The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris

Accepted Manuscript The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris Jiyeun Kate Kim, Ha Young Park, Bok Luel Lee PII:

S0145-305X(16)30033-7

DOI:

10.1016/j.dci.2016.02.009

Reference:

DCI 2555

To appear in:

Developmental and Comparative Immunology

Received Date: 7 January 2016 Revised Date:

5 February 2016

Accepted Date: 5 February 2016

Please cite this article as: Kim, J.K., Park, H.Y., Lee, B.L., The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2016.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Developmental and Comparative Immunology

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The symbiotic role of O-antigen of Burkholderia symbiont in association

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with host Riptortus pedestris

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Jiyeun Kate Kima, Ha Young Parkb and Bok Luel Leeb*

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Department of Department of Microbiology, Kosin University College of Medicine, Busan,

South Korea, bGlobal Research Laboratory, College of Pharmacy, Pusan National University,

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Busan, South Korea

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* Corresponding author. Address: Global Research Laboratory of Insect Symbiosis, College

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of

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+82-51-510-2809, FAX: +82-51-513-2801, E-mail address: [email protected]

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Pharmacy,

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Busan,

609-735,

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ACCEPTED MANUSCRIPT Abstract

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Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the

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molecular interactions between insects and bacteria. We recently reported that the

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lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus

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guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the

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Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen

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biosynthesis genes were generated and confirmed for their different patterns of the

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lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains

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initially exhibited a reduction of infectivity, having significantly lower level of symbiont

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population at the second-instar stage. However, both the wild-type and O-antigen mutant

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symbionts exhibited a similar level of symbiont population from the third-instar stage,

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indicating that the O-antigen deficiency did not affect the bacterial persistence in the host

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midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont

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plays an exclusive role in the initial symbiotic association.

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O-antigen defective mutant strains were generated for the insect gut symbiosis assay.

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LPS O-antigen is important for initial bacterial colonization in the host gut.

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O-antigen is not essential for bacterial persistence at later stage of symbiosis.

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Keywords: lipopolysaccharide / O-antigen / insect gut symbiosis / Burkholderia symbiont /

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Riptortus pedestris

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1. Introduction Bacterial lipopolysaccharide (LPS) is the major outer membrane component of

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Gram-negative bacteria. It consists of three different regions: lipid A, core-oligosaccharide

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and O-antigen (Caroff and Karibian, 2003; Raetz and Whitfield, 2002 ). The innermost lipid A

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is a hydrophobic region anchored into the membrane and generally composed of a

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di-glucosamine backbone linked with four to seven fatty acids. A 2-keto-3-deoxyoctonate

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(Kdo) unit connects the lipid A to a core-oligosaccharide. The core-oligosaccharide is linked

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to the outermost region of LPS called O-antigen. The O-antigen consists of repeating

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oligosaccharide units. Bacteria with LPS lacking O-antigen is called rough-type bacteria, and

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bacteria harboring LPS O-antigen is called smooth-type bacteria.

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LPS O-antigen provides a protective barrier against environmental and immunological

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factors for bacteria (Raetz and Whitfield, 2002). In pathogenesis, O-antigen is an important

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virulence factor that facilitates the interaction with host tissues and provides protection from

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membrane-active compounds of hosts (Trent et al., 2006). Although the roles of O-antigen

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may vary in different bacteria, many studies on pathogenic bacteria report that rough-type

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bacteria are susceptible to serum complement and antimicrobial peptides, resulting in much

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less efficiency to invade and survive in host (Burns and Hull, 1998; Gunn and Ernst, 2007;

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Murray et al., 2006; Nesper et al., 2001; VanDenBosch et al., 1997). Similarly, LPS O-antigen

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was reported to be essential for symbiotic association in some symbiotic model systems. In

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legume-Rhizobium symbiosis, O-antigen deficient mutant Rhizobium impairs root

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colonization probably due to susceptibility to antimicrobials (Ormeno-Orrillo et al., 2008). In

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squid-Vibrio symbiosis, O-antigen deficient mutant, waaL, shows a motility defect and

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significantly delayed colonization in light organ of squid (Post et al., 2012). Also pbgE mutant

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of Photorhabdus luminescens exhibiting rough-type LPS is unable to colonize the gut of the

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nematode (Bennett and Clarke, 2005). In case of leech-Aeromonas symbiosis, a

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complement resistance to the symbionts onto digestive tract of leech (Braschler et al., 2003).

