Polyketide synthase genes and the natural products potential of Dictyostelium discoideum.

MOTIVATION: The genome of the social amoeba Dictyostelium discoideum contains an unusually large number of polyketide synthase (PKS) genes. An analysis of the genes is a first step towards understanding the biological roles of their products and exploiting novel products. RESULTS: A total of 45 Type I iterative PKS genes were found, 5 of which are probably pseudogenes. Catalytic domains that are homologous with known PKS sequences as well as possible novel domains were identified. The genes often occurred in clusters of 2-5 genes, where members of the cluster had very similar sequences. The D.discoideum PKS genes formed a clade distinct from fungal and bacterial genes. All nine genes examined by RT-PCR were expressed, although at different developmental stages. The promoters of PKS genes were much more divergent than the structural genes, although we have identified motifs that are unique to some PKS gene promoters.

A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum(1).

A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary.

Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type.

Using a selection for Dictyostelium mutants that preferentially form spores, we have recovered a mutant called CheaterA. In chimeras with isogenic wild-type cells, the CheaterA mutant preferentially forms viable spores rather than inviable stalk cells. The mutant causes wild-type cells that have begun to express spore-specific genes to accumulate in the prestalk compartment of the developing organism. In the wild-type cells, the chtA transcript is absent in growing cells and appears early in development. No transcript was detected in the mutant by Northern blot. The chtA gene codes for a protein with an F-box and WD40 domains. This class of protein usually forms part of an Skp1, cullin, F-box (SCF) complex that targets specific protein substrates for ubiquitination and degradation.

A novel component involved in ubiquitination is required for development of Dictyostelium discoideum.

A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA. The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle.

Selection for spiral waves in the social amoebae Dictyostelium.

Starving Dictyostelium amoebae emit pulses of the chemoattractant cAMP that are relayed from cell to cell as circular and spiral waves. We have recently modeled spiral wave formation in Dictyostelium. Our model suggests that a secreted protein inhibitor of an extracellular cAMP phosphodiesterase selects for spirals. Herein we test the essential features of this prediction by comparing wave propagation in wild type and inhibitor mutants. We find that mutants rarely form spirals. The territory size of mutant strains is approximately 50 times smaller than wild type, and the mature fruiting bodies are smaller but otherwise normal. These results identify a mechanism for selecting one wave symmetry over another in an excitable system and suggest that the phosphodiesterase inhibitor may be under selection because it helps regulate territory size.

Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates.

Extracellular cAMP is a critical messenger in the multicellular development of the cellular slime mold Dictyostelium discoideum. The levels of cAMP are controlled by a cyclic nucleotide phosphodiesterase (PDE) that is secreted by the cells. The PDE gene (pdsA) is controlled by three promoters that permit expression during vegetative growth, during aggregation, and in prestalk cells of the older structures. Targeted disruption of the gene aborts development, and complementation with a modified pdsA restores development. Two distinct promoters must be used for full complementation, and an inhibitory domain of the PDE must be removed. We took advantage of newly isolated PDE-null cells and the natural chimerism of the organism to ask whether the absence of PDE affected individual cell behavior. PDE-null cells aggregated with isogenic wild-type cells in chimeric mixtures, but could not move in a coordinated manner in mounds. The wild-type cells move inward toward the center of the mound, leaving many of the PDE-null cells at the periphery of the aggregate. During the later stages of development, PDE-null cells in the chimera segregate to regions which correspond to the prestalk region and the rear of the slug. Participation in the prespore/spore population returns with the restoration of a modified pdsA to the null cells.

How cellular slime molds evade nematodes.

We have found a predator-prey association between the social amoeba Dictyostelium discoideum and the free soil living nematode Caenorhabditis elegans. C. elegans feeds on the amoebae and multiplies indefinitely when amoebae are the sole food source. In an environment created from soil, D. discoideum grows and develops, but not in the presence of C. elegans. During development, C. elegans feeds on amoebae until they aggregate and synthesize an extracellular matrix called the slime sheath. After the sheath forms, the aggregate and slug are protected. Adult nematodes ingest Dictyostelium spores, which pass through the gut of the worm without loss of structure and remain viable. Nematodes kill the amoebae but disperse the spores. The sheath that is constructed when the social amoebae aggregate and the spore coats of the individual cells may protect against this predator. Individual amoebae may also protect themselves by secreting compounds that repel nematodes.

Regulation of Dictyostelium early development genes in signal transduction mutants.

