Reduced social function in experimentally evolved Dictyostelium discoideum implies selection for social conflict in nature.

Many microbes interact with one another, but the difficulty of directly observing these interactions in nature makes interpreting their adaptive value complicated. The social amoeba Dictyostelium discoideum forms aggregates wherein some cells are sacrificed for the benefit of others. Within chimaeric aggregates containing multiple unrelated lineages, cheaters can gain an advantage by undercontributing, but the extent to which wild D. discoideum has adapted to cheat is not fully clear. In this study, we experimentally evolved D. discoideum in an environment where there were no selective pressures to cheat or resist cheating in chimaeras. Dictyostelium discoideum lines grown in this environment evolved reduced competitiveness within chimaeric aggregates and reduced ability to migrate during the slug stage. By contrast, we did not observe a reduction in cell number, a trait for which selection was not relaxed. The observed loss of traits that our laboratory conditions had made irrelevant suggests that these traits were adaptations driven and maintained by selective pressures D. discoideum faces in its natural environment. Our results suggest that D. discoideum faces social conflict in nature, and illustrate a general approach that could be applied to searching for social or non-social adaptations in other microbes.

Spo11-Independent Meiosis in Social Amoebae.

Sex in social amoebae (or dictyostelids) has a number of striking features. Dictyostelid zygotes do not proliferate but grow to a large size by feeding on other cells of the same species, each zygote ultimately forming a walled structure called a macrocyst. The diploid macrocyst nucleus undergoes meiosis, after which a single meiotic product survives to restart haploid vegetative growth. Meiotic recombination is generally initiated by the Spo11 enzyme, which introduces DNA double-strand breaks. Uniquely, as far as is known among sexual eukaryotes, dictyostelids lack a SPO11 gene. Despite this, recombination occurs at high frequencies during meiosis in dictyostelids, through unknown mechanisms. The molecular processes underlying these events, and the evolutionary drivers that brought them into being, may shed light on the genetic conflicts that occur within and between genomes, and how they can be resolved.

Triparental inheritance in Dictyostelium.

Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes, although our knowledge of the molecular details of sexual inheritance is scant in several major lineages. In social amoebae, sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells, but for technical reasons, sexual progeny have been difficult to obtain and study. We report here genome-wide characterization of meiotic progeny in Dictyostelium discoideum We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the Spo11 enzyme that is normally required to initiate crossover formation. Fusions of more than two gametes to form transient syncytia lead to frequent triparental inheritance, with haploid meiotic progeny bearing recombined nuclear haplotypes from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. D. discoideum mitochondrial genomes are polymorphic, and our findings raise the possibility that some of this variation might be a result of sexual selection on genes that can promote the spread of individual organelle genomes during sex. This kind of self-interested mitochondrial behavior may have had important consequences during eukaryogenesis and the initial evolution of sex.

Live cell imaging of cell movement and transdifferentiation during regeneration of an amputated multicellular body of the social amoeba Dictyostelium discoideum.

The regeneration of lost body parts is a fascinating phenomenon exhibited by some multicellular organisms. In social amoebae, such as Dictyostelium discoideum, the pseudoplasmodium is a temporary migratory multicellular structure with high regeneration ability. It consists of future stalk cells (prestalk cells) at the anterior end and future spore cells (prespore cells) at the posterior end, and if amputated, the remaining cells can rapidly regenerate the lost portion within several hours. Details of this regeneration event have been extensively documented; however, little is known about the behavior of individual cells involved in this process. In this study, we performed live cell imaging of cell behavior during regeneration of the excised anterior prestalk region. We used cells that specifically express GFP in the prestalk cell lineage to examine how the prestalk region is regenerated after this region is excised. The current model of prestalk regeneration suggests that the progenitors of prestalk cells, known as anterior-like cells (ALCs), which are sparsely distributed in the prespore region, are redistributed to form the new prestalk region. However, we found that the regenerated prestalk region was formed mainly by the transdifferentiation of prespore cells surrounding the excised anterior end, with little clustering of pre-existing ALCs. Furthermore, the movement of randomly distributed labeled cells during regeneration revealed that although the posterior end was deformed and rounded in shape, the relative position of cells along the anterior-posterior axis remained largely unchanged. These results suggest that the original anterior-posterior axis is maintained in posterior bodies and that prespore cells at the anterior side transdifferentiate and regenerate the prestalk region.

Synergistic activity of cosecreted natural products from amoebae-associated bacteria.

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Allorecognition, via TgrB1 and TgrC1, mediates the transition from unicellularity to multicellularity in the social amoeba Dictyostelium discoideum.

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.

Natural Products from Social Amoebae.

Natural products are invaluable sources of structural diversity and complexity ideally suited for the development of therapeutic agents. The search for novel bioactive molecules has prompted scientists to explore various ecological niches. Microorganisms have been shown to constitute such an important source. Despite their biosynthetic potential, social amoebae, that is, microorganisms with both a uni- and multicellular lifestyle, are underexplored regarding their secreted secondary metabolome. In this review, we present the structural diversity of amoebal natural products and discuss their biological functions as well as their total syntheses.