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In contrast to the general notion of the importance of LPS O-antigen in pathogenesis and

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symbiosis, we recently found that Burkholderia gut symbionts exist as the rough-type bacteria

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in an insect-bacteria symbiosis model (Kim et al., 2015).

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Riptortus pedestris (bean bug) harbors a single kind of gut symbiont, genus

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Burkholderia, in a specialized region of the posterior midgut (Kikuchi et al., 2005). This

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Burkholderia symbiont is not transmitted from mother to offspring, but it is orally acquired by

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Riptortus nymphs from environment. Possessing its free living ability, the symbionts isolated

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from the host midgut can be cultured in standard bacterial media and subjected to genetic

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modification (Kikuchi et al., 2007, 2011a, b). Recently we used the genetically modified

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Burkholderia symbiont strains to understand molecular cross-talks between insect and

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bacteria (Kim et al., 2014a; Kim et al., 2014b; Kim et al., 2013a; Kim et al., 2013b).

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Furthermore, we attempted to understand molecular changes occurring in the Burkholderia

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cells as they become gut symbionts in the Riptortus host. The direct comparison between the

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symbiotic Burkholderia cells and the cultured Burkholderia cells revealed striking differences

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in the cell envelope structures. The symbiotic cells isolated from Riptortus midgut exhibited

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the rough-type LPS (Kim et al., 2015).

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Because the O-antigen is important for the pathogenic bacteria to escape from the host

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immunological factors (Burns and Hull, 1998; Gunn and Ernst, 2007; Murray et al., 2006;

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Nesper et al., 2001; Trent et al., 2006; VanDenBosch et al., 1997) and for the several

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symbiotic bacteria to successfully colonize the host (Bennett and Clarke, 2005;

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Ormeno-Orrillo et al., 2008; Post et al., 2012), it was quite unexpected to find the loss of the

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O-antigen in the Burkholderia symbiont. Therefore, in this study, we investigated the role of

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the Burkholderia O-antigen in the symbiotic association with the host Riptortus host. By 4

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using the O-antigen mutant Burkholderia strains, we addressed the importance of the

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O-antigen in the initial stage as well as in the later stage of the symbiosis.

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2.1. Bacteria and media

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List of bacteria used in this study is shown in Table 1. Escherichia coli cells were

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cultured at 37°C with LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl).

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Burkholderia symbiont RPE75 cells were cultured at 30°C with YG medium (0.4% glucose,

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0.5% yeast extract, 0.1% NaCl) containing 30 µg/ml rifampicin (Kikuchi et al., 2011b).

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2.2. Isolation of symbiotic Burkholderia from midgut

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The symbiotic midguts, M4s, were dissected from fifth instar nymphs and placed in 50

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µl of 10mM phosphate buffer (PB, pH7.0). The M4 midguts were cut into pieces with fine

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scissors to break the crypts. One ml of PB was added to the M4 midgut pieces and gently

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pipetted to resuspend Burkholderia symbionts into the solution. The solution was then filtered

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through 5 µm pore to remove the gut tissues. Burkholderia cells were further washed with PB

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to remove host molecules.

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2.3. Generation of deletion mutant strains

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Chromosomal deletion mutant stains of the O-antigen biosynthesis genes were

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generated as described (Kim et al., 2013b). Briefly, allelic exchanges of target genes were

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accomplished by utilizing the suicide vector pK18mobsacB harboring 5’ region and 3’ regions 5

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Burkholderia symbiont RPE75 are indicated in Table S1. After transforming E. coli DH5α

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cells with pK18mobsacB vector containing 5’ and 3’ regions of the target genes, they were

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mixed with Burkholderia RPE75 cells along with helper cells HBL1 for the conjugal transfer

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of the cloned vector to the Burkholderia RPE75 (Table 1). Burkholderia cells with the first

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crossover were selected on the YG-agar plates containing rifampicin and kanamycin and

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cultured in YG medium for allowing the second crossover. The in-frame deletion mutant cells

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by double crossover were isolated on the YG agar plates containing rifampicin and 20% (w/v)

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sucrose.

