Development of the dictyostelid Polysphondylium violaceum does not require secreted cAMP

Multicellularity evolved independently in seven of the eight major divisions of eukaryotes (Brown et al., 2012; Du et al., 2015; Tice et al., 2016) and in prokaryotes (Lyons and Kolter, 2015). Multicellular forms range from simple clumps or mats of cells to a myriad of complex organisms composed of many specialized cells, arranged in tissues and organs. The regulation of cell-type specialization and the proper positioning of the specialized cells within the organism requires extensive cell to cell communication mechanisms.

Comparative genomics has highlighted that many proteins involved in mediating and processing intercellular communication are deeply conserved throughout multicellular lineages (Gold et al., 2019; Srivastava et al., 2010), while those involved in intracellular processing can often be retraced to their unicellular ancestors (King et al., 2008; Suga et al., 2013). Nevertheless, there are only few narratives that connect signalling mechanisms in a unicellular ancestor with developmental control in a multicellular descendant. One example is the co-option of a transcription factor, RSL1, that mediates stress response in the unicellular alga Chlamydomonas reinhardtii into a developmental role as RegA, inducing somatic cell fate in its multicellular relative Volvox carteri (König and Nedelcu, 2020). Another example is the evolution of cell-type specialization and morphogenesis in the Dictyostelia from an ancestral stress response.

Dictyostelia are members of Amoebozoa, a eukaryote division that mostly consists of single-celled amoebas, which encapsulate to form walled cysts when starved. Instead, starving Dictyostelia aggregate to form multicellular fruiting bodies that consists of walled spores and stalk cells. The model species D. discoideum (Ddis), uses cAMP as attractant to coordinate aggregation and fruiting body morphogenesis. For this role, cAMP is secreted in pulses, which are generated by a network that contains the adenylate cyclase AcaA (Pitt et al., 1992), G-protein coupled cAMP receptors (Cars) (Sun et al., 1990), an extracellular phosphodiesterase, PdsA (Sucgang et al., 1997), and some intracellular components. Secreted cAMP also regulates developmental gene expression and induces the expression of prespore genes. In addition, cAMP acts an intracellular messenger for external signals that trigger the growth to development transition and the encapsulation of spores and stalk cells. Here, cAMP activates cAMP-dependent protein kinase (PKA) and is mostly synthesized by the adenylate cyclases AcgA and AcrA and hydrolysed by the intracellular phosphodiesterase RegA (Du et al., 2015; Loomis, 2014).

Molecular phylogeny partitions Dictyostelia into four major and some minor groups (Schaap et al., 2006; Schilde et al., 2019). Many species in groups 1, 2 and 3 can still individually encyst in addition to forming fruiting bodies, but group 4, which contains Ddis, has lost encystation (Romeralo et al., 2013). Comparative studies revealed that the roles of PKA, AcgA, AcrA and RegA in Ddis spore and stalk encapsulation are evolutionary derived from roles as intermediates for stress-induced encystation in both Dictyostelia and single-celled Amoebozoa (Du et al., 2014; Kawabe et al., 2015; Ritchie et al., 2008).

Group 4 species all use cAMP as the attractant for aggregation, but most species in groups 1, 2 and 3 appear to use the dipeptide glorin (Romeralo et al., 2013), that was initially identified as the attractant of Polysphondylium violaceum (Pvio) (Shimomura et al., 1982). Deletion of pdsA and the carA and carB genes of the group 2 species Polysphondylium pallidum (Ppal) did not affect aggregation (as expected) but disrupted fruiting body morphogenesis. The carAˉcarBˉ cells also lost prespore differentiation and differentiated as cysts in the aberrant fruiting structures (Kawabe et al., 2009, 2012). These data indicated that secreted cAMP coordinated post-aggregative morphogenesis and induced prespore differentiation in Ppal, as it does in Ddis, and supported a hypothesis whereby first intracellular and next extracellular cAMP signalling in Dictyostelia evolved from cAMP-mediated encystation in the unicellular ancestor (Kin and Schaap, 2021).

