was centrifuged(15,000 � g for 20 min at 4°C), and the pellet was saved for the insolublefraction (IS), while the supernatant was transferred to a new tube. The ISfraction was washed with PBS and resuspended in 1� Laemmli samplebuffer. The liquid was incubated at 37°C for 10 min and centrifuged(10,000 � g for 10 min at 25°C). After a 10-min incubation at roomtemperature, the separated aqueous fraction (AQ) was transferred to afresh tube and supplemented with Triton X-114 solution to a final con-centration of 2% for additional phase partitioning before collecting thefinal AQ fraction. The Triton X-114 fractions (TX) from both partition-ings were combined, and an equal volume of TBSE solution (20 mMTris-HCl [pH 8], 130 mM NaCl, and 5 mM EDTA) was added. Afteragitation for 10 min at 4°C, the samples were centrifuged (10,000 � g for10 min at 25°C) and separated into upper and lower phases at roomtemperature. The upper layer was then discarded, TBSE was added, andthe procedure was repeated. Final TX fractions were precipitated withcold ethanol, and dried precipitates were resuspended in 1� Laemmlisample buffer. Proteins from different phase fractions were separated bySDS–15% PAGE and visualized by staining with Coomassie brilliant blueR250. The loading quantity for each fraction was 7 � 107 cells equivalentfor WL, 7 � 107 cells equivalent for AQ, 6 � 108 cells equivalent for TX,and 3 � 108 cells equivalent for IS.

Carbohydrate analysis. The WL samples prepared for protein analy-sis by SDS-PAGE were used for the analysis of bacterial carbohydrates.The WL sample was boiled in 1� Laemmli sample buffer, deproteinatedby incubation with 400 �g of proteinase K/ml at 60°C for 1 h, and reboiledprior to SDS-PAGE. Loading amount was 1 � 108 cells equivalent per lanefor 12% Laemmli SDS-PAGE gels and 2 � 108 cells equivalent per lane for12% Tris-Tricine SDS-PAGE gels. Bacterial carbohydrates separated inthe gels were visualized using the Pro-Q Emerald 300 lipopolysaccharidegel stain kit (Invitrogen). Briefly, the gels were fixed with 5% acetic acidand 50% methanol, washed three times with 3% acetic acid, incubated

with oxidizing solution containing periodic acid for 30 min, washed threetimes again with 3% acetic acid, and stained with Pro-Q Emerald 300staining solution for 2 h. After two washes with 3% acetic acid, the gelswere observed with the gel documentation system GDS-200.

Lysozyme susceptibility assay. Frozen mid-log phase Burkholderiacells were thawed and resuspended in PB (10 mM sodium phosphate, pH7). After washing with PB, 0.9 ml of the Burkholderia cell suspension wasprepared at an OD600 of 0.77 to 0.78 in PB and transferred to a cuvette forspectrophotometry. After an addition of 0.08 ml of lysis solution (500 �gof egg white lysozyme/ml in PB with 100 mM EDTA), the OD600 of the cellsuspension was measured every 2 min up to 28 min and then every 5 minuntil 73 min. As a control, 0.08 ml of PB containing 100 mM EDTA wasadded to the cell suspension.

Insect rearing and symbiont inoculation. R. pedestris bean bugs werereared in our insect laboratory at 28°C under a long day regime of 16 hlight and 8 h dark as described previously (38). Nymphal insects werereared in clean plastic containers (34 cm by 19.5 cm wide by 27.5 cm high)supplied with soybean seeds and DWA (distilled water containing 0.05%ascorbic acid). Upon reaching adulthood, the insects were transferred to abigger container (35 cm by 35 cm wide by 40 cm high) in which soybeanplant pots were placed for food and cotton pads were attached to the wallsas a substrate for egg laying. Eggs were collected daily and transferred tonew cages for hatching. Newly molted second instar nymphs were pro-vided with wet cotton balls soaked with a symbiont inoculum solutionconsisting of mid-log-phase Burkholderia cells suspended in DWA at aconcentration of 107 cells/ml.