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2.4. Electrophoretic analysis of LPS

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LPS were extracted from full grown bacterial cells using modified hot-phenol method

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(Westphal and Jann, 1965). In brief, full grown Burkholderia 109 cells were washed with PB

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and resuspended in 500 µl of PB. The same volume of hot phenol was added to the cell

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solution and incubated in water bath adjusted to 65°C. The cell solution was vortexed

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rigorously every 5 min. After 1 h of incubation, the solutions were cooled and 200 µl of

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chloroform was added. After vortexing, the solution was incubated at room temperature for 5

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min and then centrifuged at 15,300 x g for 15 min to separate the water and phenol phases.

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Four hundred µl of the water phase solution were transferred to a new tube and 800 µl of

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ethanol were added to the solution. After allowing LPS precipitation at -20°C overnight, the

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precipitates were collected by centrifugation at 20, 400 x g for 20 min, washed with 80%

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ethanol and air-dried. The precipitates were suspended in sample buffer (60 mM tris-HCl [pH

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6.8], 2% SDS, 10% glycerol, 0.005% bromophenol blue), boiled at 95°C for 5 min,

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de-proteinated by incubating with 400 µg/ml proteinase K at 60°C for 1 h, and boiled again

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ACCEPTED MANUSCRIPT prior to the electrophoretic analysis. The LPS extracted from 109 cells were loaded to a well

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of Tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

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(Schägger, 2006) with 12% gels. LPS separated in the gels were visualized using the Pro-Q

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Emerald 300 Lipopolysaccharide Gel Stain Kit (Invitrogen) by following manufacturer’s

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instruction.

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141 2.5. Insect rearing and symbiont inoculation

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R. pedestris was maintained in our insect laboratory at 26°C under a long day cycle of

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16 h light and 8 h dark as described (Kim et al., 2013a). Nymphal insects were reared in clean

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plastic containers with soybean seeds and distilled water containing 0.05% ascorbic acid

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(DWA). When the newborn nymphs molted to second instar, Burkholderia symbiont

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inoculum solution was provided as wet cotton balls in a small Petri dish. The inoculum

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solution consisted of mid-log phase Burkholderia cells in DWA at a concentration of 107

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cells/ml.

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2.6. Colony forming unit (CFU) assay for symbiont titer

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Individual M4 midgut dissected from R. pedestris was collected in 100 µl of PB,

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homogenized by a plastic pestle, and serially diluted with PB. The diluted samples were

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spread on rifampicin-containing YG agar plates. After two days of incubation at 30°C,

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colonies on the plates were counted. The colony forming units (CFUs) per insect were

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calculated by multiplying colony counts with dilution factor.

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2.7. Measurement of bacterial growth in liquid media Growth curves of the Burkholderia symbiont strains were examined either in YG

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medium or in minimal medium (0.6% Na2HPO4·2H2O, 0.3% KH2PO4, 0.1% NH4Cl, 0.05%

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NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 0.2% glucose). The starting cell solutions were prepared

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by adjusting OD600 to 0.05 with stationary phase cells in either YG medium or minimal

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medium. The media were incubated on a rotator shaker at 180 rpm at 30°C for 24 h, whose

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OD600 was monitored every 3 h using a spectrophotometer (Shimadzu, Japan).

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The soft agar plate was prepared with 0.3% agar in YG medium containing rifampicin.

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Three µl of mid-log Burkholderia cell solution whose OD600 was adjusted to 0.3 was

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inoculated into a spot of the agar plate. Each plate has five inoculation spots, each spot for a

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different strain: the wild-type and four mutant strains. After 26 h ofincubation at 30°C, the

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swimming radius of each inoculation was measured. Based on the swimming radius of the

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wild-type cells set as 100%, the swimming radius of mutant strains of the same plate were

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indicated by percentage. Total 10 plates were examined for motility test (each strain, n=10).

