Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts.

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola.

Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato, which is able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing that 498 protein-coding genes constitute the essential genome of Caballeronia insecticola for growth in free-living conditions. By comparing essential gene sets of Caballeronia insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified, indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of Riptortus pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by transposon-sequencing. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in Riptortus pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut. Thus, our study provides insights about specific contributions provided by the insect host to the bacterial symbiont.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (ß-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in ß-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both ß-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

Intercontinental Diversity of Caballeronia Gut Symbionts in the Conifer Pest Bug Leptoglossus occidentalis.

Many stinkbugs in the superfamily Coreoidea (Hemiptera: Heteroptera) develop crypts in the posterior midgut, harboring Caballeronia (Burkholderia) symbionts. These symbionts form a monophyletic group in Burkholderia sensu lato, called the "stinkbug-associated beneficial and environmental (SBE)" group, recently reclassified as the new genus Caballeronia. SBE symbionts are separated into the subclades SBE-α and SBE-ß. Previous studies suggested a regional effect on the symbiont infection pattern; Japanese and American bug species are more likely to be associated with SBE-α, while European bug species are almost exclusively associated with SBE-ß. However, since only a few insect species have been investigated, it remains unclear whether region-specific infection is general. We herein investigated Caballeronia gut symbionts in diverse Japanese, European, and North American populations of a cosmopolitan species, the Western conifer seed bug Leptoglossus occidentalis (Coreoidea: Coreidae). A mole-cular phylogenetic ana-lysis of the 16S rRNA gene demonstrated that SBE-ß was the most dominant in all populations. Notably, SBE-α was rarely detected in any region, while a third clade, the "Coreoidea clade" occupied one fourth of the tested populations. Although aposymbiotic bugs showed high mortality, SBE-α- and SBE-ß-inoculated insects both showed high survival rates; however, a competition assay demonstrated that SBE-ß outcompeted SBE-α in the midgut crypts of L. occidentalis. These results strongly suggest that symbiont specificity in the Leptoglossus-Caballeronia symbiotic association is influenced by the host rather than geography, while the geographic distribution of symbionts may be more important in other bugs.

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation.

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

A Peptidoglycan Amidase Mutant of Burkholderia insecticola Adapts an L-form-like Shape in the Gut Symbiotic Organ of the Bean Bug Riptortus pedestris.

Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

Burkholderia insecticola triggers midgut closure in the bean bug Riptortus pedestris to prevent secondary bacterial infections of midgut crypts.

In addition to abiotic triggers, biotic factors such as microbial symbionts can alter development of multicellular organisms. Symbiont-mediated morphogenesis is well-investigated in plants and marine invertebrates but rarely in insects despite the enormous diversity of insect-microbe symbioses. The bean bug Riptortus pedestris is associated with Burkholderia insecticola which are acquired from the environmental soil and housed in midgut crypts. To sort symbionts from soil microbiota, the bean bug develops a specific organ called the "constricted region" (CR), a narrow and symbiont-selective channel, located in the midgut immediately upstream of the crypt-bearing region. In this study, inoculation of fluorescent protein-labeled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12-18 h, blocking any potential subsequent infection events. The "midgut closure" developmental response was irreversible, even after symbiont removal from the crypts by antibiotics. It never occurred in aposymbiotic insects, nor in insects infected with nonsymbiotic bacteria or B. insecticola mutants unable to cross the CR. However, species of the genus Burkholderia and its outgroup Pandoraea that can pass the CR and partially colonize the midgut crypts induce the morphological alteration, suggesting that the molecular trigger signaling the midgut closure is conserved in this bacterial lineage. We propose that this drastic and quick alteration of the midgut morphology in response to symbiont infection is a mechanism for stabilizing the insect-microbe gut symbiosis and contributes to host-symbiont specificity in a symbiosis without vertical transmission.

Host-symbiont specificity determined by microbe-microbe competition in an insect gut.

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Burkholderia Gut Symbionts Associated with European and Japanese Populations of the Dock Bug Coreus marginatus (Coreoidea: Coreidae).

Insects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern.

Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture.

In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris.

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64T, was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei clade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314T (100 %), Burkholderia turbans LMG 29316T (99.52 %) and Burkholderia ptereochthonis LMG 29326T (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome blast Distance Phylogeny confirmed B. peredens LMG 29314T, B. ptereochthonis LMG 29326T and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64T represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C16 : 0, C18 : 1ω7c and C17 : 0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64T from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64T is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64T (=NCIMB 15023T=JCM 31142T).

Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.

Gut symbiotic bacteria stimulate insect growth and egg production by modulating hexamerin and vitellogenin gene expression.

Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont-colonized (Sym) insects, Burkholderia-non-colonized (Apo) insects, and Burkholderia-depleted (SymBurk-) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS-PAGE, the hexamerin-α, hexamerin-ß and vitellogenin-1 proteins of Sym-adults were highly expressed compared to those of Apo- and SymBurk--insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin-ß gene was specifically expressed at all nymphal stages, and its expression was detected 4-5 days earlier in Sym-insect nymphs than that in Apo- and SymBurk--insects. However, the hexamerin-α and vitellogenin-1 genes were only expressed in adult females, and they were also detected 6-7 days earlier and were 2-fold higher in Sym-adult females than those in the other insects. Depletion of hexamerin-ß by RNA interference in 2nd instar Sym-nymphs delayed adult emergence, whereas hexamerin-α and vitellogenin-1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins.

Insect's intestinal organ for symbiont sorting.

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 Riptortus pedestris, 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 Burkholderia 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 Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli 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.