A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene

Abstract

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source.

Keywords: GacA–GacS two-component system; differential metabolomics; symbiosis.

Fig. 1.

Two strains are carried by the farmer D. discoideum QS161: P. fluorescens PfA (Upper) and PfB (Lower). Though the latter serves as a food source for the farmer, the former does not. Both strains produce different secondary metabolites: PfA produces a chromene and pyrrolnitrin, whereas PfB produces the putative siderophore (iron chelator) pyochelin.

Fig. 2.

Effect of pyrrolnitrin on farmer and nonfarmer D. discoideum QS161 and QS160, respectively. Each data point shows mean ± SD at the specified concentration (three biological replicates; P < 0.05 two-tailed test paired t test of three experimental vs. three control values for each point, paired by experimental block, df = 2; SI Appendix, Table S3). For purposes of visualization, we fit the graph with a second-order smoothing polynomial with four neighbors on each side using GraphPad Prism 6 (www.graphpad.com) software.

Fig. 3.

Effect of chromene produced by PfA on the spore production of farmer D. discoideum QS161 vs. nonfarmer clone QS160. Each data point shows mean ± SD at the specified concentration (four biological replicates; P < 0.05 two-tailed test paired t test of four experimental vs. four control values for each point, paired by experimental block, df = 2; SI Appendix, Table S3). For purposes of visualization, we fit the graph with a sigmoidal curve using GraphPad Prism 6 (www.graphpad.com) software.

Fig. 4.

Amino acid sequence alignment of the gacA gene product of both PfA and PfB shows that a premature stop codon in the gacA gene of PfB leads to a truncated protein. The latter is characterized by the loss of the highly conserved helix turn helix motif, which is required for DNA binding.

Fig. 5.

HPLC (254 nm) trace of an ethyl acetate extract of PfB (Top), PfA ΔgacA (Middle), and PfA (Bottom) cultures. Though PfB and PfA ΔgacA display virtually identical traces, PfA is distinctly different: PfA produces chromene and pyrrolnitrin, whereas PfB and PfA ΔgacA both produce the enantiomers of pyochelin I and II.

Fig. 6.

Maximum-likelihood tree using 20 conserved genes. The numbers beneath branches show the branch length, and the numbers by the node show the bootstrap value of 500 replicates. Note that branch lengths are not drawn to scale and are very short in the bottom clade of five strains.