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2.9. Measurement of polymyxin B susceptibility

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The mid-log cultured Burkholderia cells and symbiotic Burkholderia cells isolated from

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a fifth instar nymph were washed with PB twice and diluted with PB to 500-1000 CFUs per

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50 µl. Different concentration of polymyxin B (Sigma) was prepared in distilled water. Fifty

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µl of polymyxin B solution and 50 µl suspension of Burkholderia cells were combined and

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incubated for 30 min at room temperature. After incubation, the sample was spread on YG 8

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agar plate containing rifampicin, cultured for two days and subjected to colony counting.

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2.10. Statistical analyses The statistical significance of differences in the data was determined using the one-way

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ANOVA with Tukey’s post-hoc test, provided in the Prism GraphPad software.

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186 187 3. Results

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3.1. Generation of the O-antigen mutant strains of Burkholderia symbiont.

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When the genome of Burkholderia symbiont strain RPE64 was searched for the

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candidate genes encoding enzymes for LPS O-antigen biosynthesis (Shibata et al., 2013), the

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gene clusters of the O-antigen biosynthesis genes were found in at least two locations in

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chromosome 1 (Fig. 1A). In order to generate mutant strains with different O-antigen

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structures, we targeted three glycosyltransferase genes (wbxA, wbxB and wbiF) and an

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epimerase gene (wbiG) involved in the LPS O-antigen synthesis. Althogh the reaction steps in

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the O-antigen biosynthesis in which WbxA, WbxB, WbiF and WbiG involved are not known,

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we confirmed that the LPSs purified from these mutant strains exhibited different O-antigen

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patterns shown in the SDS-PAGE analysis (Fig. 1B). The wild-type Burkholderia cells

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exhibited high molecular weight (HMW) O-antigen bands around 95 kDa of protein marker

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and low molecular weight (LMW) O-antigen ladder-like bands between 10-40 kDa. The

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∆wbxB mutant strain exhibits a similar O-antigen pattern like the wild-type strain. However,

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∆wbxA, ∆wbiF and ∆wbiG mutant strains exhibited almost undetectable LMW O-antigens.

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much less HMW O-antigen than the wild-type strains. The HMW O-antigen band intensity of

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∆wbxA was stronger than those of ∆wbiG, and the HMW O-antigen bands of ∆wbiF were not

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detected in the SDS-PAGE analysis. These results suggest that Burkholderia mutant strains

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with different O-antigen composition were successfully generated.

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3.2. The O-antigen mutant strains are less efficient to colonize the host midgut than the

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wild-type strain at initial stage of symbiosis.

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Using O-antigen biosynthesis mutant strains, we first examined the role of O-antigen in

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the initial infection process. The wild-type and mutant strains were orally infected to

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newly-molted Riptortus nymphs at the second-instar, the competent stage for natural

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establishment of the gut symbiotic association (Kikuchi et al., 2011b). At 24 h post-infection

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time point, the percentage of symbiotically infected insects and the symbiont population of

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the Burkholderia cells present in the M4 midgut were accessed (Fig. 2A). The ∆wbxB mutant

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strain, which exhibited smooth-type LPS, showed an infection rate of 90% and symbiont

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population size of mean 6.3 x 103 cfu, which were similar to the wild-type strain whose

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infection rate and symbiont population size were 95% and mean 8.5 x 103 cfu, respectively.

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However, ∆wbxA, ∆wbiF and ∆wbiG mutant strains, which exhibited much reduced

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O-antigen expression, revealed lower infection rate (55-67.5%) and 30-100 fold less symbiont

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population than the wild-type strain. These results indicate that the LPS O-antigen of gut

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symbiont plays a role in bacterial initial colonization in the host midgut.

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3.3. The less efficiency of colonization of the O-antigen deficient mutant strains is probably

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To address why the O-antigen deficient mutants are less efficient to colonize the host midgut,

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we performed in vitro tests on bacterial growth rate, motility and susceptibility to

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antimicrobial peptides (AMPs), which are closely related to the bacterial initial colonization.

