Pseudomonas aeruginosa and Biofilms

So I’ve taken this week to focus on information required by my microbiology professor :”Know about biofilms and quorum sensing.” No detail on *what* I should know about those two huge topics, but I need to know about them. All of it, I guess.

This is my brain. Bystanders beware!

I guess I’ll work on just knowing what I can. Let’s get to it, shall we?

First up, my plan of attack. There’s a lot of information out there about biofilms (they’re the darling of the microbiology world at the moment, and lots of time and effort is going into studying them), so there’s no way I’ll be able to memorize everything about them in the next few weeks. What I can do, however, is focus on a model specimen, and know everything about that particular model.

Pseudomonas aeruginosa is a bacterium that is frequently found in biofilms, and happens to be one of the most studied species in the biofilm world. (This is probably due to its annoying tendency to infect humans).

Skin lesions caused by aeruginosa. So gross!

This makes it the perfect subject for me to use as my model specimen. Let’s get to it!

First and foremost: what are biofilms?

Biofilms are communities of microorganisms embedded and immobilized in an extracellular polymeric substance (EPS) attached to a solid surface. The biofilm gives microorganisms protection from things like desiccation, predation, and antibiotics, and allows them to share water and nutrients. It has also been proposed that biofilms allow for the sharing of DNA, thereby facilitating the transfer of beneficial genes from cell to cell. This community organization is in direct opposition to the way we have studied microorganisms for many years: as free living single-celled organisms (called planktonic organisms).

Bacteria in a biofilm in a creek. Kinda gross looking.
Planktonic bacteria just being all lonely.

Biofilms are highly variable, and their exact make up and structure changes with species, temperature, stress, nutrients, and other environmental factors. While this is super annoying for those of us who want to know everything about biofilms, it’s actually a very good strategy for the micro communities that utilize these biofilms as environments: great diversity keeps a single organsim from decimating the world-wide biofilm population.

What makes a biofilm a biofilm is the presence of the extracellular polymeric substance (EPS for short).

"Extracellular" is outside the cell; "polymeric" is a repeated molecular unit

This stuff was originally called “extracellular polysaccharides” until scientists figured out that there were lipids, proteins and DNA involved as well. Luckily, they were able to come up with a phrase that kept the EPS acronym, so everything worked out in the end.

In order to form biofilms, planktonic bacteria go through five primary stages of biofilm development:

1. Initial attachment

2. Irreversible attachment

3. Maturation I

4. Maturation II

5. Dispersal

The five stages.

I’ll look at these in order, using P. aeruginosa as a model organism.

1. Initial attachment.

First and foremost, the planktonic bacteria need to get to the surface of something in order to form a biofilm (remember that the definition of a biofilm is a community of bacteria embedded in an external matrix attached to a solid surface). Pseudomonas aeruginosa does this via diffusive transport

Remember this type of diffusion drawing from your bio classes? Yep, bacteria move the same way.

convective transport

convective transport is when the bacteria gets carried along with the movement of a fluid...kinda like a surfer on a wave

and active transport driven by bacterium flagella.

This species has a single, polar flagellum that it uses to move.

Once the bacterial cells arrive at an appropriate spot, they attach to the surface. They are able to attach due to their flagellum, type IV pili, extracellular DNA, and Psl polysaccharides.

The flagellum is involved in attachment due to its stators.

The stator is the green stuff

You see, the flagellum works by spinning. There’s a spinning rotor portion, and a stationary stator portion, within which the rotor spins. In P. aeruginosa there are two stators, and both must be present for biofilm attachment. Truthfully, though, we don’t know exactly what the stators do to facilitate this attachment–we just know that mutants that don’t have the stators (either one or both) are unable to attach to surfaces and form biofilms.

Extra cellular DNA (eDNA) is an interesting discovery–scientists knew that it existed, but thought it was simply an artifact of lysed cells in the EPS. However, in depth studies of P. aeruginosa revealed that biofilm formation was impossible without it, and the bacterium produced such large amounts of eDNA by creating releasing vesicles from the cells which house DNA.