The care and treatment of Burkholderia cells and insects in all proce-dures strictly followed the guidelines of the Pusan National University(PNU) Institutional Animal Care and Use Committee (IACUC) and theLiving Modified Organ (LMO) Committee.

Diagnostic PCR. Insects were surface sterilized briefly with 70% eth-anol and dissected in PBS in a glass petri dish using fine scissors andforceps under a dissection microscope. Dissected samples of the posteriormidgut M4 region were individually subjected to DNA extraction usingthe QIAamp DNA minikit (Qiagen). Diagnostic PCR was conducted us-ing GoTaq Green Master Mix (Promega) with the supplied buffer systemunder a temperature profile of 95°C for 10 min, followed by 30 cycles of95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, and finally 72°C for 2 minusing the primers Burk16SF (5=-TTT TGG ACA ATG GGG GCA AC-3=)and Burk16SR (5=-GCT CTT GCG TAG CAA CTA AG-3=), which specif-ically target 16S rRNA gene of the Burkholderia symbiont (38). PCR prod-ucts were analyzed by 1% agarose gel electrophoresis and a 100-bp DNAladder was used to estimate product size.

CFU assay. Each of the M4 midgut regions dissected from secondinstar Riptortus nymphs was collected in 50 �l of PB and homogenized bya pestle mortar. The homogenized sample was diluted if necessary andspread on YG-agar plates containing chloramphenicol. After 2 days ofincubation at 30°C, colonies on the plates were counted, and the numberof symbiont cells in the sample was calculated as the CFU � the dilutionfactor.

Quantitative PCR. Quantitative PCR for estimating titers of the Burk-holderia symbiont was performed as described previously (38). Dissectedmidgut samples (either M3 or M4) were individually subjected to DNAextraction by QIAamp DNA minikit (Qiagen). DNA samples were mixedwith a master PCR solution containing 2� qPCR premix of QuantiMixSYBR kit (PhileKorea) and the primers BSdnaA-F (5=-AGC GCG AGATCA GAC GGT CGT CGA T-3=) and BSdnaA-R (5=-TCC GGC AAG TCGCGC ACG CA-3=), which target a 0.15-kb region of dnaA gene of theBurkholderia symbiont. The PCR temperature profile was 40 cycles of95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. A standard curve for dnaAgene copies was generated using a series of extracted DNA samples con-taining known numbers of Burkholderia cells.

CFU assay after stress treatments. For each test, the starting CFUwere compared to CFU after the following treatments. (i) For the M4lysate treatment, midgut M4 regions dissected from fifth-instar Riptortus

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nymphs were homogenized and heat treated at 55°C for 5 min to killintrinsic symbiont cells prior to the assay. Different concentrations of theM4 lysate, ranging from 0.0 to 0.4 mg/ml, were incubated with culturedBurkholderia cells at mid-log phase for 1 h at room temperature. Afterincubation, the samples were diluted, spread on YG-agar plates, culturedfor 2 days, and subjected to colony counting. (ii) For the hypotonic test,mid-log-phase Burkholderia cells in YG medium were washed with 10mM PB and adjusted to 107 cells/ml in PB. The cell suspensions wereincubated at 30°C for 24 h and subjected to CFU assay. (iii) For the hy-pertonic test, mid-log-phase Burkholderia cells were adjusted to an OD600

of 0.5 to 0.7 in YG medium. The cell suspension was combined with anequal volume of 2 M glucose solution, incubated at 30°C for 24 h, andsubjected to CFU assay. (iv) For the centrifugal pressure test, mid-log-phase Burkholderia cells cultured in YG medium were adjusted to 104

cells/ml, placed in 1.5-ml microcentrifuge tubes, centrifuged at 15,000rpm (20,000 � g) for 30 min, and subjected to CFU assay.