We recently developed gene knock-out procedures and generated genome and transcriptome sequence data for Pvio, a species that resides in a small sister group to group 4 (Narita et al., 2020). This position and the fact that it only has single copies of the acaA, car and pdsA genes makes Pvio uniquely suited to investigate how secreted cAMP signalling evolved to its very dominant role in group 4. The gene disruption studies led to the surprising finding that in Pvio development extracellular cAMP signalling plays no role of significance at all.

Pvio is one of the earliest identified species of Dictyostelia (Brefeld, 1884) and is characterized by aggregates with inflowing streams, the formation of whorls of side branches from cell masses that pinch off from the primary sorogen and the violet colour of its mature spores. It is the founding species of the genus Polysphondylium, which all share the whorled fruiting bodies, but not the violet colour. Classification based on molecular data later showed that the whorled phenotype was polyphyletic (Schaap et al., 2006; Schilde et al., 2019) and that white polyphondylids were members of group 2 of the 4 major dictyostelid taxon groups, while Pvio was part of a small sister clade to group 4, with the latter containing Ddis and other species that use cAMP as attractant. The Pvio attractant was identified as a dipeptide of glutamate and ornithine, called glorin (Shimomura et al., 1982) and appears to be widely used among non-group 4 species (Romeralo et al., 2013). Genome and transcriptome data for Pvio Qsvi11 are available (Narita et al., 2020) and this strain can be transformed with vectors harbouring G418 or hygromycin selection cassettes. Gene knock-out by homologous transformation proved thus far to be very efficient, usually occurring in 80-100% of transformed clones (Narita et al., 2020). The whorled phenotype of Qsvi11 is, however, variable and whorls are often sparse, making it difficult to evaluate effects of gene manipulation on whorl formation.

AcaA is encoded by a single copy gene in Pvio (Fig. S1) and to assess its role in post-aggregative morphogenesis, we deleted acaA by homologous recombination (Fig. S2A). The acaAˉ clones aggregated normally and formed fruiting bodies with somewhat thinner and longer stalks than those of wild-type cells (Fig. 1A). This was also evident in Calcofluor stained structures at higher magnification, where the length/width ratio of acaAˉ stalk cells was greater than that of wild-type stalk cells (Fig. 1B). The acaAˉ mutant formed normal elliptical spores.

Similar to other Dictyostelia, Pvio has two other adenylate cyclase genes, acrA and acgA (Fig. S1). Ddis acrAˉ cells form fruiting bodies with thin stalks and spores that germinate precociously in the spore head (Soderbom et al., 1999), while acgAˉ mutants show only sporulation defects (Alvarez-Curto et al., 2007; Van Es et al., 1996). Pvio acgA is predominantly expressed in spores in late development (Fig. S1), while acrA is like acaA upregulated during aggregation and stalk-enriched (Fig. S1). To examine the expression pattern of both genes in developing structures, we transformed wild-type Pvio with fusion constructs of the acaA or acrA promoters and the lacZ reporter. Expression of β-galactosidase from either promoter was enriched in aggregation centres and was later present throughout the primary and secondary sorogens and the stalk (Fig. S3). Expression from the acaA promoter was somewhat stronger at the sorogen tips. Because the expression pattern of acrA is almost the same as that of acaA, we investigated whether AcrA might compensate for loss of AcaA in Pvio by disrupting acrA in wild-type and acaAˉ cells (Fig. S2B).

The Pvio acrA knock-out clones formed large aggregates and robust fruiting bodies (Fig. 2A). Measurement of aggregate density showed that acrAˉ cells initiated about eight times less aggregates/mm² than wild-type cells (Fig. 2B), which is the likely cause of the increased size of their aggregates and fruiting bodies. Aggregation of acrAˉ cells also seemed a bit delayed, but because the timing of aggregation is also variable in wild-type Pvio, we cannot state this with certainty. Calcofluor staining revealed that acrAˉ cells initially formed normal spores and stalk cells in early fruiting bodies (Fig. 2C, 12 h), but that 4 h later the spore head contained many amoebas and empty spore walls, indicating that spores had precociously germinated. Precocious spore germination is also reported for Ddis acrAˉ (Soderbom et al., 1999). Expression of acrA in acrAˉ cells restored both the reduced aggregate density (Fig. 2A) and precocious germination defects of acrAˉ (Fig. 2C), indicating that these phenotypes were caused by acrA gene disruption.

