Figure 2 shows the representative results of GC–MS total ion current chromatogram. The main peak in Ax2 was the same with MPBD by GC–MS analysis. Three independent stlA null strains failed to accumulate a detectable level of MPBD, indicating that SteelyA produced MPBD. To investigate the function of MPBD in the development of Dictyostelium, we examined the phenotype of the stlA mutant. MPBD was identified as a differentiation-inducing factor that stimulated not only stalk cell differentiation but also spore
cell differentiation (Saito et al., 2006). The stlA mutant cells developed normally and produced normal fruiting bodies (data not shown). However, the spore mass differed from that of the wild-type strain and had a glassy appearance (Fig. 3a). We then examined the morphology of spores under the microscope and observed that EPZ-6438 order most of them remained in the amoebae-like form and not encapsulated spores. To confirm this observation, we stained sorus with Calcofluor, which Tamoxifen fluoresces when in contact with cellulose of mature spore cells. Figure 3b (arrows) indicates that the amoebae-like cells were not encapsulated. We then heated the cells in the sorus with 10 mM EDTA (pH 7.5) at
37 °C for 30 min and counted the number of spores (Richardson & Loomis, 1992). Table 2 shows the result of the spore maturation test. The ratio of encapsulated spores in the stlA mutant was about 20% of that in the wild-type cell (Table 2). As mentioned above, GC–MS analysis showed that the stlA mutant lacks MPBD. An alternative interpretation of this result is that SteelyA produced a polyketide that was not MPBD, but was essential for normal development and was therefore indirectly involved in MPBD production. To rule out this possibility, we attempted to compensate the defect of the stlA mutant by adding MPBD in the agar. As shown in Fig.
3b, the normal spore phenotype was restored in the stlA mutant by supplying 200 nM of MPBD in the agar. MPBD was first identified as a stalk-inducing factor and synthetic MPBD was also shown to stimulate spore cell differentiation (Saito et al., 2006). Our in vivo analysis demonstrated that SteelyA hybrid-type PKS produced MPBD in vivo and regulated the spore maturation. Because the fruiting body of Silibinin the stlA null strain produced sori, it appeared that MPBD was involved in spore maturation rather than prespore differentiation. To confirm this, we analyzed the expression of the cell-type-specific genes in the stlA null mutant (Fig. 4). The prestalk markers (ecmA and ecmB) and prespore markers (pspA and cotB) expressed normally. Unexpectedly, the expression of spiA specifically in prespore and spore cells during culmination (Richardson & Loomis, 1992) was normal. To the best of our knowledge, the hybrid-type Steely PKS has been found only in slime molds. Two types of Steely PKS occur in D. discoideum: SteelyA and SteelyB.