A genetic switch controls Pseudomonas aeruginosa surface colonization

By: Toni Dris '26

Pseudomonas aeruginosa is an extremely common, antibiotic-resistant, and opportunistic pathogen that thrives in human-made environments, but especially so in moist ones like medical equipment. These factors combined make it the bane of every hospital’s existence. P. aeruginosa is everywhere, but why and how is it everywhere? By what method does P. aeruginosa colonize various surfaces, and why is knowing this mechanism important? Manner et al. propose that a stochastic genetic switch controls the surface colonization of P. aeruginosa. If scientists can interfere with this switch, then maybe we can have some way of preventing P. aeruginosa from colonizing in places they do not belong (like medical catheters).

Image: Agar plate of bacteria with 4 antibiotic tabs. The white layer is the bacteria, and the rings of just agar around the tabs shows the level of sensitivity the bacteria has to certain antibiotics. This is called antibiotic resistance, and in this case, this bacteria is not very antibiotic resistant as it cannot grow near the tab.

P. aeruginosa is a multi-drug resistant pathogen with highly advanced anti-antibiotic mechanisms. Unfortunately, P. aeruginosa is not a harmless bacteria to humans or other animals. It can cause serious infection or death when working in conjunction with an existing disease or condition, like in patients with cystic fibrosis, immuno-compromised individuals, and traumatic burn patients. Once P. aeruginosa establishes itself, treatment can be difficult due to its extremely antibiotic-resistant nature with more advanced antibiotic drug regimens needed to combat it. However, more advanced drug regimens just make more advanced drug-resistant bacteria so it is beneficial to look at alternatives in preventing the enduring biofilm of P. aeruginosa from forming in places where vulnerable people and animals are. This is where the study of the stochastic gene switch is important. If we can find a way to interfere with the switch, then P. aeruginosa’s biofilm will not form properly. These biofilms help bacteria to survive in harsh environmental conditions and antibiotic tolerance is a hallmark for mature biofilms. So, one can see how important it could prove to be to stop biofilm formation in its tracks.

(Image sourced from NIH article on antibiotic resistance)

The study identified a stochastic genetic switch, hecR-hecE. Which is expressed bimodally, this creates bacterial subpopulations that are distinct in their function. A visual representation of a stochastic gene switch is pictured below in which Repressor 1 inhibits transcription from Promoter 1 and is induced by Inducer 1. Repressor 2 inhibits transcription from Promoter 2 and is induced by Inducer 2.

Image:A visual representation of a stochastic gene switch from Gardner et. al. 2000 

The stochastic gene switch is the basis of P. aeruginosa’s stick-and-run strategy that coordinates the surface attachment and spreading of surface colonization. For the purpose of this blog, we will mainly be looking at surface colonization and the mechanism that drives it (hecE and c-di-GMP). Cyclic di-GMP (c-di-GMP) is a global bacterial second messenger, or rather, a conserved and widely used secondary messenger, that controls a wide range of cellular processes that contribute to surface adaptation, biofilm formation, cell cycle progression, and virulence. Cellular levels of c-di-GMP are controlled by enzyme classes that have antagonistic activities. Diguanylate cyclases are one of the enzymes that synthesize c-di-GMP and phosphodiesterases are one of the enzymes that degrade c-di-GMP. Jargon aside, what needs to be understood is that the phosphodiesterase BifA (degrades), and the diguanylate cyclase WspR (synthesizes) are what are discussed in this paper. Now the question is, what or how does hecE affect c-di-GMP to drive late-stage surface colonization?

The study found that during the later stages of growth and surface colonization, P. aeruginosa generates additional differences through an entirely stochastic process that subsequently determines the cellular distribution of c-di-GMP. Already introduced, this stochastic switch is hecR-hecE and was identified to be a toxin-antitoxin module (TA module) that is only expressed in subpopulations of cells. A toxin antitoxin module sounds complicated, but it is actually a pretty simple thing to understand. Under normal conditions, antitoxin counteracts the toxicity of the toxin. Whereas during stress conditions, TA modules play a crucial role in bacterial physiology through involvement in many things, but the most important to us is biofilms. The toxin-antitoxin module expression in the specific subpopulation leads to a steep increase in c-di-GMP which modulates surface motility, biofilm production, and cellular dispersal. We can see that hecE boosts c-di-GMP levels through a two pronged activity modulation of BifA and WspR (the degrading and synthesizing enzymes). What controls hecE you might ask?