The secreted cyclic nucleotide phosphodiesterase (PDE) and its glycoprotein inhibitor (PDI) are among the first genes expressed when Dictyostelium amoebae begin their development. We used a series of mutants with defects in signal transduction to ask whether cAMP receptors 1 and 3, G alpha2, G beta, adenylyl cyclase (ACA), or the protein kinase A catalytic subunit (PKAcat) are required for the initial appearance or later regulation of the PDE and the PDI transcripts. The PDE gene produces a 1.9-kb transcript during vegetative growth and then a 2.4-kb transcript during the early hours of development. Regulation of the 2.4-kb transcript in CAR1, G alpha2, G beta, and ACA mutants is similar to that of isogenic parental strains, although its level is reduced in strains that carry the CAR1 mutation. CAR1/CAR3 double mutants also produce less PDE transcript, but the PDE gene remains inducible by cAMP. The PKAcat mutant produces the 2.4-kb PDE transcript, but in this mutant the vegetative transcript is retained in development. CAR1 and CAR3 are not required for transcription of the PDI gene, but deleting CAR1 leads to overproduction of the PDI transcript. Induction or repression of the PDI gene does not require G alpha2, G beta, or ACA. PKAcat is required for synthesis of the PDI transcript. The results suggest a two-stage regulation of these early genes through a G alpha2/G beta-independent mechanism and an absolute dependence of PDI on the PKAcat.

The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis.

Dictyostelium discoideum secretes a cyclic nucleotide phosphodiesterase to control cAMP levels during development. Three promoters control expression of the gene--one during vegetative growth, one during aggregation, and one which constrains phosphodiesterase synthesis to prestalk cells. In this report we show that the expression of phosphodiesterase (PDE) in prestalk cells is necessary for morphogenesis. A gene that codes for a specific glycoprotein inhibitor of the phosphodiesterase (Kd = 0.1 nM) was fused to the prestalk-specific promoter of the PDE gene. Transformants carrying multiple copies of this construct secreted inhibitor in 100-fold excess after the aggregation process had occurred. The first effect seen was an elongated tip, followed by a block in slug formation and an inability to culminate. Stalk and spores cells are produced but morphogenesis is uncoupled from cellular differentiation. Overproduction of inhibitor during earlier stages delayed aggregation, but did not affect fruiting body formation. A phosphodiesterase mutant was transformed with a plasmid that expresses PDE only during aggregation and not in prestalk cells. The defect in aggregation was rescued, but the defect in later development was not. The combined results indicate that PDE expression in prestalk cells is critical to morphogenesis. To ask whether the inhibitor gene under its normal regulation had a role in aggregation or later morphogenesis, it was destroyed by homologous recombination. The loss of the gene did not prevent development under the conditions used.

The role of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum during growth, aggregation, and morphogenesis: overexpression and localization studies with the separate promoters of the pde.

cAMP acts as a primary signal and is regulated by a secreted cyclic nucleotide phosphodiesterase (PDE) throughout development in Dictyostelium discoideum. Expression of the PDE gene (pde) is controlled by promoters specific to vegetative growth (prV), aggregation (prA), or late development (prL). Promoter-containing regions were individually fused to the pde coding sequence. After transformation multiple copies of each construct led to overexpression of PDE mRNA and enzyme activity with the temporal profile expected of each promoter. Overexpression of PDE from prV and prA altered the timing of aggregation compared to control transformants, but the final morphology was normal. Control transformants showed delayed aggregation compared to nontransformed cells. Cells that overexpressed PDE from prL aggregated like the control transformants, but no fruiting bodies were formed. Individual promoter regions were fused to the beta-galactosidase gene (lacZ). Cells that expressed prA-lacZ were dispersed throughout aggregation fields and mounds. Cells that expressed prL-lacZ were first seen distributed homogeneously throughout tight and tipped mounds. In slugs most of these cells are localized in the anterior region. During culmination, cells that expressed the prL-lacZ construct became incorporated into the stalk and were seen in the upper and lower cups surrounding the spore mass.

Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development.

Dictyostelium transformants that overproduce the extracellular form of cyclic nucleotide phosphodiesterase and so accumulate a reduced amount of cAMP are blocked in development after aggregation in the form of a tight mound, prior to formation of the apical tip. In such mounds, prespore cell differentiation is repressed, and the apical accumulation of prestalk cells is greatly retarded. When a source of cAMP is placed below the arrested mounds, prestalk cells that would normally migrate in an apical direction instead sort downwards to the substratum. Thus, by acting as the chemoattractant that draws prestalk cells to the apex, cAMP signaling directs the formation of a patterned structure.

Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli.

A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.

Functional cloning of a Dictyostelium discoideum cDNA encoding GMP synthetase.

A functional cloning procedure has been used to recover a cDNA coding for the GMP synthetase of Dictyostelium discoideum. The enzyme is encoded by a single gene, which is actively transcribed during growth, but not during development. The open reading frame encodes a protein of 718 amino acids with a predicted molecular mass of 79.6 kDa. The Dictyostelium enzyme has extensive homology with the GMP synthetase of Escherichia coli and regional homology to other glutamine amidotransferases.

8-Chloroadenosine 3',5'-monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite.

8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations. We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells. Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate. Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.