The Pvio acaAˉacrAˉ mutant was severely defective. It initially aggregated upon starvation but never formed sorogens (Fig. 3). Instead, the aggregated cells dispersed within a few hours and then formed aggregates again. This process was repeated several times at about 5-7 h intervals during at least 24 h. Normal development was restored by expression of either acaA or acrA from their own promoters (Fig. 4). Each of the complemented strains formed fruiting bodies, but the spores of acaAˉacrAˉ transformed with acaA germinated in spore head (Fig. 4B), as was the case for acrAˉ mutants. The restoration of fruiting body formation indicates that acaA and acrA have overlapping roles in stabilizing aggregates to initiate progression into fruiting body formation.

In Ddis, fruiting body formation requires both extracellular cAMP acting on Cars for organisation of morphogenesis and induction of prespore gene expression (Singer et al., 2019; Wang et al., 1988) and intracellular cAMP acting on PKA for induction of spore and stalk cell maturation (Harwood et al., 1992; Hopper et al., 1993). To address which cAMP signalling defect caused the acaAˉ/acrAˉ phenotype, Pvio acaAˉ/acrAˉ cells were developed on agar containing 8Br-cAMP, a membrane-permeant cAMP analogue with high affinity for PKA, but not Cars (Van Haastert and Kien, 1983). At 2.5 mM 8Br-cAMP, stable tipped aggregates were formed that did not develop further, but at 5 mM and more so at 10 mM 8Br-cAMP, the tipped mounds developed into stalked structures with spore heads, which, though morphologically aberrant, showed normal elliptical spores and vacuolated stalk cells (Fig. 5). These results show that direct activation of PKA compensates fully for defective aggregation, sporulation and stalk cell differentiation in the acaAˉacrAˉ and partially for defective morphogenesis.

The partial restoration of Pvio acaAˉacrAˉ development by 8Br-cAMP indicates that AcaA and AcrA have a shared role in providing intracellular cAMP for PKA activation. However, this does not exclude that secreted cAMP pulses are not important for Pvio development as well. In Ddis, both the cAMP receptor CarA and the extracellular cAMP phosphodiesterase PdsA are essential for generating cAMP pulses, while CarA also mediates cAMP induction of chemotaxis and prespore gene expression (Schaap and Van Driel, 1985; Sucgang et al., 1997; Sun et al., 1990). BlastP searches revealed that Pvio has only a single pdsA and a single carA gene, which were respectively amplified once and three times in group 4 (Fig. S4).

We deleted the single carA and pdsA genes to evaluate roles for secreted cAMP in Pvio (Fig. S5). Both the carAˉ and pdsAˉ mutants aggregated normally and constructed fruiting bodies (Fig. 6A). Calcofluor staining revealed that these fruiting bodies were composed of mature spores and stalk cells whose morphologies were also normal. These results suggest that secreted cAMP does not play a significant role in Pvio aggregation, fruiting body development or cell differentiation. To further test involvement of oscillatory cAMP signalling in Pvio, we developed cells on agar containing 10, 50 or 100 μM on the slowly hydrolysable cAMP analogue Sp-cAMPS, which inhibits cAMP responses like chemotaxis and cAMP relay that are subject to adaptation, and thereby inhibit Ddis development, starting from 0.5 μM (Rossier et al., 1978). Fig. S6 shows that up to 100 μM Sp-cAMPS had no effect on Pvio aggregation and development into fruiting bodies, consolidating the evidence that Pvio does not require dynamic extracellular cAMP signalling.

Phylogenetic comparative methods showed that major innovations occurred in the last common ancestor (LCA) to the group 4 dictyostelids, such as the formation of robust solitary unbranched fruiting bodies with two novel somatic cell types, the early fate mapping of spore and stalk cells and extensive slug migration. Species in groups 1, 2 and 3 form smaller grouped or branched fruiting bodies with stalk cells as the only somatic cell type, and the stalk is formed by local redifferentiation of prespore cells. The changes in group 4 are accompanied by the loss of encystation as an alternative survival strategy and the use of secreted cAMP pulses to coordinate chemotactic aggregation (Romeralo et al., 2013; Schilde et al., 2014).

