Dictyostelium discoideum

Abstract: The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Abstract: 1. A simple axenic medium suitable for the growth of the myxamoebae of a strain of the cellular slime mould Dictyostelium discoideum is described. 2. Procedures suitable for the growth of this strain in liquid and on solid media are described. 3. Conditions suitable for initiating the cell differentiation of myxamoebae grown axenically are described.

Abstract: The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.

Abstract: The bacterium Vibrio cholerae, like other human pathogens that reside in environmental reservoirs, survives predation by unicellular eukaryotes. Strains of the O1 and O139 serogroups cause cholera, whereas non-O1/non-O139 strains cause human infections through poorly defined mechanisms. Using Dictyostelium discoideum as a model host, we have identified a virulence mechanism in a non-O1/non-O139 V. cholerae strain that involves extracellular translocation of proteins that lack N-terminal hydrophobic leader sequences. Accordingly, we have named these genes “VAS” genes for virulence-associated secretion, and we propose that these genes encode a prototypic “type VI” secretion system. We show that vas genes are required for cytotoxicity of V. cholerae cells toward Dictyostelium amoebae and mammalian J774 macrophages by a contact-dependent mechanism. A large number of Gram-negative bacterial pathogens carry genes homologous to vas genes and potential effector proteins secreted by this pathway (i.e., hemolysin-coregulated protein and VgrG). Mutations in vas homologs in other bacterial species have been reported to attenuate virulence in animals and cultured macrophages. Thus, the genes encoding the VAS-related, type VI secretion system likely play an important conserved function in microbial pathogenesis and represent an additional class of targets for vaccine and antimicrobial drug-based therapies.

Abstract: In eukaryotic cells directional sensing is mediated by heterotrimeric guanine nucleotide-binding protein (G protein)-linked signaling pathways. In Dictyostelium discoideum amoebae and mammalian leukocytes, the receptors and G-protein subunits are uniformly distributed around the cell perimeter. Chemoattractants induce the transient appearance of binding sites for several pleckstrin homology domain-containing proteins on the inner face of the membrane. In gradients of attractant these sites are persistently present on the side of the cell facing the higher concentration, even in the absence of a functional actin cytoskeleton or cell movement. Thus, the cell senses direction by spatially regulating the activity of the signal transduction pathway.