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Upon oral uptake, bacteria have to survive against the AMPs in saliva and gut, be able to

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move toward the symbiotic organ and proliferate in the M4 midgut. Because the growth rates

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of the mutant strains were similar to that of the wild-type strain in both nutrient rich (YG)

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medium and minimal medium (Fig. 2B), we assumed that the in vivo lower infection density

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of the mutant strains may not be related to the ability to proliferate. In motility test, the

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∆wbxB mutant strain exhibited bigger swimming radius than the wild-type strain, while the

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∆wbxA and ∆wbiF mutant strains exhibited smaller swimming radius than the wild-type strain

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(Fig. 2C). However, the ∆wbiG mutant strain exhibited similar motility to the wild-type strain,

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suggesting that motility may not be the key reason for the less efficiency of colonization of

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these O-antigen deficient mutant strains. Lastly, Burkholderia strains were tested for their

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susceptibility to the cationic polymyxin B. The ∆wbxA, ∆wbiF and ∆wbiG mutant strains

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exhibited more susceptibility to the polymyxin B than the wild-type strain (Fig. 2D). These

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results suggest that the LPS O-antigen deficient mutant strains are probably more susceptible

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to host cationic AMPs, and their AMP susceptibility may result in the lower level of initial

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colonization in the host midgut than the wild-type strain.

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3.4. The wild-type and LPS O-antigen mutant strains exhibit the similar level of population

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in later stages of symbiosis.

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Next we investigated the role of the LPS O-antigen in the later stages of symbiosis by

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examining the symbiont’s population size of the wild-type and LPS O-antigen mutant strains 11

ACCEPTED MANUSCRIPT at the third, fourth and fifth-instar insects. On the contrary to the low infection density in the

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early infection stage (Fig. 2A), the O-antigen deficient mutant symbionts exhibited no

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significantly different size of symbiont population in the later stages of symbiotic association

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(Fig. 3A). In order to understand molecular basis of the similar infection densities among

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strains, the symbiotic cells of the wild-type and LPS O-antigen mutant strains were isolated

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from the fifth-instar nymphs and their LPS patterns were analyzed by the SDS-PAGE (Fig.

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3B). As the symbiotic Burkholderia cells lost their O-antigen, all the symbiotic cells of the

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wild-type and O-antigen mutant strains revealed the similar pattern of LPS: almost

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undetectable LMW O-antigens and greatly reduced HMW O-antigens. We further tested the

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polymyxin B susceptibility of these symbiotic cells. In contrast to the results of the cultured

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Burkholderia cells (Fig. 2D), the symbiotic wild-type and ∆wbxB cells increased their

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susceptibility to polymyxin B, similarly to the symbiotic ∆wbxA, ∆wbiF and ∆wbiG cells (Fig.

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3C). These results demonstrate that (i) the LPS of the wild-type and LPS O-antigen mutant

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cells harboring in the symbiotic midgut shares the structural and functional similarities, and

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that (ii) the LPS O-antigen of Burkholderia symbiont is not a critical factor for maintaining

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infection density in the symbiotic midgut.

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4. Discussion

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In this study, we investigated the role of the LPS O-antigen in the Riptortus-Burkholderia

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symbiosis using O-antigen biosynthesis mutant strains. Because LPS O-antigen is shown to

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be critical for bacterial colonization to host in other symbiotic model systems, we expected

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that O-antigen deficient mutant strains would exhibit impairment in the colonization to the

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midgut of R. pedestris. Our results demonstrated that the O-antigen deficient strains exhibit 12

ACCEPTED MANUSCRIPT the lower level of colonization at the initial stage of infection than the wild-type strain. The

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low infection densities of the LPS O-antigen mutant strains during the second-instar stage

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seem to be attributed to their susceptibility to the cationic AMPs. In the laboratory, we

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provide 107 cells/ml Burkholderia solution to bean bug to ensure the symbiosis. However,

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bean bugs probably encounter much less number of Burkholderia cells in the fields. In case of

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other Burkholderia spp. such as B. cepacia complex, their abundance in the field is estimated

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102 to 104 cell/g of soil (Ramette et al., 2005). Therefore, in reality, the LPS O-antigen of