Deletion of car and pdsA genes in the group 2 species Ppal indicated that non-group 4 species still used secreted cAMP to organize post-aggregative morphogenesis and prespore differentiation (Alvarez-Curto et al., 2005; Kawabe et al., 2002, 2009, 2012). This notion was supported by findings that the group 3 species D. minutum, displayed cAMP stimulated cAMP synthesis (Schaap, 1985) and optical density waves after aggregation, with the latter being disrupted by the non-hydrolysable cAMP analogue Sp-cAMPS (Schaap et al., 1984). Sp-cAMPS also disrupted post-aggregative morphogenesis of most other non-group 4 species (Romeralo et al., 2013).

With its position in the closest sister clade to group 4, Pvio is well placed for investigating the molecular changes that occurred in the LCA to group 4 that caused the dramatic innovations in this group. An added advantage is the efficiency of gene knock-out in Pvio (Narita et al., 2020) and that its attractant, glorin, is known (Shimomura et al., 1982). To particularly understand the evolution of secreted cAMP signalling, we deleted the single gene encoding AcaA, the pivotal enzyme in the network that generates the secreted cAMP pulses that control Ddis aggregation and post-aggregative morphogenesis (Patel et al., 2000; Pitt et al., 1992; Singer et al., 2019).

The loss of AcaA function in Pvio did not markedly affect aggregation, fruiting body morphogenesis or spore and stalk cell differentiation. Only stalks appeared somewhat thinner with the individual stalk cells showing a lower width to length ratio (Fig. 1). This suggested that either secreted cAMP played no mayor role in Pvio or the role of AcaA was shared with another adenylate cyclase.

Apart from a sporulation defect, Ddis acrAˉ mutants also make long thin stalks (Soderbom et al., 1999), while Pvio acrA showed similar developmental regulation and cell type specificity as acaA (Figs S1 and S3), suggesting that AcrA and AcaA functions may overlap. A Pvio acrAˉ mutant showed the same sporulation defect as Ddis acrAˉ, but its fruiting bodies were actually more robust than those of wild type (Fig. 2). This was due to the formation of less closely spaced and therefore larger aggregates, suggesting a requirement for AcrA in aggregation centre initiation. A Pvio acaAˉacrAˉ double mutant showed a more severe phenotype. It formed aggregates that never progressed into sorogens but disaggregated instead, only to repeat a cycle of aggregation and disaggregation several times over (Fig. 3). Sorogen and fruiting body formation of Pvio acaAˉacrAˉ was partially restored by developing mutants with the PKA agonist 8Br-cAMP (Fig. 5). While fruiting structures were morphologically aberrant, normal spores and vacuolated stalk cells were formed. Pvio acaAˉacrAˉ fruiting body morphogenesis was also restored by overexpression of either acaA or acrA (Fig. 4). Since coordination of morphogenetic cell movement is attributed to the propagating cAMP waves that are produced by AcaA, this raised the question, whether cAMP waves are not required for Pvio morphogenesis.

To address the question whether cAMP pulses or just secreted cAMP play any role in Pvio development, we deleted pdsA, which is essential for pulsatile cAMP signalling in Ddis (Sucgang et al., 1997) and carA, the only cAMP receptor gene that we could detect in the separately assembled genome and transcriptome of Pvio (Fig. S7). Both the carAˉ and the pdsAˉ mutants aggregated normally and formed normal fruiting bodies with mature spore and stalk cells. Unless other unrelated proteins have taken over CarA and PdsA function, this suggests that unlike Ddis and Ppal, secreted cAMP has no major role in coordination of cell movement or in the induction of prespore differentiation in Pvio. This notion was substantiated by Pvio aggregation and development proceeding normally in the presence of Sp-cAMPS (Fig. S6). This is a striking result considering that Pvio is more closely related to Ddis, with its multiple roles for secreted cAMP, than Ppal, where deletion of its two car genes or pdsA gene disrupts fruiting body morphogenesis and of car genes also spore differentiation (Kawabe et al., 2002, 2009, 2012). Evidently, not only group 4 but also Pvio underwent significant innovations in developmental control.

