Background Amoebae and bacteria interact within predator/prey and host/pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by diverse bacterial species including Gram-positive [Gram(+)] and Gram-negative [Gram(−)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. Results Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+), or with Gram(−) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell surface protein gp130, as well as several genes that are only required for growth on Gram(−) bacteria including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. Conclusions We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(−), bacteria which we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival.

BackgroundDevelopment of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under both developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods.ResultsHere, we combine the superior depth and specificity of RNA-seq-based analysis of mRNA abundance with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large shifts. For each time point we treated the entire transcriptome as single phenotype, and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represented gradual changes in mRNA abundance, or molecular phenotype, and the gaps represented times during which many genes are differentially expressed rapidly, and thus the phenotype changes dramatically. Comparing developmental experiments revealed that gene expression in filter developed cells lagged behind those treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis suggested that the transition to multicellularity coincided with rapid accumulation of transcripts associated with DNA processes and mitosis. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. Our analysis also demonstrated a high level of synchrony among the developing structures throughout development.ConclusionsOur data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and among multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for studies of developmental gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1491-7) contains supplementary material, which is available to authorized users.

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/ nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.terpene synthases | amoebae | volatiles | evolution | chemical ecology

Biological oscillations are observed at many levels of cellular organization. In the social amoebae Dictyostelium discoideum, starvation-triggered multicellular development is organized by periodic cAMP waves, which provide both chemoattractant gradients and developmental signals. We report that GtaC, a GATA transcription factor, exhibits rapid nucleocytoplasmic shuttling in response to cAMP waves. This behavior requires coordinated action of a nuclear localization signal and reversible G protein-coupled receptor (GPCR)-mediated phosphorylation. While both are required for developmental gene expression, receptor occupancy promotes nuclear exit of GtaC, which leads to a transient burst of transcription at each cAMP cycle. We demonstrate that this biological circuit, like an “edge trigger”, filters out high frequency signals and counts those admitted, thereby enabling cells to modulate gene expression according to the dynamic pattern of the external stimuli.

The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP), and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprising the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism, and this acts as a gating step for multicellularity.

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The approach employed the advanced intermediate 3-O-acetyl-6-O-(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-d-glucopyrano-syl)-2-azido-4-O-benzyl-2-deoxy-1-thio-alpha-d-glucopyranoside (5), which is protected in such a way that the anomeric center, the C-2 and C-2' amino groups, and the C-3 and C-3' hydroxyls can be selectively functionalized. The synthetic strategy was used for the preparation of 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-alpha-d-glucopyranose (11) and 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-d-glucono-1,5-lactone (13), which contain an unusual octacosanoic acid moiety and differ in the oxidation state of the anomeric center. The results of biological studies indicate that 11 and 13 lack the proinflammatory effects of Escherichia coli lipopolysaccharides (LPS). Furthermore, 13 emulated the ability of heterogeneous R. sin-1 LPS to antagonize enteric LPS, providing evidence for the critical role of the gluconolactone moiety of R. sin-1 LPS in mediating this antagonistic effect. Compound 13 is the first example of a lipid A derivative that is devoid of phosphate but possesses antagonistic properties, making it an attractive lead compound for development of a drug to use in the treatment of Gram-negative septicemia.

Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum. Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost-and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods.

In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism.