Comparative genomics of the social amoebae <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> and <Emphasis Type="Italic">Dictyostelium purpureum</Emphasis>

Background

The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.

Results

We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.

Conclusions

The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Isolation and characterization of the major histocompatibility complex <Emphasis Type="BoldItalic">DQA1</Emphasis> and <Emphasis Type="BoldItalic">DQA2</Emphasis> genes in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identified MHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest that duplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.     

Subcellular localization of ammonium transporters in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists.     

Isolation and Hybridization Kinetics of Messenger RNA from <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Messenger RNA from the cellular slime mould Dictyostelium discoideum contains a sequence of poly A residues and more than 95% hybridizes to the non-reiterated DNA.     

Arachidonic acid is a chemoattractant for <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> cells

Cyclic AMP (cAMP) is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP₃), Ca²⁺ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca²⁺ concentration by causing the release of Ca²⁺ from intracellular stores and activating influx of extracellular Ca²⁺. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8–9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca²⁺ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycolbis(b-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P₃-receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca²⁺ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.     

Understanding Streaming in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: Theory Versus Experiments

Recent experimental work involving Dictyostelium discoideum seems to contradict several theoretical models. Experiments suggest that localization of the release of the chemoattractant cyclic adenosine monophosphate to the uropod of the cell is important for stream formation during aggregation. Yet several mathematical models are able to reproduce streaming as the cells aggregate without taking into account localization of the chemoattractant. A careful analysis of the experiments and the theory suggests the two major features of the system which are important to stream formation are random cell motion and chemotaxis to regions of higher cell density. Random cell motion acts to reduce streaming, whereas chemotaxis to regions of higher cell density reinforces streaming. With this understanding, the experimental results can be explained in a manner consistent with the theoretical results. In all the experiments, alterations in the two main factors of random motion and chemotaxis to regions of higher cell density, not the localization of the release of the chemoattractant, can explain the results as they relate to streaming. Additionally, a comparison of results from a mathematical model that simulates cells which localize the chemoattractant and cells which do not shows little difference in the streaming patterns.     

The Microbial <Emphasis Type="BoldItalic">Phyllo</Emphasis>geography of the Carnivorous Plant <Emphasis Type="BoldItalic">Sarracenia alata</Emphasis>

Carnivorous pitcher plants host diverse microbial communities. This plant–microbe association provides a unique opportunity to investigate the evolutionary processes that influence the spatial diversity of microbial communities. Using next-generation sequencing of environmental samples, we surveyed microbial communities from 29 pitcher plants (Sarracenia alata) and compare community composition with plant genetic diversity in order to explore the influence of historical processes on the population structure of each lineage. Analyses reveal that there is a core S. alata microbiome, and that it is similar in composition to animal gut microfaunas. The spatial structure of community composition in S. alata (phyllogeography) is congruent at the deepest level with the dominant features of the landscape, including the Mississippi river and the discrete habitat boundaries that the plants occupy. Intriguingly, the microbial community structure reflects the phylogeographic structure of the host plant, suggesting that the phylogenetic structure of bacterial communities and population genetic structure of their host plant are influenced by similar historical processes.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Marker-assisted pyramiding of <Emphasis Type="BoldItalic">Thinopyrum-</Emphasis>derived leaf rust resistance genes <Emphasis Type="BoldItalic">Lr19</Emphasis> and <Emphasis Type="BoldItalic">Lr24</Emphasis> in bread wheat variety HD2733

This study was undertaken to pyramid two effective leaf rust resistance genes (Lr19 and Lr24) derived from Thinopyrum (syn. Agropyron), in the susceptible, but agronomically superior wheat cultivar HD2733 using marker-assisted selection. In the year 2001, HD2733 was released for irrigated timely sown conditions of the north eastern plains zone (NEPZ) of India became susceptible to leaf rust, a major disease of the region. Background selection helped in developing near-isogenic lines (NILs) of HD2733 with Lr19 and Lr24 with 97.27 and \(98.94\%\), respectively, of genomic similarity with the parent cultivar, after two backcrossing and one generation of selfing. NILs were intercrossed to combine the genes Lr19 and Lr24. The combination of these two genes in the cultivar HD2733 is expected to provide durable leaf rust resistance in farmers’ fields.     

Autonomous and non-autonomous traits mediate social cooperation in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

In the trishanku (triA ⁻ ) mutant of the social amoeba Dictyostelium discoideum, aggregates are smaller than usual and the spore mass is located mid-way up the stalk, not at the apex. We have monitored aggregate territory size, spore allocation and fruiting body morphology in chimaeric groups of (quasi-wild-type) Ax2 and triA ⁻ cells. Developmental canalisation breaks down in chimaeras and leads to an increase in phenotypic variation. A minority of triA ⁻ cells causes largely Ax2 aggregation streams to break up; the effect is not due to the counting factor. Most chimaeric fruiting bodies resemble those of Ax2 or triA ⁻ . Others are double-deckers with a single stalk and two spore masses, one each at the terminus and midway along the stalk. The relative number of spores belonging to the two genotypes depends both on the mixing ratio and on the fruiting body morphology. In double-deckers formed from 1:1 chimaeras, the upper spore mass has more Ax2 spores, and the lower spore mass more triA ⁻ spores, than expected. Thus, the traits under study depend partly on the cells’ own genotype and partly on the phenotypes, and so genotypes, of other cells: they are both autonomous and non-autonomous. These findings strengthen the parallels between multicellular development and behaviour in social groups. Besides that, they reinforce the point that a trait can be associated with a genotype only in a specified context.     

Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

<Emphasis Type="Bold">Report from the 33rd</Emphasis><Emphasis Type="BoldItalic">Mycotoxin-Workshop</Emphasis><Emphasis Type="Bold">(Freising,Germany)</Emphasis>

Society for Mycotoxin Research – News and Announcements 2011/II

Report from the 33rd Mycotoxin-Workshop (Freising, Germany)     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

cAMP controls cytosolic Ca<Superscript>2+</Superscript> levels in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) that is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca²⁺]_i.