If You Cheat Pseudomonas, You Get Gassed

A paper published last week in the Proceedings of the National Academy of Sciences reveals an interesting new twist in the complicated social lives of bacteria. No, I'm not anthropomorphizing. By any reasonable definition, bacteria really do have social lives. Indeed, in recent years bacteriologists have focused intently on understanding the manifold ways in which these seemingly simple organisms compete and cooperate with each other, and the story keeps getting more convoluted.

Of course we've known for decades about antibiotics, those complex small molecules microbes produce to kill off competitors, and which we've co-opted clumsily to save ourselves from infections. But closer inspection reveals a far more sophisticated set of microbial interactions. For many species, highly evolved sensory systems can tell a bacterium when enough of its brethren are nearby, triggering changes in the cell's physiology and behavior. This allows communities of bacteria to cooperate, exploiting food sources and resisting stresses that individuals couldn't handle on their own.

For example, a bacterium might carry a secreted enzyme that can break down an otherwise indigestible food. If a single cell secretes the enzyme, it will just diffuse into the environment; throwing a pinch of salt in the general direction of a pig doesn't yield bacon. However, if enough other bacteria are around, and they all start producing the same enzyme, the whole group suddenly finds itself in a delicious stew of processed nutrients. Similarly, bacteria in groups can form biofilms on surfaces, linking together and protecting themselves from attacks like Roman soldiers behind a shield wall. Biofilms cause some of the most serious and intractable bacterial infections in humans, often resisting antibiotics that could easily kill the isolated cells. The collective can do things the individual can't.

Making energetically expensive enzymes and mucus shields isn't a task to be undertaken lightly, so bacteria only turn these systems on when they sense that there are enough of their species around for the collective strategies to work. This quorum sensing ability usually relies on secreting and detecting distinctive molecules. They smell each other.

Dude, simplify!

The opportunistic pathogen Pseudomonas aeruginosa is an infamous and fairly well studied cooperative species. It senses its colleagues with two linked gene regulation systems, called LasR-LasI and RhlR-RhlI. At first glance, the setup seems unnecessarily complex. LasI catalyzes the production of a small secreted molecule. When that molecule builds up in the solution, indicating that there are a good number of P. aeruginosa in the neighborhood, it binds LasR to turn on numerous genes useful for collective activities. That should be enough, but apparently it isn't. In addition to the genes for cooperative food digestion and such, LasR also turns on the RhlI gene. RhlI then produces another small soluble molecule, as if it's double-checking the whole quorum sensing response. If this second molecule is abundant enough, it causes RhlR to turn on its own set of genes, partially overlapping with the ones already being turned on by LasR. Biologists are trained no to ask "why" questions, but ... seriously, Pseudomonas, why?

It turns out there's at least one good reason: cheaters. In any cooperative system, there's a chance for some individuals to benefit without contributing to the community effort. Evolution mandates that individuals constantly struggle against each other, so such cheaters are virtually inevitable. In P. aeruginosa, one could imagine an individual that acquires a mutation inactivating the LasR quorum sensing gene. If its neighbors all start producing an enzyme to collectively digest a tough food, the LasR mutant can simply eat the resulting nutrients without having to invest energy in producing the enzyme. It gets free bacon.

A cheater gains an individual advantage at the expense of the community, at least until its progeny overtake the whole population. At that point, the population of freeloaders will be more than the few remaining cooperators can feed, and the whole culture will crash. A species with cooperative behavior must therefore have some mechanism for dealing with cheating, or the strategy will inevitably fail.

In the new work, the researchers started by growing P. aeruginosa in a medium where casein was the only carbon source. To eat casein, Pseudomonas must use a secreted enzyme controlled by the quorum sensing system. In these cultures, mutants arise with defective LasR genes, exactly as predicted by evolutionary theory. However, the mutants don't overtake the population. Instead, they reach an equilibrium where they make up somewhere between a third and half the total population. There's apparently some mechanism for punishing cheaters.

If you read my post a couple of years ago about Pseudomonas, it probably won't surprise you that this bug's handling of cheaters is exactly as brutal as the rest of its adaptations. It uses cyanide.

Through an elegant series of co-culturing experiments, the scientists show that cheater control depends on RhlR, the second quorum-sensing regulator. Specifically, RhlR turns on genes for cyanide synthesis and also cyanide resistance. When cheaters arise in cultures of bacteria lacking RhlR or the cyanide synthesis genes, the cheaters overgrow the cooperators and the culture crashes. Pitting different cultures against each other across a dialysis membrane shows that the cheater-combating factor is diffusible, consistent with cyanide.

What seems to happen is that the LasR mutants can't activate the RhlI-RhlR system, so while they gain an energetic advantage by not having to produce the casein-digesting enzyme, they also get a penalty because they're not resistant to the cyanide their cooperating neighbors are all producing. Cheaters could theoretically get around that by adding a second mutation to activate the RhlI-RhlR system, or just the cyanide resistance gene, but the tightly linked nature of the whole locus seems to make that unlikely. Also, activating part of the system would incur an energy cost that could very well offset the advantage of cheating. At some point it's easier just to follow the rules.

It's a good bet that Pseudomonas isn't the only genus to discover the need for law enforcement. Understanding how other cooperative bacterial species handle cheating could reveal even more levels of social regulation. In the meantime, if you find yourself living among Pseudomonas, don't try to pull one over on them.

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