Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility

Abstract

Swarming motility is a flagella-driven multicellular behaviour that allows bacteria to colonize new niches and escape competition. Here, we investigated the evolution of specific mutations in the GacS/GacA two-component regulatory system in swarming colonies of Pseudomonas protegens Pf-5. Experimental evolution assays showed that repeated rounds of swarming by wildtype Pf-5 drives the accumulation of gacS/gacA spontaneous mutants on the swarming edge. These mutants cannot swarm on their own because they lack production of the biosurfactant orfamide A, but they do co-swarm with orfamide-producing wildtype Pf-5. These co-swarming assays further demonstrated that ΔgacA mutant cells indeed predominate on the edge and that initial ΔgacA:wildtype Pf-5 ratios of at least 2:1 lead to a collapse of the swarming colony. Subsequent whole-genome transcriptome analyses revealed that genes associated with motility, resource acquisition, chemotaxis and efflux were significantly upregulated in ΔgacA mutant on swarming medium. Moreover, transmission electron microscopy showed that ΔgacA mutant cells were longer and more flagellated than wildtype cells, which may explain their predominance on the swarming edge. We postulate that adaptive evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these mutations during dispersal of bacteria into new territories are frequency-dependent.

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

“…Of the 24 exoprotease-deficient colonies, 23 have a single missense or nonsense mutation, an insertion, or a short deletion in the open reading frame (ORF) or ribosome binding site (RBS) of gacA or gacS (Table 1). This result substantiates that counting colonies lacking exoprotease production provided an accurate estimate of the number of gacS and gacA mutants in a culture of P. protegens Pf-5, as shown earlier (17). The mutations of 23 colonies mapped to 17 different sites of gacS or gacA.…”

“…(17,21,22,25,26,(38)(39)(40)(41), but the reasons for this phenomenon have remained obscure. In this study, we focused on P. protegens Pf-5, which accumulated a very high proportion of Gac Ϫ mutants in NYB, outnumbering wild-type cells by at least 9:1 in 6-day cultures ( Fig.…”

“…In the presence of an unknown signal(s), the sensor kinase GacS autophosphorylates and transfers a phosphate group to the response regulator GacA, which governs a complex signal transduction pathway involving regulatory RNAs and translational repression (14). The GacS-GacA system controls the expression of hundreds of genes, including those required for the production of secondary metabolites and extracellular enzymes (15)(16)(17)(18)(19). Accordingly, mutation of gacA and/or gacS has pleiotropic effects on the bacterial cell, influencing motility, iron acquisition, biofilm formation, aspects of primary metabolism, and many other phenotypes (15,16,20).…”

“…Given the broad influence of the GacS-GacA system on bacterial physiology, it is striking that spontaneous gacS and gacA mutants (i.e., Gac Ϫ mutants) emerge and accumulate in cultures of Pseudomonas spp. (17,(21)(22)(23)(24)(25)(26)(27). Colonies of these mutants can be distinguished from those of the wild type visually by their expanded colony size, flattened appearance, increased fluorescence under UV light, and lack of extracellular protease production (21,24).…”

“…activity. Production of exoprotease requires the GacS-GacA system (33) and is a readily visualized phenotype (27) that has been used reliably to estimate the proportion of Gac Ϫ mutants in cultures of P. protegens Pf-5 (17). As such, colonies deficient in exoprotease activity are referred to as Gac Ϫ mutants throughout this report.…”