Molecular mechanisms underlying metamorphosis in the most-ancestral winged insect.

Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 (Kr-h1), broad, and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonataE93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant (Met), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1, broad, and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1, broad, and E93 in the evolution of insect metamorphosis.

Characterizing an amidase and its operon from actinomycete bacteria responsible for paraben catabolism.

Hydrazidase from Microbacterium hydrocarbonoxydans was revealed to catalyze synthetic hydrazide compounds, enabling the bacteria to grow with them as sole carbon source, but natural substrates have remained unknown. In this study, kinetic analyses of hydrazidase with parabens showed that the compounds can be substrates. Then, methylparaben induced gene expressions of the operon containing hydrazidase and ABC transporter, and the compound as sole carbon source was able to grow the bacteria. Furthermore, homology search was carried out revealing that several actinomycetes possess hydrazidase-homolog in the operon. Among those bacteria, an amidase from Pseudonocardia acaciae was subjected to a kinetic analysis and a structure determination revealing similar but not identical to those of hydrazidase. Since parabens are reported to exist in plants and soil, and several actinomycetes codes the homologous operon, the enzymes with those operons may play a physiologically important role for bacterial survival with use of parabens.

Involvement of cyclic nucleotide-gated channels in soybean cyst nematode chemotaxis and thermotaxis.

The soybean cyst nematode (SCN) is one of the most damaging pests affecting soybean production. SCN displays important host recognition behaviors, such as hatching and infection, by recognizing several compounds produced by the host. Therefore, controlling SCN behaviors such as chemotaxis and thermotaxis is an attractive pest control strategy. In this study, we found that cyclic nucleotide-gated channels (CNG channels) regulate SCN chemotaxis and thermotaxis and Hg-tax-2, a gene encoding a CNG channel, is an important regulator of SCN behavior. Gene silencing of Hg-tax-2 and treatment with a CNG channel inhibitor reduced the attraction of second-stage juveniles to nitrate, an attractant with a different recognition mechanism from the host-derived chemoattractant(s), and to host soybean roots, as well as their avoidance behavior toward high temperatures. Co-treatment of ds Hg-tax-2 with the CNG channel inhibitor indicated that Hg-tax-2 is a major regulator of SCN chemotaxis and thermotaxis. These results suggest new avenues for research on control of SCN.

Mycothiol maintains the homeostasis and signalling of nitric oxide in Streptomyces coelicolor A3(2) M145.

BACKGROUND: Previous studies have revealed a nitric oxide (NO) metabolic cycle in which NO, nitrate (NO3-), and nitrite (NO2-) circulate. The NO produced in this cycle serves as a signalling molecule that regulates actinorhodin (ACT) production via the DevS/DevR NO-dependent two-component system (TCS) in Streptomyces coelicolor A3(2) M145. However, the mechanisms involved in the regulation of NO signalling in S. coelicolor have not yet been elucidated. Mycothiol (MSH), a thiol molecule produced by Actinomyces, is involved in the defence mechanisms against oxidative stress. Therefore, this study focused on the correlation between intracellular NO and MSH levels. RESULTS: To investigate the interaction of MSH with endogenously produced NO, we generated an S. coelicolor A3(2) strain deficient in MSH biosynthesis. This mutant strain exhibited a decrease in low-molecular-weight S-nitrosothiols and intracellular NO levels during culture compared to those of the wild-type strain. Moreover, the mutant strain exhibited reduced activity of the DevS/DevR TCS, a regulator of NO homeostasis and ACT production, from the early stage of culture, along with a decrease in ACT production compared to those of the wild-type strain. CONCLUSIONS: This study suggests that MSH maintains intracellular NO homeostasis by forming S-nitrosomycothiol, which induces NO signalling. Finally, we propose a metabolic model in which MSH from endogenously produced NO facilitates the maintenance of both NO homeostasis and signalling in S. coelicolor A3(2) M145.

Genomic traces of Japanese malting barley breeding in two modern high-quality cultivars, 'Sukai Golden' and 'Sachiho Golden'.