RESULTSGrowth rates of wild-type and mutant Burkholderia symbiontstrains. We disrupted the uppP gene of the wild-type Burkholderiasymbiont strain RPE161, thereby establishing a �uppP mutantBurkholderia symbiont strain BBL005. By transforming the �uppPmutant strain with a plasmid encoding a functional uppP gene, wealso generated a �uppP/uppP-complemented mutant Burkhold-eria symbiont strain BBL105. Growth curves of these Burkholderiastrains in nutritionally rich yeast-glucose (YG) medium revealedthat the wild-type strain and the �uppP mutant exhibited similargrowth rates, while the �uppP/uppP-complemented mutant grewa little slower (Fig. 1A). Growth curves in nutritionally limitedminimal medium exhibited similar patterns, although growthrates overall were much slower in minimal medium than in YGmedium (Fig. 1B). These results indicate that deletion of the uppPgene does not affect growth of the Burkholderia symbiont under invitro culture conditions. The slower growth of the �uppP/uppP-complemented mutant may be due to a cost of harboring theplasmid.

Susceptibility of the �uppP mutant to lysozyme. Previousstudies have shown that the product of the UppP-mediated enzy-matic reaction, C55-P, is involved in biosynthesis of various cellwall components including peptidoglycan, LPS, colanic acid, andteichoic acid (30–34, 39). Hence, we compared protein composi-tion, carbohydrate expression and lysozyme susceptibility of thewild-type Burkholderia symbiont strain and the �uppP mutantstrain. In sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE) of proteins extracted from the cultured Burk-holderia cells, the whole lysates (WL) were partitioned into water-soluble (aqueous [AQ]), Triton X-114-soluble (TX), and water/Triton X-114-insoluble (IS) fractions. No notable differences inprotein profiles were detected between the wild-type strain and�uppP mutant (Fig. 2A). Carbohydrates of proteinase K-treatedbacterial lysates were separated by SDS-PAGE and subjected toperiodic acid oxidation and fluorescent staining. Ladder patternsrepresenting repeating units of LPS O-antigen and high-molecu-lar-weight bacterial carbohydrates were commonly detected in thewild-type strain and �uppP mutant, and the profiles exhibited noapparent differences between them (Fig. 2B). On the other hand,when lysozyme was added to bacterial cell suspensions, the �uppPmutant exhibited a much greater reduction in turbidity than thewild-type strain, and the reduction in turbidity was restored in the�uppP/uppP-complemented mutant to the level of the wild-type

strain (Fig. 2C). These results indicate that cell wall integrity of the�uppP mutant is impaired by disruption of the uppP gene.

Atrophied host symbiotic organ and symbiosis defect in theRiptortus host infected with the �uppP mutant. To examine thesymbiotic properties of the �uppP mutant, wild-type, �uppP, or�uppP/uppP Burkholderia cells were orally administered to earlysecond instar Riptortus nymphs. The insects were reared to thefourth instar, and their midgut symbiotic organs were dissectedand inspected morphologically. In wild-type-infected insects, thesymbiotic organs were well developed and hazy in color, whichwas indicative of bacterial cells filling the midgut crypts (Fig. 3A).In �uppP-infected insects, by contrast, the symbiotic organs wereatrophied and translucent in color (Fig. 3B), which was reminis-cent of the symbiotic organs of uninfected control insects (Fig.3C). In �uppP/uppP-infected insects, the well-developed hazysymbiotic organs were restored (Fig. 3D). Diagnostic PCR of thedissected symbiotic organs confirmed the absence of symbiontinfection in the �uppP-infected insects (Fig. 3E). These resultsindicate that the �uppP mutant strain is deficient in symbiosis andthat disruption of the uppP gene is responsible for this phenotype.

Initial infection but no proliferation of the �uppP mutant inthe host symbiotic organ. To compare the initial infection pro-cesses of the wild-type strain and the �uppP mutant, second-in-star Riptortus nymphs were orally administered with the culturedsymbiont strains and maintained for 10, 15, 20, or 25 h after inoc-

FIG 1 Growth curves of the wild-type Burkholderia symbiont strain (RPE161),the �uppP mutant strain (BBL005), and the �uppP/uppP-complemented mutantstrain (BBL105) in YG medium (A) and in minimal medium (B).

