The genome of Dictyostelium contains two novel hybrid-type polyketide synthases (PKSs) known as 'Steely'; the Steely enzyme is formed by the fusion of type I and type III PKSs. One of these enzymes, SteelyB, is known to be responsible for the production of the stalk cell-inducing factor DIF-1 in vivo. On the other hand, the product(s) and expression pattern of SteelyA are not clearly understood, because there are two different reports associated with the in vitro products of SteelyA and its expression pattern. To solve this problem, we first examined the expression pattern using two different primer sets and found that it was quite similar to that shown in the dictyExpress database. stlA expression peaked at approximately 3 h and declined, but showed a small peak around the end of development. Next, we examined the in vivo product of SteelyA using a stlA null mutant and found that the mutant lacked 4-methyl-5-pentylbenzene-1,3-diol (MPBD). This null mutant showed aberrant, glassy sori, and most of the cells in the sori remained amoeba-like without a cell wall. This defect was restored by adding 200 nM of MPBD to the agar. These results indicate that SteelyA produces MPBD in vivo and induces spore maturation.

BackgroundIn our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development.Principal FindingsOur stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation.ConclusionOur data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB− mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB− sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB− by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.