Two modern high-quality Japanese malting barley cultivars, 'Sukai Golden' and 'Sachiho Golden', were subjected to RNA-sequencing of transcripts extracted from 20-day-old immature seeds. Despite their close relation, 2,419 Sukai Golden-specific and 3,058 Sachiho Golden-specific SNPs were detected in comparison to the genome sequences of two reference cultivars: 'Morex' and 'Haruna Nijo'. Two single nucleotide polymorphism (SNP) clusters respectively showing the incorporation of (1) the barley yellow mosaic virus (BaYMV) resistance gene rym5 from six-row non-malting Chinese landrace Mokusekko 3 on the long arm of 3H, and (2) the anthocyanin-less ant2 gene from a two-row Dutch cultivar on the long arm of 2H were detected specifically in 'Sukai Golden'. Using 221 recombinant inbred lines of a cross between 'Ishukushirazu' and 'Nishinochikara', another BaYMV resistance rym3 gene derived from six-row non-malting Japanese cultivar 'Haganemugi' was mapped to a 0.4-cM interval on the proximal region of 5H. Haplotype analysis of progenitor accessions of the two modern malting cultivars revealed that rym3 of 'Haganemugi' was independently introduced into 'Sukai Golden' and 'Sachiho Golden'. Residual chromosome 5H segments of 'Haganemugi' surrounding rym3 were larger in 'Sukai Golden'. Available results suggest possibilities for malting quality improvement by minimizing residual segments surrounding rym3.

Targeted in vivo mutagenesis of a sensor histidine kinase playing an essential role in ABA signaling of the moss Physcomitrium patens.

Land plants exhibit various adaptation responses to unfavorable water environments, such as drought and flooding. The phytohormone abscisic acid (ABA) and ethylene play essential roles in plant adaptation to drought and flooding, respectively. It remains largely unknown how plants integrate environmental information for water availability. In the moss Physcomitrium patens, we recently reported that not only ethylene/flooding signaling but also ABA/osmostress signaling are mediated by ethylene receptor-related sensor histidine kinases (ETR-HKs). Subfamily I ETR-HKs of this moss were found to interact with a RAF kinase (ARK) and were required for ABA-dependent activation of SNF1-related protein kinase 2 (SnRK2) via ARK activation. To elucidate the mechanisms of ARK regulation by ETR-HKs, here we employed targeted in vivo mutagenesis of PpHK5, a member of subfamily I ETR-HKs. Analyses of ABA-insensitive Pphk5 mutants indicated that PpHK5 mutations affecting the interaction with ARK resulted in loss of PpHK5 function to activate ABA signaling. We also identified a PpHK5 mutation that does not affect ARK interaction but resulted in loss of PpHK5 function. These results suggest that physical interaction between ETR-HK and ARK is essential but not sufficient for the regulation of ARK activity, and the C-terminal response regulator domain is involved in regulating ARK activation.

Nitric Oxide Signaling for Aerial Mycelium Formation in Streptomyces coelicolor A3(2) M145.

Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes. IMPORTANCE Eukaryotic and prokaryotic cells utilize nitric oxide (NO) to regulate physiological functions. Besides its role as a producer of different bioactive substances, Streptomyces is suggested to be involved in mycelial development regulated by endogenously produced NO. However, the regulatory mechanisms are unclear. In this study, we proposed that NO signaling is involved in aerial mycelium formation in S. coelicolor A3(2) M145. NO serves as a signaling molecule for the regulation of intracellular cyclic 3',5'-diguanylate (c-di-GMP) levels, resulting in aerial mycelium formation controlled by a c-di-GMP receptor, BldD. As the abundant production of valuable secondary metabolites is closely related to the initiation of morphological differentiation in Streptomyces, NO may provide value for application in industrial fermentation by serving as a tool for regulating secondary metabolism.

Parallel evolution of opsin visual pigments in hawkmoths by tuning of spectral sensitivities during transition from a nocturnal to a diurnal ecology.