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ulation. Subsequently, their midguts were dissected, individuallysubjected to DNA extraction, and analyzed by quantitative PCRtargeting dnaA gene of the Burkholderia symbiont strain (Fig. 4A).The wild-type strain was already detectable in both the M3 regionand the M4 symbiotic region of the host midgut at 10 h afterinoculation, and the symbiont population steadily increased at 15,20, and 25 h after inoculation. In contrast, the �uppP mutant wasalso detected in both the M3 region and the M4 symbiotic regionof the host midgut at 10 h after inoculation, but the symbiontpopulation exhibited no increase at 15, 20, or 25 h after inocula-tion (Fig. 4A). We also performed a CFU assay for the wild-typestrain, �uppP mutant, and �uppP/uppP-complemented mutanton dissected midgut samples from second instar Riptortus nymphsat 36 and 63 h after inoculation (Fig. 4B). At these later stages, theinfection titers of the �uppP mutant (102 per insect) were dras-tically lower than those of the wild-type strain (104 to 105 perinsect). Notably, infection titers of the �uppP/uppP-comple-mented mutant exhibited significant restoration to 103 to 104 perinsect (Fig. 4B). These results indicate that the �uppP mutant iscertainly incorporated into the host midgut but cannot proliferateand survive in the symbiotic organ, thereby failing to establish thesymbiotic association with the Riptortus host.

Effect of symbiotic organ lysate on the �uppP mutant. Con-sidering the lysozyme susceptibility of the �uppP mutant (Fig. 2C)and its incapability of survival in the host symbiotic organ (Fig. 3and 4), we hypothesized that the host symbiotic organ may possessbactericidal activities to which the wild-type strain is resistant butthe �uppP mutant is susceptible. To explore this possibility, we

dissected fifth-instar Riptortus nymphs and collected their mid-guts. The dissected symbiotic organs were homogenized and heat-treated to inactivate intrinsic Burkholderia cells, and the lysates atdifferent concentrations were applied to cultured wild-type Burk-holderia cells and �uppP mutant cells. No significant effect of themidgut lysate was observed on either the wild-type strain or the�uppP mutant (Fig. 5A).

Survival of the �uppP mutant under environmental stressconditions. The wild-type strain, the �uppP mutant, and the�uppP/uppP-complemented mutant of the Burkholderia symbi-ont were exposed to several stressful conditions in vitro, and theirsurvival was evaluated by CFU assay. Under a hypotonic condi-tion in 10 mM phosphate buffer for 24 h, the �uppP mutant ex-hibited a significantly lower survival rate than the wild-type strainand the �uppP/uppP-complemented mutant (Fig. 5B). Under ahypertonic condition in 1 M glucose for 24 h, the �uppP mutantalso showed a significantly lower survival rate than the wild-typestrain and the �uppP/uppP-complemented mutant (Fig. 5C).When centrifugal pressure at 20,000 � g for 30 min was applied tothe cultured Burkholderia cells, the �uppP mutant again showed asignificantly lower survival rate than the wild-type strain and the�uppP/uppP-complemented mutant (Fig. 5D). These resultsstrongly suggest that the �uppP mutant is susceptible to environ-mental stresses, which is likely attributable to the impaired cellwall integrity caused by the disruption of uppP gene.

DISCUSSION

In this study, we show that the �uppP mutant of the Burkholderiasymbiont fails to establish symbiosis in the host midgut M4 re-

FIG 2 In vitro characterization of the �uppP mutant strain BBL005. (A) Pro-tein analysis of the Burkholderia symbiont strains by SDS-PAGE. WL, wholelysate; AQ, aqueous soluble fraction; TX, Triton X-114 soluble fraction; IS,insoluble fraction. (B) Carbohydrate analysis by SDS-PAGE. (C) Lysozymesusceptibility assay of the Burkholderia strains. Error bars indicate standarddeviations.

