A mutant called chtB (cheater B) was recovered at the end of a selection for mutants that preferentially produce spores rather than stalks in a mixed population [21].
In the parental strain AX4, the chtB transcript appears early in development and is completely absent in chtB mutant cells. This mutant produces a higher number of spores than AX4 in chimeras that are made with equal numbers of cells of the two strains. When it is plated clonally it shows a normal developmental phenotype, so it is not dependent on parental cells in a social stage chimera. The loss of function of the chtB gene also increases expression of cotB, a prespore marker, early in development, indicating the mechanism of action is early specialization as spore over the ancestral strain. On the other hand, parental cells reduce the expression of cotB and differentiate a lower number of spores in chimera. We tested chtB mutants to detect whether fitness costs were associated with its cheating ability. Not only did we not detect any fitness cost, but we also found that the mutant presents the same sporulation efficiency and faster doubling time when grown in liquid, than AX4. Finally, the presence of chtB mutant cells in chimera with parental strain limits the expression of a pre-spore marker in the latter. As a consequence the parental strain is unable to produce its fair share of spores.
Isolation of the chtBmutants
We isolated the chtB mutant during a selection for cheaters from a pool of 10,000 mutants [21]. This pool of mutants was subjected to 20 rounds of growth, development, and spore germination in a mixed population so that cheaters that differentiate into spores with a higher efficiency would become enriched in the evolving population. At the end of the selection, chtB was one of the mutants that were randomly chosen to be tested for cheating properties. To confirm that chtB really increased its frequency during the selection, we used quantitative PCR (Q-PCR) to obtain information about the abundance of the chtB allele. We extracted genomic DNA from the entire population after the 1st, 10th, and 20th generations of the selection and used gene-specific primers to quantify the chtB allele. The mutant chtB increased 7.4 fold at the 10th generation and 26.4 fold at the 20th (Figure 1), thus supporting the hypothesis that this cheater increased in frequency during the selection.
The chtBgene
The mutant chtB was generated by insertional mutation of the pBSR1 plasmid [29] in chromosome 5 at position 4377789 towards the 3’ end of the ORF of the gene DDB_G0290617 (Figure 2). The predicted protein has not been studied before and it consists of a FNIP repeat (named FNIP after the pattern of a conserved motif found only in D. discoideum). We named the gene chtB because its social behavior resembles the previously described chtA mutant [19]. To verify that the insertion was responsible for the mutant phenotype, the mutation was recapitulated by homologous recombination in the AX4 genetic background using the rescued plasmid as a knockout vector [30]. We confirmed the mutation by Southern blot hybridization using a gene-specific probe and tested the strain for cheating.
RT-PCR analysis showed that the parental strain AX4 expressed the chtB mRNA at 0 hours of development and mRNA abundance was greatly increased at 4–24 hours (Figure 3). In the mutant cells, chtB mRNA was not expressed at any time.
The chtBmutant cheats on the parental strain
To be defined as a cheater, the mutant chtB must produce significantly more than 50% of the spores in a pairwise mixing experiment with the parental strain, and this was the case. When mixed at equal proportions with AX4, chtB differentiated 59.9±3.3% of the total number of spores. This result differed significantly from the control chimera between AX4 and AX4 [act15/GFP] (p<.0001, T-test; Figure 4).
Analysis of fitness cost associated with cheating
chtBhas no overt morphological defects
To test whether chtB shows any morphological difference relative to its ancestor, we observed cells of both strains at different stages of development. In both parent and knockout, we observed loose aggregates at about 10 hours, tight aggregates at 12 hours, fingers at 16 hours and Mexican hats at 20 hours. Between 20 and 24 hours both strains culminated, leading to the formation of well-proportioned fruiting bodies consisting of stalks cells and spores (Figure 5). We saw no noticeable differences between the phenotypes of AX4 and chtB developed on filters.
chtBdifferentiates a similar number of spores than AX4 in a pure population
A fitness cost for a cheater could be manifested as reduced sporulation when developing in a pure population. To test that possibility we developed chtB cells in a pure population or mixed with AX4 cells. The results (see Additional file 1: Figure S1) show that the sporulation efficiency of chtB (83.11±4.69%, n=10) is not significantly different from AX4 (71.53±5.82%, n=10, t-test p<0.14), suggesting that the mutation does not have a sporulation-related fitness cost. The chimera sporulation efficiency was similar to both pure populations (84.16±7.26%, n=10, t-test p=0.19 vs. AX4, p=0.90 vs. chtB).