Image: Helpful visualization of a toxin-antitoxin module (TA module)

This meaty figure is the first figure shown in the Manner et. al study and is the culmination of all their work and data that show how exactly hecE influences c-di-GMP.

At first glance, this figure may be intimidating, but if you break it into pieces it becomes more manageable. Full disclosure, since this figure is so jam-packed I will only be covering a small portion. Not because the other parts of the figure are not interesting or important, but because I would need a much longer blog to properly explain what is going on. Now we can go straight to discussing part B with the pretty image of the actual P. aeruginosa colonies used in the study (how cool!).

This figure is showing us how the hecE system regulates c-di-GMP. WT means the “wild type” population. EV means “empty vector”, and is another control, but is one for the plasmids used (explained more below!). ΔhecRE means the mutant population is lacking in both hecR and hecE. However, for this study, the mutant strain was genetically modified to carry a plasmid by an IPTG-inducible promoter. This means that the addition of IPTG, a chemical inducer, activates the promoter to turn on or increase the expression of the hecR, hecE, or both genes on the plasmid. Researchers can control when and how much these genes are expressed by adding IPTG. In essence, the colony appearance of the normal P. aeruginosa and a mutant missing hecR and hecE genes (but carrying a plasmid with either gene(s) controlled by an IPTG-inducible system) is being examined to study the effect of these genes on colony morphology by selectively turning gene expression on or off with IPTG. This figure shows that if you delete hecR, hecE, or both there is no change in overall colony morphology. However, if you overexpress hecE, you see something called a small colony variant (SCV) morphotype and in which you see an increase in surface attachment. 

Figure C is like the numerical equivalent of figure B, but it also shows us some cool stuff. Now, you can see that overexpression of hecE = increased surface attachment (yellow bar). You can also see that the overexpression of hecR by itself is not doing much (blue bar). Therefore we can conclude that hecR-mediated surface attachment was dependent on an intact chromosomal copy of hecE ( red bar), based on how poorly hecR was doing on its own. At the same time, we can also see that hecE does the best while being supported by hecR (red bar). The big conclusion is that expression of hecE mediates SCV formation and stimulates P. aeruginosa surface attachment. This conclusion is important because it gives researchers insight into avenues of thought in how to combat P. aeruginosa surface colonization and biofilm formation.

Based on this conclusion, researchers were able to come up with more experiments to test how exactly hecR promotes hecE, and were even able to hypothesize that this stochastic gene switch may be a bet hedging strategy to avoid bacteriophages (a virus for bacteria) that target the sessile (non-motile) population of P. aeruginosa. Figure G is a schematic of the Kedl bacteriophage. Knedl was used to make a stressful environment for the bacteria while also doing knockdown experiments of our old friend enzymes BifA and WspR to see if they had any influence on hecE mediated biofilm formation while the bacteria was stressed. Simply, it is a more complex continuation of Figure B where you have mutants that do not produce Bifa, WspR, or both and see how that affects hecE and what the colony does or looks like (morphology). This was all shown in figure H. DMS3vir is also a bacteriophage and was used as a control for phage infection that is not dependent on c-di-GMP. This figure is so very important because it gives researchers an idea on how to mess with hecE to reduce biofilm formation. It was shown that hecE mediates c-di-GMP levels exclusively via BifA and WspR as biofilm formation was completely abolished in the double deletion (both) Bifa and WspR mutant. These findings demonstrated that hecE tunes the levels of c-di-GMP in P. aeruginosa by simultaneously modulating the activities of BifA and WspR. 

To conclude, the results of this study shows that surface colonization of P. aeruginosa is controlled by a stochastic genetic switch. This switch is mainly controlled by the bimodal expression of hecE. hecE does this by using BifA and WspR to control c-di-GMP levels (the stick and run strategy). It was shown that hecE directly binds to BifA, and that BifA may actually be the proverbial Achilles heel of P. aeruginosa. This knowledge used in combination with analogous studies of similar pathways in other model organisms, may aid us in the fight against antibiotic resistant bacteria by giving us a better understanding of these pathways and how we may manipulate them to our advantage.

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