Which secreted signal(s) might have taken over the roles of secreted cAMP in Pvio? Here the Pvio chemoattractant glorin comes first to mind, since it was also reported to trigger gene expression in early Ppal development (Asghar et al., 2011). However, Dictyostelids are known to synthesize a range of other secondary metabolites that affect cell differentiation (Araki and Saito, 2019; Kikuchi et al., 2013; Kondo et al., 2019; Saito et al., 2022; Sasaki et al., 2020; Tsujioka et al., 2004) and focusing on one or a few compounds is therefore premature.

Comparative analysis of adenylate cyclase function across Dictyostelia highlight considerable refunctionalisation of AcaA, AcrA and AcgA in the course of dictyostelid evolution. AcaA is essential in Ddis for producing the secreted cAMP pulses that organize aggregation and post-aggregative morphogenesis (Patel et al., 2000; Pitt et al., 1992), although a Ddis acrAˉacgAˉacaAˉ mutant that overexpresses PKA can still form mounds at high cell density (Hirose et al., 2021). Ddis AcaA also acts intracellularly in response to the stalk-inducer c-di-GMP to activate PKA and thereby stalk maturation (Chen and Schaap, 2012; Chen et al., 2017). However, apart from a reduction in stalk thickness, Pvio acaAˉ mutants showed no developmental abnormalities. Ppal aca1ˉ cells also form fruiting bodies with thinner stalks. However, even knock-outs in all three Ppal aca genes still form some fruiting bodies after a long delay (Kawabe and Schaap, 2022).

AcrA is required for robust stalk formation in Ddis and has an overlapping role with AcgA in induction of prespore differentiation and spore maturation (Alvarez-Curto et al., 2007; Soderbom et al., 1999). AcgA on its own mediates inhibition of spore germination by high osmolarity in the Ddis spore head (Van Es et al., 1996). However, neither AcrA nor AcgA or both together are essential for Ppal prespore and spore differentiation. They do have overlapping roles in mediating stress-induced encystation in Ppal and inhibition of spore and cyst germination by high osmolarity (Kawabe et al., 2015). Induction of prespore differentiation and spore maturation is dependent on both Cars and PKA in Ppal (Funamoto et al., 2003; Kawabe et al., 2009, 2015) as it is in Ddis (Hopper et al., 1993; Schaap and Van Driel, 1985). We surmised that in the Ppal acrAˉacgAˉ cells, any or all of the three Ppal Aca enzymes may provide cAMP for spore differentiation.

Similar to Ddis AcrA (Soderbom et al., 1999), Pvio AcrA is required for the differentiation of stable spores (Soderbom et al., 1999) (Fig. 2). Pvio AcrA is additionally required for efficient aggregation centre initiation and has an overlapping role with AcaA in the formation of stable aggregates. For these roles both AcaA and AcrA act upstream of PKA, since stable aggregation and fruiting body formation are restored in acaAˉacrAˉ mutants by 8Br-cAMP (Fig. 5). The distinction in Ddis between roles of AcaA in mostly extracellular Car activation, and roles of AcrA and AcgA in mostly PKA activation is therefore blurred in Ppal and Pvio.

Compared to other Amoebozoa, such as Physarum polycephalum, Protostelium fungivorum and Acanthamoeba castellani with, respectively, 64, 52 and 67 adenylate cyclases each (Clarke et al., 2013; Hillmann et al., 2018; Schaap et al., 2015), the number of adenylate cyclases in Dictyostelia is low. It is possible that in the unicellular ancestor of Dictyostelia the roles of their three adenylate cyclases were not highly specialized, i.e. they all responded to environmental stressors to induce the transition of amoebas into dormant cysts. In the newly emerging Dictyostelid taxon groups, the enzymes and their regulation may then have evolved independently to take on group-specific developmental roles.