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Burkholderia could be a critical factor for the symbiotic association with host bean bug. The

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low infection density of LPS O-antigen mutant symbiont at the second-instar stage recovered

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to the normal level from the third-instar stage. In later stage of infection, the wild-type

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Burkholderia cells lose their O-antigen, and hence both the wild-type and O-antigen mutants

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exhibit the same rough-type LPS. The AMP susceptibility of the rough-type symbionts seems

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to have no effect to the persistence of the symbionts in the midgut. The persistence of the

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AMP-susceptible symbionts is probably due to the suppression of AMP expression in the M4

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midgut (Kim et al., 2015). Previous report demonstrated that small number of Burkholderia

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cell (even 5 cells) colonization at the second-instar is sufficient enough to reach a normal

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level of population in the M4 midgut by the third-instar stage (Kikuchi and Yumoto, 2013).

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Our study additionally shows that the LPS O-antigen does not affect bacterial survival and

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proliferation once bacteria cells are colonized in the midgut.

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While many studies on pathogenic bacteria have demonstrated that O-antigen protects

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bacteria against complement and antimicrobial peptides (Burns and Hull, 1998; Gunn and

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Ernst, 2007; Murray et al., 2006; Nesper et al., 2001; VanDenBosch et al., 1997), there are

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cases where the LPS O-antigen deficiency is involved in the pathogenesis. Yersinia pestis,

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which is a cause for deadly infectious plague, does not have O-antigen by carrying mutations

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in genes of the O-antigen biosynthesis (Perry and Fetherston, 1997; Skurnik et al., 2000). Y. 13

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plasminogen and mediates adhesion to host cells (Sodeinde et al., 1992). The presence of the

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O-antigen on Y. pseudotuberculosis, from which Y. pestis evolved, prevented of Pla-mediated

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adhesion and plasminogen activation, suggesting the loss of O-antigen facilitates invasiveness

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of Y. pestis (Kukkonen et al., 2004). It would be interesting to examine whether the loss of

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O-antigen in the Burkholderia symbiont is able to expose other bacterial surface molecules

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that are important for the symbiotic interaction with host.

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Pseudomonas aeruginosa is an opportunistic pathogen infecting immunocompromised

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population including cystic fibrosis (CF) patients. P. aeruginosa isolates from chronic CF

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infection often exhibit loss of O-antigen (Govan and Deretic, 1996). Whole genome

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sequencing of CF P. aeruginosa isolates revealed that mutations were prone to occur in the

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O-antigen biosynthesis genes (Spencer et al., 2003). In case of pathogen Burkholderia

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pseudomallei, some isolates from chronic lung infected patients also exhibited rough-type

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LPS phenotype by O-antigen biosynthesis mutations (Tuanyok et al., 2012). Although these

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O-antigen defective mutants were sensitive to serum, they might be able to survive in special

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niches, and the absence of O-antigen might support to escape from the antibody recognition.

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For the long-term association with host, such as chronic infection and symbiosis, bacteria

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require molecular switches from free-living status and even from the short-term association

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with host. Characteristics of those molecular switches include down-regulation of flagella/pili

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and up-regulation of biofilm components (Jenal and Malone, 2006; Jimenez et al., 2012).

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Since LPS O-antigen deficient mutant bacteria are prone to be found in the chronic diseases,

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although speculative, the loss of O-antigen might be another molecular switch that grants an

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advantage on lineages to persist in the chronic state of the infection.

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On the ground of our findings, three perspectives for future studies could be proposed.

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Firstly, molecular mechanisms of the loss of the O-antigen in the Burkholderia symbiont 14

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outer-memebrane proteins and core-oligosaccharide of LPS to the symbiotic interfaces, the

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role of outer-membrane proteins and core-oligosaccharide should be pursued to understand

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molecular interactions between symbionts and host. Lastly, since rough-type mutant bacteria

328

are prevalent in isolates from the chronic infections, it is of great interest to investigate

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whether pathogenic bacteria lose their O-antigen during chronic state of infection as shown in

330

symbiotic Burkholderia in the Riptortus midgut.

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331 Acknowledgements

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We thank Naruo Kikoh (Open University, Japan) and Takema Fukatsu (AIST, Japan) for the

334

resources and advice. This study was supported by the Global Research Laboratory Program

335

(grant

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2014R1A1A4A01007507) of the National Research Foundation of Korea, and a grant from

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Kosin University College of Medicine (2015).

2011-0021535),

Basic

Science

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Westphal, O., Jann, K., 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and

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further applications of the procedure, in: Whistler, R.L. (Ed.), Methods in Carbohydrate

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Figure Legends

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Fig 1. Generation of LPS O-antigen mutant strains. (A) Gene maps of O-antigen

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biosynthesis genes. Two of LPS biosynthesis gene clusters in chromosome 1 are shown here.

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Four genes indicated by dark arrow are targeted to generate deletion mutants. (B) LPS

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profiles of mutant strains. LPS extracted from 109 cells per strains was resolved in the Tricine

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SDS-PAGE and visualized by Pro-Q Emerald 300 staining. Protein size marker was used as

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size reference.

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density of Burkholderia strains at early stage of infection. A total of forty insects per group

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were examined. Infection rates are indicated at the top of the graph. Horizontal lines in the

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graph indicate mean values of Burkholderia cfu. Different letters (a and b) indicate

460

statistically significant differences (P < 0.05; one-way ANOVA with Tukey’s correction). (B)

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Growth rates of the wild-type and O-antigen mutant strains in YG medium (left) and minimal

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medium (right). (C) Motility test. Swimming radius in the 0.3% YG-agar plate was measured

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for each strain and normalized based on the swimming radius of wild-type strain. Means and

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SDs (n=10) are shown as columns and error bars, respectively. Different letters (a, b, c) on the

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top of the columns indicate statistically significant differences (P < 0.05; one-way ANOVA

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with Tukey’s correction). (D) Polymyxin B susceptibility test. Different concentrations of

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polymyxin B were incubated with Burkholderia cells and subjected to the CFU assay. Means

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and SDs (n=3) are shown as points and error bars, respectively. This is a representative data

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from three independent experiments.

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Fig 3. The LPS O-antigen mutant symbiont in later stage of infection. (A) Infection

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density of Burkholderia symbionts at third, fourth and fifth instar stages. Means and SDs

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(n=10) are shown as columns and error bars, respectively. NS means not significant (one-way

474

ANOVA with Tukey’s correction). (B) LPS profiles of the wild-type and mutant symbionts.

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LPS extracted from isolated symbionts was resolved in the Tricine SDS-PAGE and visualized

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by Pro-Q Emerald 300 staining. Protein size marker was used as size reference. (C)

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Polymyxin B susceptibility test. Different concentrations of polymyxin B were incubated with

478

Burkholderia symbionts and subjected to the CFU assay. Means and SDs (n=3) are shown as

479

points sand error bars, respectively. This is a representative data from three independent

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Tables

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Table 1. Bacterial strains and plasmids used in this study.

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Bacterial strain

References

Burkholderia symbiont

Burkholderia symbiont (RPE64); Rif RPE75 ∆wbxA; RifR

BBL012

RPE75 ∆wbxB; RifR

BBL013

RPE75 ∆wbiF; RifR

BBL014

RPE75 ∆wbiG; RifR

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BBL011

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Characteristics or plasmid

Escherichia coli

(Kikuchi et al., 2011b)

This study This study This study (Kim et al., 2015)

F–Φ80lacZ∆M15 ∆(lacZYA-argF) U169 recA1 endA1 DH5α

Invitrogen

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hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1 F- ∆lac169 rpoS(am) robA1 creC510 hsdR514 endA Invitrogen

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PIR1

recA1 uidA(∆Mlu I)::pir-116

HBL1

(Kim et al.,

PIR1 carrying pEVS104; CmR, KmR 2013b)

Plasmid (Stabb and oriR6K helper plasmid containing conjugal tra and trb; pEVS104

Ruby, KmR 2002)

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pMB1ori allelic exchange vector containing oriT; KmR al., 1994)

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