FIG 3 (A to D) Morphology of host symbiotic midgut inoculated with Burk-holderia symbiont strains: wild-type strain RPE161 (A), �uppP mutant strainBBL005 (B), uninfected control (C), and �uppP/uppP-complemented mutantstrain BBL105 (D). Insects were orally administered with the Burkholderia cellsat the second instar and dissected for inspection of the midgut at the fourthinstar. (E) Diagnostic PCR detection of the Burkholderia infection in midgutdissected from third-, fourth-, and fifth-instar nymphs.

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gion, while the �uppP/uppP-complemented mutant restores nor-mal association with the host midgut (Fig. 3 and 4). These resultsindicate that uppP gene of the Burkholderia symbiont is essentialfor establishing normal gut symbiotic association with the Riptor-tus host.

In E. coli and other bacteria, uppP gene is involved in the bio-synthesis of various cell wall components, including peptidogly-can, LPS, and others (29–34). The �uppP mutant of the Burkhold-eria symbiont exhibits higher susceptibility to lysozyme than thewild-type strain, whereas the �uppP/uppP-complemented mu-tant shows a restored level of lysozyme susceptibility comparableto that of the wild-type strain (Fig. 2C). These results indicate thatdisruption of the uppP gene impairs integrity of the symbiont cellwall and suggest that the cell wall defect is likely relevant to thesymbiosis defect of the �uppP mutant.

Why the �uppP mutant cannot establish infection in the host’ssymbiotic organ is currently elusive. Considering the lysozyme

susceptibility and the impaired cell wall integrity of the �uppPmutant (Fig. 2C), a hypothesis is that the symbiotic midgut isproducing bactericidal factors such as lysozymes or antimicrobialpeptides, to which the wild-type symbiont is resistant but the�uppP mutant (and possibly also nonsymbiotic bacteria) is sus-ceptible. Our results that lysates of the midgut M4 region of fifth-instar Riptortus nymphs affected neither the wild-type symbiontnor the �uppP mutant (Fig. 5A) do not support this hypothesis,but it should be noted that the lysate was heat treated to kill in-trinsic Burkholderia cells and thus heat-sensitive bactericidal fac-tors may have been inactivated by the treatment. Although themidgut lysate was prepared from fifth-instar nymphs due to diffi-culty in collecting sufficient amount of the sample from youngernymphs, it should also be noted that the Burkholderia infectioninitially establishes in the host midgut at the second instar, not thefifth. Interestingly, a recent transcriptomic analysis of the midgutregions of Riptortus nymphs revealed that host antimicrobialgenes, such as a c-type lysozyme gene and a defensin-like gene, arehighly expressed in asymbiotic insects but scarcely expressed insymbiotic insects (40). In the bacteriocytes of the grain weevils, anantimicrobial peptide, coleoptericin A, regulates the populationand proliferation of the Sodalis-allied endosymbiont (41). In thebacteriocytes of the pea aphid, two i-type lysozyme genes are spe-cifically expressed and represent the most abundant transcripts inthe symbiotic cells, presumably regulating the population andproliferation of the Buchnera endosymbiont (42). Hence, the pos-sibility cannot be ruled out that such bactericidal gene productsare preferentially expressed in the Riptortus midgut, act on thesymbiont cell wall, and result in the infection failure of the �uppPmutant of the Burkholderia symbiont.

Considering the susceptibility of the �uppP mutant to envi-ronmental stresses, such as low osmolality, high osmolality, andhigh centrifugal pressure (Fig. 5B to D), an alternative hypothesisis that the symbiotic conditions within the host midgut entailsome environmental stresses, to which the wild-type symbiont isresistant but the �uppP mutant is susceptible. Although the na-ture of the “symbiotic stress” is unknown, it may be osmotic,anoxic, nutritional, immunological, or a combination of these. Inthis context, a recent study demonstrated a crucial involvement ofbacterial stress-related genes in the Riptortus-Burkholderia symbi-osis: disruption of symbiont genes for synthesizing an endocellu-lar storage polyester, polyhydroxyalkanoate (PHA), which confersbacterial resistance to nutritional depletion and other environ-mental stresses, resulted in failure of normal symbiotic associa-tion, while complementation of the PHA synthesis genes rescuedthe symbiosis defect (54). It should be noted that the “bactericidalfactor hypothesis” and the “symbiotic stress hypothesis” may notnecessarily be mutually exclusive, on the ground that the bacteri-cidal factors could be regarded as comprising host-derived immu-nological stresses.

