Caste Differentiation, Division of Labor and Chemical Communication in a Social Aphid

Harunobu Shibao,Mayako Kutsukake,Shigeru Matsuyama,Takahisa Suzuki,Takema Fukatsu 한국응용곤충학회 2005 한국응용곤충학회 학술대회논문집 Vol.2005 No.05

Bacterial Cell Wall Synthesis Gene <i>uppP</i> Is Required for <i>Burkholderia</i> Colonization of the Stinkbug Gut

Kim, Jiyeun Kate,Lee, Ho Jin,Kikuchi, Yoshitomo,Kitagawa, Wataru,Nikoh, Naruo,Fukatsu, Takema,Lee, Bok Luel American Society for Microbiology 2013 Applied and environmental microbiology Vol.79 No.16

<P>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, <I>Riptortus pedestris</I>, and its beneficial gut bacterium, <I>Burkholderia</I> 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 <I>Burkholderia</I> symbiont by disruption of <I>uppP</I> gene, which encodes undecaprenyl pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall components. Under culture conditions, the Δ<I>uppP</I> 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 Δ<I>uppP</I> mutant failed to establish normal symbiotic association: the bacterial cells reached to the symbiotic midgut but neither proliferated nor persisted there. Transformation of the Δ<I>uppP</I> mutant with <I>uppP</I>-encoding plasmid complemented these phenotypic defects: lysozyme susceptibility <I>in vitro</I> was restored, and normal infection and proliferation in the midgut symbiotic organ were observed <I>in vivo</I>. The Δ<I>uppP</I> mutant 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 <I>Burkholderia</I> symbiont.</P>

Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug

Kim, Jiyeun Kate,Jang, Ho Am,Won, Yeo Jin,Kikuchi, Yoshitomo,Heum Han, Sang,Kim, Chan-Hee,Nikoh, Naruo,Fukatsu, Takema,Lee, Bok Luel Springer Science and Business Media LLC 2014 The ISME journal Vol.8 No.3

<P>The Riptortus-Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect-bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host's symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host's symbiotic organ at early instar stages and reduced the host's fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host's symbiotic organ and enhancing the host's fitness values.</P>

Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

Kim, Jiyeun Kate,Kim, Na Hyang,Jang, Ho Am,Kikuchi, Yoshitomo,Kim, Chan-Hee,Fukatsu, Takema,Lee, Bok Luel American Society for Microbiology 2013 Applied and environmental microbiology Vol.79 No.23

<P>Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host's control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug <I>Riptortus pedestris</I> harbors a betaproteobacterial extracellular symbiont of the genus <I>Burkholderia</I> in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors <I>Burkholderia</I> symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the <I>Burkholderia</I> symbiont but not the cultured <I>Burkholderia</I> and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of <I>R. pedestris</I> that plays a critical role in controlling the population of the <I>Burkholderia</I> gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.</P>

Purine Biosynthesis, Biofilm Formation, and Persistence of an Insect-Microbe Gut Symbiosis

Kim, Jiyeun Kate,Kwon, Jeong Yun,Kim, Soo Kyoung,Han, Sang Heum,Won, Yeo Jin,Lee, Joon Hee,Kim, Chan-Hee,Fukatsu, Takema,Lee, Bok Luel American Society for Microbiology 2014 Applied and environmental microbiology Vol.80 No.14

<P>The <I>Riptortus-Burkholderia</I> symbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut of <I>Riptortus pedestris</I> was investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizing <I>Burkholderia</I> symbionts were occupied by an extracellular matrix consisting of polysaccharides. This observation prompted us to search for symbiont genes involved in the induction of biofilm formation and to examine whether the biofilms are necessary for the symbiont to establish a successful symbiotic association with the host. To answer these questions, we focused on <I>purN</I> and <I>purT</I>, which independently catalyze the same step of bacterial purine biosynthesis. When we disrupted <I>purN</I> and <I>purT</I> in the <I>Burkholderia</I> symbiont, the Δ<I>purN</I> and Δ<I>purT</I> mutants grew normally, and only the Δ<I>purT</I> mutant failed to form biofilms. Notably, the Δ<I>purT</I> mutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the Δ<I>purN</I> mutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the Δ<I>purT</I> mutant, which might operate via impaired purine biosynthesis. The host insects infected with the Δ<I>purT</I> mutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the Δ<I>purN</I> mutant. These results show that the function of <I>purT</I> of the gut symbiont is important for the persistence of the insect gut symbiont, suggesting the intricate biological relevance of purine biosynthesis, biofilm formation, and symbiosis.</P>

Polyester synthesis genes associated with stress resistance are involved in an insect–bacterium symbiosis

Kim, Jiyeun Kate,Won, Yeo Jin,Nikoh, Naruo,Nakayama, Hiroshi,Han, Sang Heum,Kikuchi, Yoshitomo,Rhee, Young Ha,Park, Ha Young,Kwon, Jeong Yun,Kurokawa, Kenji,Dohmae, Naoshi,Fukatsu, Takema,Lee, Bok Lue National Academy of Sciences 2013 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.110 No.26

<P>Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect–bacterium symbiotic association. The bean bug <I>Riptortus pedestris</I> harbors a beneficial and specific gut symbiont of the β-proteobacterial genus <I>Burkholderia</I>, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured <I>Burkholderia</I> detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic <I>Burkholderia</I>. Among major PHA synthesis genes, <I>phaB</I> and <I>phaC</I> were disrupted by homologous recombination together with the <I>phaP</I> gene, whereby <I>ΔphaB</I>, <I>ΔphaC</I>, and <I>ΔphaP</I> mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in <I>ΔphaB</I> and <I>ΔphaC</I>, but only moderately in <I>ΔphaP.</I> In symbiosis, the host insects infected with <I>ΔphaB</I> and <I>ΔphaC</I> exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with <I>ΔphaB</I> and <I>ΔphaC</I> were restored by complementation of the mutants with plasmids encoding a functional <I>phaB</I>/<I>phaC</I> gene. Retention analysis of the plasmids revealed positive selection acting on the functional <I>phaB</I>/<I>phaC</I> in symbiosis. These results indicate that the PHA synthesis genes of the <I>Burkholderia</I> symbiont are required for normal symbiotic association with the <I>Riptortus</I> host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.</P>