P. violaceum QSvi11, (Pvio) (Kalla et al., 2011), gift from J. E. Strassmann (Washington University in St. Louis, USA) was routinely grown in KK2 (16 mM KH₂PO₄ and 4 mM K₂HPO₄), containing autoclaved Klebsiella aerogenes (K.aer) (final OD₆₀₀=8.5) and 10% HL5 shaken at 150 rpm. For some experiments cells were grown in association with Escherichia coli on 1/5^th SM agar (Formedium, UK). For multicellular development, cells were harvested from growth media and spread at 10⁶ cells/cm² on KK2 agar (1.5% agar in KK2), incubated at 4°C overnight and then at 22°C until the desired developmental stage had been reached.

To construct a gene fusion of the promoter of Pvio acaA (Pvio_g2213, NCBI id.: KAF2076473) and lacZ, a fragment comprising the full 1.8 kb acaA 5'intergenic region and 0.1 kb 5′ coding sequence was amplified from Pvio gDNA, using primer pair Pv-ACA-P51 K and Pv-ACA-P31B (Table S1) that harbour KpnI and BamHI sites, respectively. After KpnI/BamHI digestion, the fragment was ligated into KpnI/BamHI digested pDdGal16 (Harwood and Drury, 1990), yielding vector pPv-acaA-LacZ.

To construct an acrA_LacZ fusion, a fragment from −822 to +125 nt relative to the start ATG of acrA (Pvio_g1249, KAF2077476) and containing the full 5′ intergenic region was amplified from Pvio gDNA, using primers Pv-ACB-P51X and Pv-ACB-P31B (Table S1) that harbour XbaI and BamHI sites, respectively. After digestion the fragment was ligated into XbaI/BamHI digested pDdGal16, yielding vector pPv-acrA-LacZ.

Both plasmids were validated by DNA sequencing and transformed into Pvio cells by electroporation. Transformants were selected at 50 µg/ml G418 on growth plates with G418 resistant E. coli (Narita et al., 2020). Transformed cells were developed on dialysis membrane supported by KK2 agar and β-galactosidase activity was visualised with X-gal in developing structures as described previously (Dingermann et al., 1989).

To disrupt Pvio acaA, an acaA fragment was amplified from Pvio gDNA using primers Pv-aca-51S2 and Pv-aca-31K that harbour SacII and KpnI sites (Table S1), respectively. After digestion, the fragment was cloned into SacII/KpnI digested pBluescript SK+, which was next digested with BamHI and SalI. The LoxP-NeoR cassette was excised from pLoxNeoIII (Kawabe et al., 2012) with BamHI/SalI and ligated into the digested acaA-pBluescript vector. This yielded pPv-acaA-KO in which LoxP-NeoR was flanked by 1870 bp of 5′UTR and 5′ acaA sequence and 1825 bp of 3′ acaA sequence (Fig. S2A).

To disrupt Pvio acrA, two acrA fragments, KO-A and KO-B, were amplified from Pvio genomic DNA using primer pair Pv-ACB-51K/Pv-ACB-31S with KpnI and SacII sites for KO-A and Pv-ACB-52 K/Pv-ACB-32 with a KpnI site for KO-B (Table S1). After KpnI/SacII digestion, KO-A was ligated into KpnI/SacII digested pBluescript SK+, which was next digested with KpnI/XbaI and ligated to LoxP-NeoR, which was excised from pLoxNeoIII with KpnI/XbaI. Fragment KO-B was digested with KpnI/XhoI and cloned into this vector, yielding vector pPv-acrA-KO with LoxP-NeoR flanked by 1483 bp 5′acrA sequence and 1373 bp 3′acrA and 3′UTR sequence (Fig. S2B).

The linearized KO vectors were transformed in Pvio (Narita et al., 2020). Genomic DNA was isolated from G418 resistant clones and screened by PCR to diagnose gene disruption by homologous recombination (Fig. S2). To generate a double acaAˉacrAˉ knock-out, acaAˉ cells were transformed with pA15NLS.Cre for transient expression of Cre-recombinase (Faix et al., 2004). G418 sensitive clones were selected and transformed with pPv-acrA-KO and diagnosed for acrA gene disruption by PCR (Fig. S2B).