chtBspores are viable
We also tested whether a fitness cost may be associated with the spore germination efficiency. Our results show that chtB was able to germinate a number of spores (72.3±16.2%) comparable to AX4 (75.5±15.1%, t-test, p=0.08, n=6). This result indicates that chtB does not produce fewer viable spores than its parent (Additional file 2: Figure S2).
chtBgrows faster in liquid medium
To compare the growth rates of the chtB mutant and its ancestor, we grew cultures of chtB and AX4 in liquid media starting at a cell density of 1x105 cells/ml. chtB reached log phase after about 40 hours, while AX4 took about 10 hours longer. When in logarithmic phase, chtB cells showed a doubling time of 7.6±0.7 hours (Figure 6) while AX4 cells showed a doubling time of 10.3±1.5 hours (t-test, p<0.05, n=3), showing that chtB has a faster doubling time than AX4 in liquid medium.
Alteration of cell type proportioning as cheating mechanism
We assessed cell-type proportioning by measuring beta-galactosidase activity in cells that express the marker under the promoters of either cotB[31] (a prespore marker) or ecmA[32] (a prestalk marker). We measured the overall enzyme activity in the population using an ONPG-assay and the number of cells of each type using X-gal staining as described [33]. We compared the level of lacZ expression in chimerae between the reporter strains and AX4 or chtB to determine the effect of chtB on prespore and prestalk differentiation in the AX4 victim.
Prespore differentiation
ONPG analysis of AX4 [cotB/lacZ] showed that cotB is not expressed until 12 hours. Then it starts increasing and reaches saturation at 16 hours (Figure 7). If AX4 [cotB/lacZ] cells were mixed at a 1:1 ratio with AX4 cells that do not express lacZ, the level of β-galactosidase activity is about half of that produced by AX4 [cotB/lacZ] alone. When AX4 [cotB/lacZ] cells were mixed at the same ratio with chtB cells, the β-galactosidase activity was significantly lower than in the mix with AX4 (16, 20 and 24 hour time points, t-test, p<0.05, n=3). These results show that in chimeras chtB is able to reduce the expression of cotB in the wild type cells and that AX4 cells are forced to form fewer prespore-cells than their fair share.
Prestalk differentiation
To test whether the presence of chtB cells in chimeras affects prestalk cell formation in the victim, we performed a similar analysis using the strain AX4[ecmA/lacZ] alone and in pairwise mixes with AX4 and chtB (Figure 8). In this case, no significant differences were seen in the β-galactosidase activity when AX4[ecmA/lacZ] cells are mixed with AX4 or with chtB. We conclude that the presence of chtB cells in chimeras does not influence the expression of the prestalk marker ecmA in the wild type cells.
Developmental cell fate
The results obtained with ONPG analysis show that the presence of chtB cells in a mix with AX4 reduces the promoter activity of the pre-spore gene cotB. This observation could be due to reduced cotB expression in all the wild type prespore cells, or to a reduction in the number of prespore cells in the wild type. We addressed this issue by counting the number of cells that expressed the marker gene. When AX4 [cotB/lacZ] were mixed in equal proportions with chtB (Figure 9A) they produced a significantly lower percentage of stained cells than when mixed with AX4 (for 16 and 20 hour time points, t-test, p<0.05, n=3). Therefore we conclude that, in chimera with wild type, chtB forces the parental cells to reduce the proportion of prespore cells and, as a consequence, to produce fewer spores. When the same test was performed using the AX4 [ecmA/lacZ] strain (Figure 9B), there was no difference between the number of stained cells observed in mixes of AX4[ecmA/lacZ] with AX4 or chtB (for 16, 20 and 24 hours time points, t-test, p>0.26, n=3). We conclude that the presence of chtB in chimera did not affect the number of prestalk A cells produced by the victim strain, but could have made them become prestalk B (pstB) cells, which normally produces the basal disc of the fruiting body, or pstO cells, which occupy the rear half of the prestalk region.