Versatile synthesis of the signaling peptide glorin.

We present a versatile synthesis of the eukaryotic signaling peptide glorin as well as glorinamide, a synthetic analog. The ability of these compounds to activate glorin-induced genes in the social amoeba Polysphondylium pallidum was evaluated by quantitative reverse transcription PCR, whereby both compounds showed bioactivity comparable to a glorin standard. This synthetic route will be useful in conducting detailed structure-activity relationship studies as well as in the design of chemical probes to dissect glorin-mediated signaling pathways.

New species of Polysphondylium from Madagascar.

Two series of samples collected for isolation of dictyostelid cellular slime molds (dictyostelids) in Madagascar yielded a relatively large number of isolates of Polysphondylium. Most of these turned out to be species new to science that show varying degrees of clustering from unclustered to coremiform as well as an ability to migrate. Migratory ability (phototaxis) is a common feature of species assigned to Group 2 of the Polysphondylia and is common in the new species from Madagascar. Another common feature, clustering, appears to be a strategy for keeping fruiting bodies erect for a longer time in a climate that is relatively dry, whereas migratory ability may function seasonally when there is more rainfall. Thirteen species are described herein. Each of these is characterized by a particular set of distinguishing features, and collectively they expand our concept of the genus Polysphondylium.

Terpene Cyclases from Social Amoebae.

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)-(-)-protoillud-7-ene and (3S)-(+)-asterisca-2(9),6-diene. The enzyme mechanisms and EI-MS fragmentations of the products were studied by labeling experiments.

Stress and development in Dictyostelium discoideum: the involvement of the catalytic calcineurin A subunit.

Calcium signaling is one of the most important signaling-pathways in all eukaryotes. One important target activated by an increased intracellular calcium concentration via calmodulin is the protein phosphatase calcineurin, which is composed of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B). The importance of calcium and calcineurin for the differentiation and development of the social amoeba Dictyostelium discoideum has already been shown by pharmacological approaches. However, so far only a RNAi-silenced calcineurin B mutant has been investigated on a molecular level. Here, we describe the construction and phenotypic investigation of a RNAi-silenced calcineurin A mutant. Phenotypic aberrations during development resemble those produced by silencing of calcineurin B with ectopic tip formation of the fruiting bodies. Additionally, we tested the response of the mutants under various stress conditions in liquid culture as well as during development. Both, calcineurin A and B RNAi-mutants, are hypersensitive during development towards cation stress. Besides its role in development, calcineurin is thus also involved in the stress response in D. discoideum. Further, our data imply that many functions of calcineurin are conserved among the eukaryotes.

New small dictyostelids from seasonal rainforests of Central America.

Ten small dictyostelids isolated from samples collected from the surface humus layer of seasonal rainforests of Belize and Guatemala were studied morphologically, and nine were found to represent distinct species, all with an average height of < 2 mm (0.5-3.5 mm). Although their fruiting bodies (sorocarps) closely resemble one another, the nine species differ in their patterns of aggregation, stream pattern, branching development, formation of microcysts, spore shape, presence or absence of spore granules and their distribution, as well as in the shapes and behavior of their sorogens and myxamoebae. These stable morphological features were sufficient to recognize nine new species of small dictyostelids, one with two varieties. SSU rDNA sequences were generated for all these new isolates, and phylogenetic analyses of these sequences show these new isolates belong to Dictyostelid group 3. As a result of this and other recent studies, the concept of what constitutes a species in the dictyostelids has become much more restricted and well defined, in as much as some of the morphological and behavioral patterns now being observed were overlooked in the past. The extent, flow direction and conformation of streaming within the group varies from simple aggregation mounds with no streams to short streams, to somewhat longer streams and finally to well developed streams. Each of these is characterized by a particular set of distinguishing features.

Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Paraburkholderia agricolaris sp. nov., Paraburkholderia hayleyella sp. nov., and Paraburkholderia bonniea sp. nov.

Here we give names to three new species of Paraburkholderia that can remain in symbiosis indefinitely in the spores of a soil dwelling eukaryote, Dictyostelium discoideum. The new species P. agricolaris sp. nov., P. hayleyella sp. nov., and P. bonniea sp. nov. are widespread across the eastern USA and were isolated as internal symbionts of wild-collected D. discoideum. We describe these sp. nov. using several approaches. Evidence that they are each a distinct new species comes from their phylogenetic position, average nucleotide identity, genome-genome distance, carbon usage, reduced length, cooler optimal growth temperature, metabolic tests, and their previously described ability to invade D. discoideum amoebae and form a symbiotic relationship. All three of these new species facilitate the prolonged carriage of food bacteria by D. discoideum, though they themselves are not food. Further studies of the interactions of these three new species with D. discoideum should be fruitful for understanding the ecology and evolution of symbioses.