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  • Bacterial Cell Wall Synthesis Gene uppP Is Required for Burkholderia Colonization 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, National Institute 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 coordinated manner. In insects, bacterial factors for symbiosis have been poorly characterized at the molecular and biochemical levels, since many symbionts have not yet been cultured or are as yet genetically intractable. Recently, the symbiotic association between a stinkbug, Riptortus pedestris, and its beneficial gut bacterium, Burkholderia sp., has emerged as a promising experimental model system, providing opportunities to study insect symbiosis using genetically manipulated symbiotic bacteria. Here, in search of bacterial symbiotic factors, we targeted cell wall components of the Burkholderia symbiont by disruption of uppP gene, which encodes undecaprenyl pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall components. Under culture conditions, the �uppP mutant showed higher susceptibility to lysozyme than the wild-type strain, indicating impaired integrity of peptidoglycan of the mutant. When administered to the host insect, the �uppP mutant failed to establish normal symbiotic 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 in vitro 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 cell wall integrity is a stress resistance factor relevant to the successful colonization of the stinkbug midgut by Burkholderia symbiont.

    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-symbiont interactions, several model symbiotic systems have been used to identify novel symbiotic factors and to determine their molecular functions (1). For example, the legume-Rhizobium nitrogen-fix- ing symbiosis and the squid-Vibrio luminescent symbiosis have been studied in depth. In both systems, the symbiotic bacteria are easily cultivable and genetically manipulatable and are thus suit- able for elucidating the molecular properties of their symbiotic factors (2–8).

    However, among insect-microbe symbiotic systems, molecu- lar factors relevant to symbiosis have been poorly characterized except 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 insect hosts. Consequently, powerful mutant-based molecular genetic approaches have not been effectively applied to insect-microbe symbiotic systems in general. Obligate insect symbionts, such as Buchnera in aphids and Wigglesworthia in tsetse flies, have been associated with their hosts over evolutionary time and are incapa- ble of independent living and thus are uncultivable (9, 12). As for facultative insect symbionts, such as Wolbachia in various insects and Sodalis in tsetse flies, which are transmitted through host gen- erations not only vertically but also horizontally, at least some of them are cultivable outside their host insects and thus potentially genetically 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, and the symbionts grow very slowly, are prone to contamination, and are 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 family Alydidae in the insect order Hemiptera. In contrast to previously known insect-bacterium symbiotic systems, nymphal R. pedestris acquires a betaproteobacterial symbiont of the genus Burkholderia not vertically but from the soil environment every generation (22). A posterior region of the insect midgut bears numerous crypts whose lumens are filled with bacterial cells of the symbiotic Burkholderia (23). Reflecting its free-living origin in the environ- ment, the symbiotic Burkholderia is easily cultivable on standard microbiological media and can be experimentally reinfected into the host insect by oral administration (24, 25). Comparisons be- tween symbiotic and asymbiotic insects showed beneficial fitness consequences of Burkholderia infection to the host insect (22, 26). These features of the Riptortus-Burkholderia gut symbiotic system provide unprecedented opportunities to study insect symbiosis at molecular 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

    August 2013 Volume 79 Number 16 Applied and Environmental Microbiology p. 4879–4886 aem.asm.org 4879

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    http://dx.doi.org/10.1128/AEM.01269-13 http://aem.asm.org http://aem.asm.org/

  • 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 the peptidoglycan layer resides (27). Bacterial cell wall components such as LPS and peptidoglycan are essential for maintaining the structural integrity of bacterial cells and generally required for viability (27, 28). In addition, these cell wall components most likely play a role in bacterial association with host and hence, may function as symbiotic factors. Biosynthesis of bacterial cell wall components, such as LPS and peptidoglycan, requires a key lipid carrier, undecaprenyl phosphate (C55-P), which is generated from dephosphorylation of undecaprenyl pyrophosphate (C55-PP) (29–34). C55-P is a precursor of various cell wall components that are synthesized in the cytoplasm and transported to the periplasm, where further polymerization occurs. After release from the cell wall component precursors, the lipid carrier is in a pyrophosphate form (C55-PP) and requires another dephosphorylation step be- fore being reused as a lipid carrier (35). This dephosphorylation step is catalyzed by C55-PP phosphatase enzymes. Four C55-PP phosphatases have been identified in Escherichia coli: UppP (also called BacA), YbjG, YeiU and PgpB, of which UppP is regarded as the major phosphatase (36, 37).

    To identify bacterial symbiotic factors in the Riptortus-Burk- holderia symbiosis, we targeted the bacterial cell wall-related uppP gene. We generated an uppP-deficient mutant (�uppP) of the Burkholderia symbiont by allelic exchange and homologous re- combination. Because the �uppP mutant shows 75% reduction of C55-PP phosphatase activity in E. coli (36), we hypothesized that the decrease of C55-PP phosphatase activity affects the cell wall component synthesis, resulting in defected cell wall. Since the ac- tual effects on the cell wall by the uppP mutation are not well characterized, we first examined cell wall components of a �uppP Burkholderia strain. Furthermore, the growth phenotypes in vitro and 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 plasmid encoding a functional uppP gene.

    MATERIALS AND METHODS

    Bacteria, plasmids, and culture media. Bacterial strains and plasmids used in the present study are listed in Table 1. E. coli cells were cultured at 37°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, a spontaneous 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 at the following concentrations unless otherwise described: kanamycin at 30 �g/ml and chloramphenicol at 10 �g/ml.

    Generation of �uppP mutant. Deletion of the chromosomal uppP gene from the Burkholderia symbiont was accomplished by allelic ex- change and homologous recombination using a suicide vector pK18mobsacB containing the 5= (uppP-L) and 3= (uppP-R) regions of uppP gene. The wild-type Burkholderia symbiont strain RPE161 was sub- jected to PCR using the primers uppP-L-P1 (5=-TTT AAG CTT GAG TTC GAC TTC GAG CGT GT-3=) and uppP-L-P2 (5=-TTT GGA TCC AAG ACT GCT GAC CGG AAA AA-3=) for the uppP-L region, and the primers up

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