Generating polyketide diversity in Dictyostelium: a Steely hybrid polyketide synthase produces alternate products at different developmental stages

Abstract

The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba Dictyostelium discoideum has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique 'Steely' enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that D. discoideum further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.

Keywords: Dictyostelium discoideum; antibacterial compound; dibenzofuran; differentiation-inducing factor (DIF); polyketide synthase.

Figure 1.

The polyketide synthase and chlorinating enzyme that produce DIF-1 also produce the abundant chlorinated compounds of fruiting bodies. (a) Biosynthetic pathway for DIF-1. DIF-1 is made from the polyketide THPH, which is produced by the SteelyB hybrid PKS, and then chlorinated and methylated by ChlA and DmtA. (b,c,d) Metabolic labelling experiments in which cells are developed on agarose containing ³⁶Cl⁻ and extracts analysed by TLC and autoradiography. (b) Time course of chlorinated compound production during development. At 16 h, when cells have formed fingers and slugs, only DIF-1 is detectable. By 24 h fruiting bodies have formed and remain for the rest of the time course. (c) Genetic evidence that StlB and ChlA are required to produce the chlorinated compounds of fruiting bodies. ‘No addition’ lanes show that parental Ax2 cells produce abundant chlorinated compounds, while mutants lacking StlB (HM1154) or ChlA (HM1522 and HM1523) do not. ‘Mix’ lanes show that the mutants can synergize to restore production. ‘+THPH’ lanes show that production is rescued by supplying the polyketide THPH to stlB⁻ but not to chlA⁻ cells. ‘+DIF-1’ lanes show that when proper development of the mutants is rescued by adding 100 nM DIF-1 to the agarose [13,14], production of the chlorinated compounds is not rescued. (d) The mutant lacking the DmtA methylase (HM1030) catalyzing the final step in DIF biosynthesis can still produce the late chlorinated compounds.

Figure 2.

Purification and characterization of CDF-1. (a) Purification scheme. (b) HPLC profile from the last step of the purification. The acetone fraction from SiO₂ column chromatography was analysed by reverse-phase HPLC (TSK gel ODS-120T) eluting at 1 ml min⁻¹ with a gradient of 50–80% acetonitrile in 60 min (c) Mass-spectroscopic analysis of CDF-1 (FAB-MS in negative ion mode).

Figure 3.

Structure of CDF-1 and related natural products. (a) Space-filling model of CDF-1 as the sodium salt. Ethyl acetate (the solvent) is omitted for clarity. (b) CDF-1 and dibenzofurans from other slime mould species.

Figure 4.

Cells can utilize the polyketide THPH or its chlorinated derivative Cl-THPH to make CDF-1. Axenically grown stlB null cells were collected, washed and spread on 10% DIFlab agar containing 100 nM DIF-1, with or without THPH or Cl-THPH. Cells were allowed to make fruiting bodies on agar for 3 days, then extracted with ethanol, partitioned between ethyl acetate and water and the extracted molecules purified through SiO₂ column chromatography and HPLC to detect CDF-1. (a) HPLC profile of stlB null stain, (b) HPLC profile of stlB null strain incubated with 8 µM THPH, (c) HPLC profile of stlB null strain incubated with 8 µM Cl-THPH, (d) ESI-MS with negative ion mode analysis of CDF-1 peak isolated in (c).

Figure 5.

Spatial expression of the stlB and chlA genes. Both genes are expressed in the prespore region of slugs, but in the stalk and basal disc of fruiting bodies. Ax2, the parental strain, was used as negative control. Promoters of the two genes were used to drive EGFP expression. Red arrow indicates the basal disc.

Figure 6.

Stalk cells release the chalcone synthase domain of Steely-B. Western blots probed for the TAP tag of the StlB-TAP protein. (a) Developmental expression of the TAP-tagged StlB protein (about 352 kDa). StlB expression peaked at 9–12 h of development, as aggregates consolidated and developed tips, then declined, before a second peak of strong expression as fruiting bodies formed. In this second phase a smaller band (about 64 kDa) is also detected, corresponding to the released C-terminal chalcone synthase domain. (b) StlB-TAP expression is limited to the stalk of mature fruiting bodies, where the shorter, chalcone synthase domain dominates. Stalk and spores were separated from mature fruiting bodies by washing through a mesh. st: stalk; sp: spore. (c) Expression of the isolated, TAP-tagged chalcone synthase domain (indicated as ‘chalcone-TAP’) of StlB in a stlB null strain. The expressed protein is of the expected size and expression occurs late in development. Asterisk indicates unknown degradation product.

Figure 7.

The isolated chalcone synthase domain of Steely-B supports synthesis of CDF-1. Extracts from fruiting bodies of cells expressing the chalcone synthase domain of StlB were analysed for production of CDF-1. (a) HPLC profile of the partially purified extract, (b) Negative ion mode ESI-MS analysis of each peak from (a). (c) Calculated mass and deduced formula of CDF-1 and other chlorinated compounds produced by stlB null cells expressing the isolated chalcone synthase domain. Peak 1 corresponds to CDF-1, while peaks 2 and 3 are consistent with derivatives lacking one or two methylene moieties. The TAP-tagged chalcone synthase domain was expressed in a stlB null mutant under the control of the stalk-specific ST promoter. Cells were grown with K. aerogenes on 0.5xSM agar in 25 × 25 cm plastic plates, with 120 plates per experiment. Neither DIF, nor DIF precursors, were added in the agar. Mature fruiting bodies were collected, extracted with ethanol and partitioned between ethyl acetate and water. The ethyl acetate extract was separated by preparative SiO₂ column chromatography to yield the partially purified extract, which was analysed by HPLC and mass-spectroscopy.