Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.     

Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi.

The NMR structures of the homologous pheromones Er-1, Er-10, and Er-2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3-helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half-cystine residues in the individual sequences within the preserved 3-dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er-2, which is accommodated by a transition from an alpha-helix in Er-1 and Er-10 to a 3(10)-helix in Er-2. The most significant structural differences are located in the C-terminal part of the proteins, which may have an important role in specific receptor recognition.     

The NMR solution structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi.

The NMR structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40-residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 3-37 was 0.31 A. The molecular architecture is dominated by an up-down-up bundle of 3 short helices of residues 5-11, 14-20, and 23-33, which is similar to the structures of the homologous pheromones Er-1 and Er-10. Novel structural features include a well-defined N-cap on the first helix, a 1-residue deletion in the second helix resulting in the formation of a 3(10)-helix rather than an alpha-helix as found in Er-1 and Er-10, and the simultaneous presence of 2 different conformations for the C-terminal tetrapeptide segment, i.e., a major conformation with the Leu 39-Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.     

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae

Background and Aims The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the ‘viscid sepals’ of Specklinia endotrachys were visited by a ‘small fly’. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species.Methods Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography–mass spectometry using those same pheromones as standards.Key Results Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species.Conclusions Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.     

游仆虫信息素Er-1和Er-2对四膜虫的种间作用

信息素是生物体向外释放的化学物质,在细胞及生物体中具有种内信息传递的生理学功能。信息素这一类分子广泛分布于系统发生史中,它们的特异活性在单细胞生物、昆虫以及脊椎动物中均有报道。脊椎动物中信息素的信号传输已被证实是一嗅觉依赖过程,7TM-受体被认为是信号传输过程中的信号转换器。在低等单细胞生物(例如:来可夫游仆虫)的细胞膜上存在有信息素异构体,作为信息素分子的有效结合位点而行使其功能。本研究主要探讨单细胞的信息素(Er-1和Er-2)的基础细胞生理学作用是仅限于产生该信息素的物种,还是对其它的原生动物(例如:四膜虫)或对系统发育中分类地位较高的细胞(例如:MRC5成纤维细胞或J774巨噬细胞)均具有调节活性。研究结果表明,游仆虫的两种信息素对梨形四膜虫GL的生长调节有显著不同的作用:当信息素浓度为10-11M时,Er-1具有正调控作用,而Er-2具有抑制剂的作用。这两种配体的趋化作用也有很不同:Er-1具有一种广范的化学排斥特性,而Er-2具有一个双峰的化学吸引剂的性质。计算机检测发现,与Er-2的作用不同,Er-1可略微降低被测细胞的游动速率。趋化现象的选择特性表明Er-2信息素的受体有一种“短期”的特性;而Er-1是不能选择任何亚种群的,这也支持了我们先前的研究数据,即这两种信息素在四膜虫GL内产生两种不同的信号。四膜虫对信息素特异性的反应表明四膜虫能辨别非常近似但带有微小差异的配体(如Er-1和Er-2的电荷差异)。     

Chemical Signaling in Ciliates

ABSTRACT. For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated "gamones" identified in culture filtrates of a Blepharisma species. However, the characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone-receptor interactions and thus, provide important clues into pheromone mechanisms of action.     

Structural characterization of a protein pheromone from a cold-adapted (Antarctic) single-cell eukaryote, the ciliate Euplotes nobilii

Free-living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type-specific protein signals (pheromones). One of these molecules, designated En-2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA(20)WDTCSGGTGNCGTVCCGQCF(40)SFPVSQSCAGMADSNDCPNA(60). The En-2 structure appears to be characterized by an adaptive insertion of a glycine-rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.     

cDNA cloning and in situ hybridization of Delta11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae)

Female sex pheromones are considered to be produced in a "pheromone gland" located in the terminal abdominal segments (8th-10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for Delta11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a Delta11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th-9th intersegmental membrane.     

Perspectives on the Behavior of Entomopathogenic Nematodes from Dispersal to Reproduction: Traits Contributing to Nematode Fitness and Biocontrol Efficacy

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema are widely used for the biological control of insect pests and are gaining importance as model organisms for studying parasitism and symbiosis. In this paper recent advances in the understanding of EPN behavior are reviewed. The “foraging strategy” paradigm (distinction between species with ambush and cruise strategies) as applied to EPN is being challenged and alternative paradigms proposed. Infection decisions are based on condition of the potential host, and it is becoming clear that already-infected and even long-dead hosts may be invaded, as well as healthy live hosts. The state of the infective juvenile (IJ) also influences infection, and evidence for a phased increase in infectivity of EPN species is mounting. The possibility of social behavior - adaptive interactions between IJs outside the host - is discussed. EPNs’ symbiotic bacteria (Photorhabdus and Xenorhabdus) are important for killing the host and rendering it suitable for nematode reproduction, but may reduce survival of IJs, resulting in a trade-off between survival and reproduction. The symbiont also contributes to defence of the cadaver by affecting food-choice decisions of insect and avian scavengers. I review EPN reproductive behavior (including sperm competition, copulation and evidence for attractive and organizational effects of pheromones), and consider the role of endotokia matricida as parental behavior exploited by the symbiont for transmission.     

