Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

Abstract

cAs biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O 2 . We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O 2 -replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities. Most bacteria form multicellular communities called biofilms by producing and encasing themselves in matrices of secreted polymers (1). The National Institutes of Health has estimated that more than half of all bacterial infections involve such biofilm formation, a feature that complicates treatment due to a variety of associated mechanisms that confer increased antibiotic resistance and tolerance in these communities (2). Steep chemical gradients that form within cellular aggregates give rise to microenvironmental heterogeneity; consequently, cells in biofilms exist in diverse physiological states, at least some of which are unique to this lifestyle. For example, in the opportunistic pathogen Pseudomonas aeruginosa PA14, some genes expressed specifically in biofilms are critical for the establishment of lung infections in a mouse model (3). A better understanding of the physiology of biofilm development is required for rational approaches to new therapies for many types of bacterial infections.Using a colony biofilm model, we have found that a primary factor influencing P. aeruginosa PA14 community morphology is the availability of electron acceptors (4-6). As colonies increase in thickness, the formation of O 2 gradients renders the community anoxic at depth and leads to an increase in intr...

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa

“…EPS was measured in the quantitative Congo red (CR) binding assay, as previously described (28). Cultures were grown in BM2 with 20 M Mg 2ϩ containing a 10 M concentration of the reordered compounds in 5-ml glass tubes for 24 h at 37°C with shaking (150 rpm).…”

“…Cultures were grown in BM2 with 20 M Mg 2ϩ containing a 10 M concentration of the reordered compounds in 5-ml glass tubes for 24 h at 37°C with shaking (150 rpm). For EPS quantification, CR was added at a final concentration of 40 g/ml to 2-ml cultures and bound dye was indirectly calculated by determination of remaining unbound dye still in solution (28).…”

Laboratory Evolution of Microbial Interactions in Bacterial Biofilms

“…Experimental evolution studies provide evidence for the prediction that evolution in structured environments can favor adaptations to sociality (34,35,(42)(43)(44). For example, Koch and colleagues demonstrated how the emergence of a strain with an increased level of cell-cell communication can eventually lead to evolution of antibiotic resistance to vancomycin, a last-resort antibiotic (27).…”

“…Lenski's experiment is still in progress, reaching 64,000 generations in January 2016, and many similar studies have been performed by other researchers on different microbial species (not only bacteria but also yeasts, phages, or protists) covering basic scientific questions (29,30) or even in directly applicative projects (31). While planktonic cultures offer a simple setup to study the evolution of bacteria (23,28,31,32), several studies have shown that the selective pressures in unstructured conditions differ from those in biofilms (33)(34)(35)(36). For example, homogeneous, well-mixed environments may select against "social" genotypes that secrete costly metabolites, so-called "public goods," and rather favor fast-reproducing selfish individuals (37).…”

“…Therefore, spatial assortment not only promotes stability of cooperation in biofilms (22,40) but also supports the evolution of novel public good producers that results in increased biofilm matrix production (34,45). Spatial structure can also promote diversification into strong and weak secretors as shown in Pseudomonas aeruginosa colonies, where repetitive reinoculation of P. aeruginosa from the edge or the middle of colony biofilms resulted in clones with reduced or diversified matrix production levels, respectively (34). Structured environments can also support stable coexistence of cooperative and noncooperative strains, thereby promoting diversity within species (46).…”