The Ultimate Guide to Bacterial Swarming: An Experimental Model to Study the Evolution of Cooperative Behavior

Abstract

Cooperation has fascinated biologists since Darwin. How did cooperative behaviors evolve despite the fitness cost to the cooperator? Bacteria have cooperative behaviors that make excellent models to take on this age-old problem from both proximate (molecular) and ultimate (evolutionary) angles. We delve into Pseudomonas aeruginosa swarming, a phenomenon where billions of bacteria move cooperatively across distances of centimeters in a matter of a few hours. Experiments with swarming have unveiled a strategy called metabolic prudence that stabilizes cooperation, have showed the importance of spatial structure, and have revealed a regulatory network that integrates environmental stimuli and direct cooperative behavior, similar to a machine learning algorithm. The study of swarming elucidates more than proximate mechanisms: It exposes ultimate mechanisms valid to all scales, from cells in cancerous tumors to animals in large communities.

Keywords: biofilm; cheater; metabolic prudence; rhamnolipids; sociomicrobiology.

Figure 1.. P. aeruginosa swarms on soft agar using its flagella and rhamnolipid secretion; this cooperative behavior benefits the colony which can harvest more nutrients and grow to larger numbers.

(A) The swarm started to expand around 5 h and reached the edge of a petri dish before 24 h (Supplementary video 1). (B) Swarming on soft agar enabled P. aeruginosa to use available nutrients and increase to a population size of more than 15 billion cells. The strain inoculated on the same nutrient medium but in hard agar cannot swarm, and yields less than 3 billion cells. Deletion either flgK or rhlA gene prevented swarming even on soft agar, resulting in final population sizes of less than 5 billion cells. (C) Two P. aeruginosa swarms expel each other through rhamnolipid secretion (Supplementary video 2).

Figure 2.. The four types of social behavior, showing P. aeruginosa mutants according to the type of social behavior they display: mutualism, altruism, selfishness or spite.

Wild type bacteria benefit themselves by swarming but also others, such as the rhamnolipid deficient mutants, without that impacting their fitness; this is an example of mutualistic cooperation. The hyperswarmer (faced against the wild-type) moves to the front of swarm and consumes fresh nutrients, selfishly increasing its own frequency and harming the wild-type. The ΔflgK (faced against the wild-type) is spiteful: at low frequencies it cannot free-ride and is left behind, but drags down the wild type or even blocks it if its initial frequency exceeds 1:1. The constitutive rhamnolipid producer P_BAD:rhlAB, the deletion strain ΔcbrA and the compensatory mutant ΔcbrA crc* overproduce rhamnolipid at different levels, but for each one the secretion is highly costly to their own fitness and those behaviors altruistically help ΔrhlA swarm to increase its frequency. The ΔcbrA hfq* helps ΔrhlA without a cost to itself, which is a mutualistic behavior similar to the wild type.

Figure 3. Swarming was robust against flagella-less cheaters, but flagella-less mutants could block swarming when they started at large frequencies.

(A) The wild type (in red) swarmed well when mixed with a flagella-less ΔflgK (in green) when the starting ΔflgK frequency did not exceed 1:1; the ΔflgK could not free-ride and was left behind at the nutrient-exhausted colony center. (B) The ΔflgK could compromise swarming when its initial frequency was 5:1 or higher; at 50:1 it blocked the wild type from swarming entirely.

Figure 4.. Repeated rounds of growth in soft agar evolved multi-flagellated hyperswarmers.

A) Scheme of experimental evolution which started from a single flagellated ancestor strain (B) which was repeatedly passaged to fresh soft agar plate everyday for ~9 days (C) and evolved into a robust hyperswarming phenotype with multi-flagellated bacterium (D).

Figure 5. P. aeruginosa uses a strategy called metabolic prudence to avoid cheating by free-riders that do not produce rhamnolipids.

(A) ΔrhlA could free-ride on a wild type swarm. (B) Experimental evolution of ΔrhlA:wild type mixture (initially 1:1) showed that the wild type is robust to cheating because its numbers did not decrease over time. (C) This robustness was absent in a strain that could be induced to express rhlAB constitutively (also initially at 1:1 ratio with ΔrhlA) because this strain lacked metabolic prudence. (D) Metabolic prudence ensures that cells use their carbon source for growth when they have all reagents needed to make new biomass (e.g. nitrogen source or iron) and start rhamnolipids synthesis to consume excess carbon when growth is limited by another nutrient (e.g. once nitrogen or iron are depleted).

Figure 6.. The c-di-GMP network functions as a classifier that determines whether bacteria should stay in that environment to form a biofilm or move away.

(A) The c-di-GMP signaling network is shaped like a bow-tie. (B, E) Wild type cells could classify the environment properly and form biofilm when they are supposed to. (C) The hyperswarmer with mutation in fleN fails to respond to high c-di-GMP level and therefore is locked in swarming phenotype. (D) Mutation in the wspF gene resulted in high c-di-GMP levels even in swarming conditions and locked the mutant in a biofilm phenotype. Additional mutation to fleN* could change its phenotype to either mimic wild type (fleN*dipA*, B, E), or mimic wspF* (fleN*wspF*, D)