To disrupt Pvio carA (Pvio_g6080, KAF2072602), two carA fragments, A and B, were amplified from Pvio gDNA using primer pair Pv-cAR-51K/Pv-cAR-31C with KpnI and ClaI sites for A and Pv-cAR-52B/Pv-cAR-32X with BamHI and XbaI sites for B (Table S1). After digestion with KpnI and ClaI, fragment A was ligated into KpnI/ClaI digested pLoxNeoIII. Fragment B was digested with BamHI/XbaI and cloned into this vector, yielding vector pPv-carA-KO in which LoxP-NeoR is flanked by 1183 bp 5′UTR and 5′carA sequence and 1068 bp 3′ carA and 3′UTR sequence (Fig. S5A).

To disrupt Pvio pdsA (Pvio_g5708, KAF2072968), two pdsA fragments, A and B with internal HindIII and SacI sites, respectively, were amplified from Pvio gDNA using primer pair Pv-pdsA-51K with KpnI site and Pv-pdsA-31 for A and Pv-pdsA-52B with BamHI site and Pv-pdsA-32 for B (Table S1). Fragment B was digested with BamHI/SacI and ligated into BamHI/SacI digested pLoxNeoIII. Fragment A was digested with KpnI/HindIII and cloned into this vector, yielding vector pPv-pdsA-KO that contained 1391 bp 5′UTR and 5′pdsA sequence and 1205 bp 3′pdsA and 3′UTR sequence (Fig. S5B).

To express Pvio acaA from its own promoter, a 5833 bp segment containing the Pv-acaA promoter, coding and terminator regions was amplified from gDNA in two fragments, A and B, using primer set Pv-aca-P51K (with KpnI site) and Pv-aca-C31 for A and Pv-aca-C51 and Pv-aca-31C (with ClaI site) for B. The fragments were cloned into pCR-BluntII-TOPO for sequence validation. Using an acaA internal BamHI site, fragment A was isolated from its TOPO plasmid using KpnI and BamHI and ligated into the KpnI/BamHI digested plasmid that contained fragment B, thus reconstructing the entire 5.8 kb acaA genomic segment. This segment was excised with KpnI/ClaI and ligated into KpnI/ClaI digested vector pHygTm(plus)/pG7 (http://dictybase.org/db/cgi-bin/dictyBase/SC/plasmid_details.pl?id=453), which contains a hygromycin resistance cassette, yielding vector pPv-acaA-Exp.

To express Pvio acrA from its own promoter, a segment containing the acrA coding region, part of its promoter and the terminator were amplified from gDNA using primers Pv-ACB-P51X/Pv-ACB-31S. The fragment was digested with SpeI/SacII and cloned into pBluescript SK+ for sequence validation, yielding plasmid pBs-PvAcrA. The remaining part of the promoter region was excised with XbaI/SmaI from pPv-acrA-LacZ (see above) and ligated into the XbaI/SmaI digested vector pHygTm(plus)/pG7, which was subsequently digested with SmaI and SpeI. The PvAcrA fragment from pBs-PvAcrA was excised with SpeI/HpaI and ligated into the SpeI/SmaI digested vector, yielding pPv-acrA-Exp, which now harboured a 6.4 kb region encompassing the acrA promoter, coding region and terminator. pPv-acrA-Exp was introduced into both acrAˉ and acaAˉ/acrAˉ cells by electroporation and pPv-acaA-Exp into acaAˉ/acrAˉ only. Transformants were incubated with autoclaved Klebsiella aerogenes in 10% HL5 with 30 μg/ml of hygromycin in petri dishes for 48 h, and next distributed with E. coli on 1/5th SM plates supplemented with 30 µg/ml of hygromycin. The plasmids and knock-out cell lines prepared for the study are in the Dictyostelium Stock Center http://dictybase.org/StockCenter/StockCenter.html.

Quantitative data were collected in Excel (Microsoft). Statistical analysis and graph preparation was performed in Sigmaplot v14.5 (Systat Software, Inc.).

DNA sequence validation was performed by MRC PPU DNA Sequencing and Services at the University of Dundee School of Life Sciences.