Isolation of iridoid and secoiridoid glycosides and comparative study on Radix gentianae and related adulterants by HPLC analysis

HPLC profile guided study led to the isolation of an acylated secoiridoid glycoside, named gentiotrifloroside (1), together with six known compounds, i.e., loganic acid (2), 6-O-beta-d-glucopyranosylgentiopicroside (3), swertiamarin (4), gentiopicroside (5), sweroside (6) and 2 -(o,m-dihydroxybenzyl)-sweroside (7) from Gentiana triflora and Gentiana rigescens. The structure of 1 was deduced from one- and two-dimensional NMR spectroscopic experiments. Compounds 1-7 were used successfully as chemical markers for the comparison of the four species of Gentiana used as Radix gentianae. Additionally, differentiation of Gentiana species mentioned and those used as adulterants was evaluated. The close similarity of chemical composition among the four genuine Gentiana species explain their popular usage as R. gentianae in Chinese medicine. We have also shown that the variation of chemical composition in R. gentianae and related adulterants agree well with their botanical phylogeny.     

The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The 3-dimensional structure of the pheromone Er-1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40-residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin-spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er-1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 A for the backbone atoms N, C alpha, and C', and 0.93 A for all heavy atoms of the complete polypeptide chain, residues 1-40. The molecular architecture is dominated by an up-down-up bundle of 3 alpha-helices formed by residues 2-9, 12-19, and 24-33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er-10, the C-terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.     

Phylogenetic analysis of the genus Sorghum based on combined sequence data from cpDNA regions and ITS generate well-supported trees with two major lineages

Background and Aims

Wild Sorghum species provide novel traits for both biotic and abiotic stress resistance and yield for the improvement of cultivated sorghum. A better understanding of the phylogeny in the genus Sorghum will enhance use of the valuable agronomic traits found in wild sorghum.

Methods

Four regions of chloroplast DNA (cpDNA; psbZ-trnG, trnY-trnD, trnY-psbM and trnT-trnL) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA were used to analyse the phylogeny of sorghum based on maximum-parsimony analyses.

Key Results

Parsimony analyses of the ITS and cpDNA regions as separate or combined sequence datasets formed trees with strong bootstrap support with two lineages: the Eu-sorghum species S. laxiflorum and S. macrospermum in one and Stiposorghum and Para-sorghum in the other. Within Eu-sorghum, S. bicolor-3, -11 and -14 originating from southern Africa form a distinct clade. S. bicolor-2, originally from Yemen, is distantly related to other S. bicolor accessions.

Conclusions

Eu-sorghum species are more closely related to S. macrospermum and S. laxiflorum than to any other Australian wild Sorghum species. S. macrospermum and S. laxiflorum are so closely related that it is inappropriate to classify them in separate sections. S. almum is closely associated with S. bicolor, suggesting that the latter is the maternal parent of the former given that cpDNA is maternally inherited in angiosperms. S. bicolor-3, -11 and -14, from southern Africa, are closely related to each other but distantly related to S. bicolor-2.     

Structural characterization of En-1, a cold-adapted protein pheromone isolated from the Antarctic ciliate Euplotes nobilii

The second of two diffusible cell signal proteins (pheromones) purified from a wild-type strain of the Antarctic ciliate, Euplotes nobilii, has been determined by automated Edman degradation of the whole molecule and peptides generated by its chymotryptic digestion. The proposed sequence of 52 amino acids of this new pheromone, designated En-1, is: NPEDWFTPDT(10)CAYGDSNTAW(20)TTCTTPGQTC(30)YTCCSSCFDV(40)VGEQACQMSA(50)QC. In common with the previously determined 60-amino-acid sequence of the other pheromone, En-2, it bears eight cysteines in conserved positions (presumably linked into four conserved intrachain disulfide bonds), and physicochemical features of potential significance for cold adaptation, such as a reduced hydrophobicity, an increased solvent accessibility, and an improved local backbone flexibility. However, En-1 diverges from En-2 for having evolved a threonine cluster in the place of a glycine cluster to apparently make more flexible a region that is likely functionally important.     

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.     

NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7

Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.     

Anhydrobiotic Coiling of Nematodes in Soil

Nematodes of three genera (Acrobeloides sp., Aphelenchus avenae, and Scutellonema brachyurum) were induced to coil and enter anhydrobiosis in drying soil of two types: sandy loam and loamy sand. Coiling was studied in relationship to soil moisture characteristics. Coiling and the physiological state of anhydrobiosis occurred before the water in sandy soils reached a water potential of -15 bars. Coiling was maximum at 3-6 bars, depending on the soil type and nematode species. It appeared that induction of coiling and anhydrohiosis were determined by the physical forces exerted by the water film surrounding the nematode, which, for these three species, was 6-9 monomolecular layers of water, rather than the % moisture and relative humidity of the soil per se.     

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.     

Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera,Drosophilidae): Cytochemical,structural and ultrastructural characterization

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1–3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.