The cell wall is located on the outer surface of bacterial cells asa front line of host-symbiont interactions. Therefore, consider-able attention has been paid to the possible relevance of the sym-biont cell wall to symbiosis, particularly to interactions with host’sinnate immunity. For example, some endosymbiotic bacteria,such as Spiroplasma and Wolbachia, exhibit remarkable degener-ation in their cell wall, thereby eliciting no or little innate immuneresponses of their host insects (43–46). Transcriptomic compari-sons between symbiotic and asymbiotic host insects have revealedthat a variety of immunity-related genes, including lysozyme

FIG 4 Quantitative analyses of the Burkholderia symbiont strains in the hostsymbiotic organs of second instar Riptortus nymphs. (A) Quantitative PCRanalysis of infection densities of the wild-type strain RPE161 and the �uppPmutant BBL005 at 10, 15, 20, and 25 h after inoculation. (B) CFU quantifica-tion of infection densities of the wild-type strain RPE161, the �uppP mutantBBL005, and the �uppP/uppP-complemented mutant BBL105 at 36 and 63 hafter inoculation. Different letters (a and b) indicate statistically significantdifferences (unpaired Student t test; *, P 0.05; **, P 0.01; ****, P 0.0001).

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genes and antimicrobial peptide genes, are upregulated in symbi-osis-associated patterns (40, 42, 47–50). To our knowledge, apartfrom general studies of bacterial cell wall changes and host im-mune responses, this study is the first to unequivocally identifythat a specific cell wall biosynthesis-related symbiont gene is re-quired for an insect-bacterium symbiotic association.

On the basis of previous studies in squid-Vibrio, nematode-Photorhabdus/Xenorhabdus, and other model symbiotic systems,Ruby in 2008 classified symbiosis-deficient bacterial mutants into(i) initiation mutants, which are unable to establish infection inthe host, (ii) accommodation mutants, which can establish infec-tion but fail to reach the usual infection density, and (iii) persis-tence mutants, which at first establish infection normally but areunable to maintain the normal infection level (1). Under thesecriteria, the �uppP mutant can be regarded as a mutant betweenan initiation mutant and accommodation mutant, because it isable to infect initially but fails to establish colonization in theRiptortus host. The cell wall deficiency of the �uppP mutant mostlikely affects the initial host-symbiont association, which high-lights a previously underexplored aspect of insect-bacterium sym-biotic associations.

ACKNOWLEDGMENTS

This study was supported by the Global Research Laboratory Grant of theNational Research Foundation of Korea (grant 2011-0021535) to B.L.L.and T.F.

We thank Joerg Graf (University of Connecticut) for providing plas-mids.

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FIG 5 (A) Survival of the wild-type Burkholderia symbiont strain RPE161 andthe �uppP mutant strain BBL005 when symbiotic midgut lysates from fifthinstar Riptortus nymphs were added to the cultured bacteria. (B to D) Survivalof the wild-type strain RPE161, the �uppP mutant BBL005, and the �uppP/uppP-complemented mutant BBL105 under environmental stress conditions.(B) Under a hypotonic condition in 10 mM phosphate buffer for 24 h. (C)Under a hypertonic condition in 1 M glucose for 24 h. (D) Under a high gravitycondition of centrifugation at 20,000 � g for 30 min. Different letters (a and b)indicate statistically significant differences (unpaired Student t test with a Bon-ferroni correction; P 0.05).

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