Light environments differ dramatically between day and night. The transition between diurnal and nocturnal visual ecology has happened repeatedly throughout evolution in many species. However, the molecular mechanism underlying the evolution of vision in recent diurnal-nocturnal transition is poorly understood. Here, we focus on hawkmoths (Lepidoptera: Sphingidae) to address this question by investigating five nocturnal and five diurnal species. We performed RNA-sequencing analysis and identified opsin genes corresponding to the ultraviolet (UV), short-wavelength (SW) and long-wavelength (LW)-absorbing visual pigments. We found no significant differences in the expression patterns of opsin genes between the nocturnal and diurnal species. We then constructed the phylogenetic trees of hawkmoth species and opsins. The diurnal lineages had emerged at least three times from the nocturnal ancestors. The evolutionary rates of amino acid substitutions in the three opsins differed between the nocturnal and diurnal species. We found an excess number of parallel amino acid substitutions in the opsins in three independent diurnal lineages. The numbers were significantly more than those inferred from neutral evolution, suggesting that positive selection acted on these parallel substitutions. Moreover, we predicted the visual pigment absorption spectra based on electrophysiologically determined spectral sensitivity in two nocturnal and two diurnal species belonging to different clades. In the diurnal species, the LW pigments shift 10 nm towards shorter wavelengths, and the SW pigments shift 10 nm in the opposite direction. Taken together, our results suggest that parallel evolution of opsins may have enhanced the colour discrimination properties of diurnal hawkmoths in ambient light.

Nitric Oxide Signaling for Actinorhodin Production in Streptomyces coelicolor A3(2) via the DevS/R Two-Component System.

Nitric oxide (NO) is an important signaling molecule in eukaryotic and prokaryotic cells. A previous study revealed an NO synthase-independent NO production metabolic cycle in which the three nitrogen oxides, nitrate (NO3-), nitrite (NO2-), and NO, were generated in the actinobacterium Streptomyces coelicolor A3(2). NO was suggested to act as a signaling molecule, functioning as a hormone that regulates secondary metabolism. Here, we demonstrate the NO-mediated regulation of the production of the blue-pigmented antibiotic actinorhodin (ACT), via the heme-based DevS/R two-component system (TCS). Intracellular NO controls the stabilization or inactivation of DevS, depending on the NO concentration. An electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR analysis revealed the direct binding between DevR and the promoter region of actII-ORF4, resulting in gene expression. Our results indicate that NO regulates the DevS/R TCS, thereby strictly controlling the secondary metabolism of S. coelicolor A3(2). IMPORTANCE Diverse organisms, such as mammals, plants, and bacteria, utilize NO via well-known signal transduction mechanisms. Many useful secondary metabolite-producing bacteria of the Streptomyces genus had been also suggested for the metabolism regulated by endogenously produced NO; however, the regulatory mechanisms remain to be elucidated. In this study, we demonstrated the molecular mechanism by which endogenously produced NO regulates antibiotic production via the DevS/R TCS in S. coelicolor A3(2). NO serves as both a stabilizer and a repressor in the regulation of antibiotic production. This report shows the mechanism by which Streptomyces utilizes endogenously produced NO to modulate its normal life cycle. Moreover, this study implies that studying NO signaling in actinobacteria can help in the development of both clinical strategies against pathogenic actinomycetes and the actinobacterial industries.

Structural basis of substrate recognition by the substrate binding protein (SBP) of a hydrazide transporter, obtained from Microbacterium hydrocarbonoxydans.

Microbacterium hydrocarbonoxydans was isolated, using hydrazide compounds as its sole carbon source. The key enzyme that metabolizes these compounds was identified as hydrazidase, and the operon containing the gene coding for the enzyme, was revealed by genome sequencing. The operon also contained genes coding for an ATP-binding cassette transporter (ABC transporter), which was expected to transport the hydrazide compounds. Substrate binding protein (SBP), a component subunit of the transporter, plays an important role in recognizing the correct substrates for transport. Therefore, to elucidate the mechanism of recognition of the unnatural hydrazide compounds, we determined the crystal structures of the SBP, obtained from M. hydrocarbonoxydans (Mh-SBP), complexed with and without the hydrazide compound, at 2.2 Å and 1.75 Å resolutions, respectively. The overall structures of Mh-SBP were similar to those of the SBP in oligopeptide transporters such as OppA. On comparison, the liganded and unliganded structures of Mh-SBP showed an open - close conformation change. Interestingly, the binding mode of the compound to Mh-SBP was almost identical to that of the compound to hydrazidase, suggesting that the ABC transporter served transporting these compounds. Furthermore, based on the hydrazide complex structure, paraben, the other putative substrate of the protein, was successfully used with Mh-SBP to obtain the paraben complex structure.

Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans.

scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as L-glucose, converting L-glucose to L-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and L-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for L-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.Abbreviations: IDH: inositol dehydrogenase; LB: Luria-Bertani; kcat: catalyst rate constant; Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL.

Triflumizole as a Novel Lead Compound for Strigolactone Biosynthesis Inhibitor.

Strigolactones (SLs) are carotenoid-derived plant hormones involved in the development of various plants. SLs also stimulate seed germination of the root parasitic plants, Striga spp. and Orobanche spp., which reduce crop yield. Therefore, regulating SL biosynthesis may lessen the damage of root parasitic plants. Biosynthetic inhibitors effectively control biological processes by targeted regulation of biologically active compounds. In addition, biosynthetic inhibitors regulate endogenous levels in developmental stage- and tissue-specific manners. To date, although some chemicals have been found as SL biosynthesis inhibitor, these are derived from only three lead chemicals. In this study, to find a novel lead chemical for SL biosynthesis inhibitor, 27 nitrogen-containing heterocyclic derivatives were screened for inhibition of SL biosynthesis. Triflumizole most effectively reduced the levels of rice SL, 4-deoxyorobanchol (4DO), in root exudates. In addition, triflumizole inhibited endogenous 4DO biosynthesis in rice roots by inhibiting the enzymatic activity of Os900, a rice enzyme that converts the SL intermediate carlactone to 4DO. A Striga germination assay revealed that triflumizole-treated rice displayed a reduced level of germination stimulation for Striga. These results identify triflumizole as a novel lead compound for inhibition of SL biosynthesis.

Crystal structure of the delta-class glutathione transferase in Musca domestica.

Among the various glutathione transferase (GST) isozymes in insects, the delta- and epsilon-class GSTs fulfill critical functions during the detoxification of insecticides. We crystalized MdGSTD1, the major delta-class GST isozyme in the housefly (Musca domestica), in complex with glutathione (GSH) and solved its structure at a resolution of 1.4 Å. The overall folding of MdGSTD1 resembled other known delta-class GSTs. Its substrate binding pocket was exposed to solvent and considerably more open than in the epsilon-class GST from M. domestica (MdGSTE2). However, their C-terminal structures differed the most because of the different lengths of the C-terminal regions. Although this region does not seem to directly interact with substrates, its deletion reduced the enzymatic activity by more than 70%, indicating a function in maintaining the proper conformation of the binding pocket. Binding of GSH to the GSH-binding region of MdGSTD1 results in a rigid conformation of this region. Although MdGSTD1 has a higher affinity for GSH than the epsilon class enzymes, the thiol group of the GSH molecule was not close enough to serine residue 9 to form a hydrogen-bond with this residue, which is predicted to act as the catalytic center for thiol group deprotonation in GSH.

Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation.

Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.

Regulation of Strigolactone Biosynthesis by Gibberellin Signaling.

Strigolactones (SLs) are a class of plant hormones that regulate diverse physiological processes, including shoot branching and root development. They also act as rhizosphere signaling molecules to stimulate the germination of root parasitic weeds and the branching of arbuscular mycorrhizal fungi. Although various types of cross talk between SLs and other hormones have been reported in physiological analyses, the cross talk between gibberellin (GA) and SLs is poorly understood. We screened for chemicals that regulate the level of SLs in rice (Oryza sativa) and identified GA as, to our knowledge, a novel SL-regulating molecule. The regulation of SL biosynthesis by GA is dependent on the GA receptor GID1 and F-box protein GID2. GA treatment also reduced the infection of rice plants by the parasitic plant witchers weed (Striga hermonthica). These data not only demonstrate, to our knowledge, the novel plant hormone cross talk between SL and GA, but also suggest that GA can be used to control parasitic weed infections.

Extraordinary diversity of visual opsin genes in dragonflies.

Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.

Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source.

