Insect’s intestinal organ for symbiont sorting

Ohbayashi, Tsubasa,Takeshita, Kazutaka,Kitagawa, Wataru,Nikoh, Naruo,Koga, Ryuichi,Meng, Xian-Ying,Tago, Kanako,Hori, Tomoyuki,Hayatsu, Masahito,Asano, Kozo,Kamagata, Yoichi,Lee, Bok Luel,Fukatsu, Tak National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.37

<P><B>Significance</B></P><P>In general, animals have a mouth for feeding, an anus for defecation, and a gut connecting them for digestion and absorption. However, we discovered that the stinkbug’s gut is functionally disconnected in the middle by a previously unrecognized organ for symbiont sorting, which blocks food fluid and nonsymbiotic bacteria but selectively allows passing of a specific bacterial symbiont. Though very tiny and inconspicuous, the organ governs the configuration and specificity of stinkbug gut symbiosis, wherein the posterior gut region is devoid of food flow, populated by a specific bacterial symbiont, and transformed into an isolated organ for symbiosis. Mutant analyses showed that the symbiont’s flagellar motility is needed for passing the host organ, highlighting intricate host–symbiont interactions underpinning the symbiont sorting process.</P><P>Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug <I>Riptortus pedestris</I>, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled <I>Burkholderia</I> symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled <I>Escherichia coli</I> unveiled selective passage of the symbiont and blockage of <I>E. coli</I> at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.</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>

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>