Hydrazidase was an enzyme that remained unidentified for a half century. However, recently, it was purified, and its encoding gene was cloned. Microbacterium sp. strain HM58-2 grows with acylhydrazides as its sole carbon source; it produces hydrazidase and degrades acylhydrazides to acetate and hydrazides. The bacterial hydrazidase belongs to the amidase signature enzyme family and contains a Ser-cisSer-Lys catalytic motif. The condensation of hydrazine and carbonic acid produces various hydrazides, some of which are raw materials for synthesizing pharmaceuticals and other useful chemicals. Although natural hydrazide compounds have been identified, the metabolic systems for hydrazides are not fully understood. Here, we report the crystal structure of hydrazidase from Microbacterium sp. strain HM58-2. The active site was revealed to consist of a Ser-cisSer-Lys catalytic triad, in which Ser179 forms a covalent bond with a carbonyl carbon of the substrate. 4-Hydroxybenzoic acid hydrazide bound to the S179A mutant, showing an oxyanion hole composed of the three backbone amide groups. Furthermore, H336 in the non-conserved region in the amidase family may define the substrate specificity, which was confirmed by mutation analysis. A wild-type apoenzyme structure revealed an unidentified molecule covalently bound to S179, representing a tetrahedral intermediate.

Nitrate analogs as attractants for soybean cyst nematode.

Soybean cyst nematode (SCN) Heterodera glycines Ichinohe, a plant parasite, is one of the most serious pests of soybean. In this paper, we report that SCN is attracted to nitrate and its analogs. We performed attraction assays to screen for novel attractants for SCN and found that nitrates were attractants for SCN and SCN recognized nitrate gradients. However, attraction of SCN to nitrates was not observed on agar containing nitrate. To further elucidate the attraction mechanism in SCN, we performed attraction assays using nitrate analogs ([Formula: see text], [Formula: see text], [Formula: see text]). SCN was attracted to all nitrate analogs; however, attraction of SCN to nitrate analogs was not observed on agar containing nitrate. In contrast, SCN was attracted to azuki root, irrespective of presence or absence of nitrate in agar media. Our results suggest that the attraction mechanisms differ between plant-derived attractant and nitrate.

Expression analysis of Egr-1 ortholog in metamorphic brain of honeybee (Apis mellifera L.): Possible evolutionary conservation of roles of Egr in eye development in vertebrates and insects.

Specific genes quickly transcribed after extracellular stimuli without de novo protein synthesis are known as immediate early genes (IEGs) and are thought to contribute to learning and memory processes in the mature nervous system of vertebrates. A recent study revealed that the homolog of Early growth response protein-1 (Egr-1), which is one of the best-characterized vertebrate IEGs, shared similar properties as a neural activity-dependent gene in the adult brain of insects. With regard to the roles of vertebrate Egr-1 in neural development, the contribution to the development and growth of visual systems has been reported. However, in insects, the expression dynamics of the Egr-1 homologous gene during neural development remains poorly understood. Our expression analysis demonstrated that AmEgr, a honeybee homolog of Egr-1, was transiently upregulated in the developing brain during the early to mid pupal stages. In situ hybridization and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry revealed that AmEgr was mainly expressed in post-mitotic cells in optic lobes, the primary visual center of the insect brain. These findings suggest the evolutionarily conserved role of Egr homologs in the development of visual systems in vertebrates and insects.

Conformational divergence in the HA-33/HA-17 trimer of serotype C and D botulinum toxin complex.

Clostridium botulinum produces a large toxin complex (L-TC) comprising botulinum neurotoxin associated with auxiliary nontoxic proteins. A complex of 33- and 17-kDa hemagglutinins (an HA-33/HA-17 trimer) enhances L-TC transport across the intestinal epithelial cell layer via binding HA-33 to a sugar on the cell surface. At least two subtypes of serotype C/D HA-33 exhibit differing preferences for the sugars sialic acid and galactose. Here, we compared the three-dimensional structures of the galactose-binding HA-33 and HA-33/HA-17 trimers produced by the C-Yoichi strain. Comparisons of serotype C/D HA-33 sequences reveal a variable region with relatively low sequence similarity across the C. botulinum strains; the variability of this region may influence the manner of sugar-recognition by HA-33. Crystal structures of sialic acid- and galactose-binding HA-33 are broadly similar in appearance. However, small-angle X-ray scattering revealed distinct solution structures for HA-33/HA-17 trimers. A structural change in the C-terminal variable region of HA-33 might cause a dramatic shift in the conformation and sugar-recognition mode of HA-33/HA-17 trimer.