The yellow is the eDNA, the green is living cells, and the red is dead cells

So far, eDNA is simply refereed to as an “adhesion factor.” This is science code for “we know it’s important, but we don’t know how or why.” Basically, scientists have found that when cells don’t make eDNA, or when bacteria are flooded with something that breaks down the eDNA, these cells cannot form biofilms. If the eDNA is present, biofilms are present. Pretty convincing argument that eDNA is required, but there’s little information as to what it does exactly.

One article mentioned that eDNA may react with N-acyl-glucosamine, a major component of cell walls in bacteria. This reaction would cause a a bond to form between the eDNA and the cell walls, which would cause the cells to stick together. This may be the mechanism by which eDNA causes biofilm formation–flagella and type IV pilli are used to attach to a solid surface, while eDNA is used for cell to cell attachment. I wonder then if biofilms that are not attached to a solid surface (although some purists don’t consider these flocs biofilms at all) have a disproportionately high amount of eDNA? This is what’s neat about science…there’s so much to find out!

During this stage of initial attachment, some cells attach to the substrate and some don’t.

2. Irreversible attachment

This stage is characterized by most cells being attached, and the community differentiating into two parts: parts of the new community that are motile, and parts that are immotile. This seems to be a function of the nutrient availability of the environment where the bacteria are forming a biofilm. If there’s plenty of glucose in the environment (which P. aeruginosa uses as a carbon source), then part of the bacterial cells become immediately nonmotile, while another part are motile.

At this point, the Las quorum sensing system became active (that is the subject of the next blog, so I’ll let you wait for an explanation).

The nonmotile cells form microcolonies, which are used as “stalks” for mushroom-shaped structures. The motile bacteria climb up the stalks and form the “caps.”

Mushroom-shaped colonies formed from a bunch of motile and nonmotile cells

If citrate is used as a carbon source, however, the cells are all motile to begin with, and none of them stop to form these stalks. The resulting biofilm is flat.

The top ones have the mushroom shapes, while the bottom ones are flat

Man, phenotypic variation due to differences in nutrient availability! How much more complex can we get?!?

During this time, the extracellular polymeric substance is produced. The EPS is involved in attachment (naturally), interactions between subpopulations and therefore cell-t0-cell intercommunications, tolerance, and exchange of genes. As I said above, the matrix is made of polysaccharides, lipids, proteins and eDNA. We know the most about the polysaccharides, proteins and eDNA (it only has recently be discovered that lipids are more prevalent than we thought…that’s always how it goes, isn’t it?). The exact make up of this matrix is, once again, dependent upon environmental conditions. That makes sense, doesn’t it? The armor you wear, the communication you use, and the way you attach to things is always going to be dependent upon what is going on around you.

These different environmental conditions are sensed and regulated by a series of kinase/response regulators called LadS, RetS, and GacS.

Oh dear, new terms. Ok, what the heck are kinase/response regulators?

Let’s start with a kinase. Notice that -ase suffix on the word? That means it’s an enzyme. This particular enzyme is responsible for transferring phosphate groups from high-energy donor molecules (such as ATP) to some sort of substrate.

A phosphate group is just a phosphorus molecule with some oxygen and hydrogen attached.

When a phosphate group is added to another molecule or substrate, that substrate changes (duh…it’s got something added to it!). That change can change the shape of the molecule, opening up some receptor site that was closed before. Or it can allow the molecule to react with a new substrate. Whatever happens, the phosphorilation (addition of a phosphate group) causes a change in the substrate which starts a cascade of reactions within a cell.

It’s a neat system, actually…since cells don’t want reactions happening all the time, they can regulate them by requiring a phosphate group inorder to make a substance reactive. That phosphate group won’t be added with out kinase, and that kinase won’t even be around until the right time. Regulation at it’s finest!

Ok, now we know what kinase is, what about response regulators?

Response regulators are exactly what they sound like–proteins that regulate a response, usually the transcription/translation of a gene. They work with kinases by accepting the phosphate groups.

HDS=histadine kinsase; RR=response regulator; P=phosphate group

So, a kinase is usually in contact with the outside of the cell somehow. When it gets the signal it’s looking for, it takes a phosphate group and attaches it to a response regulator. The response regulator changes shape and becomes active, when then causes something in the cell to happen.

The kinase/response regulators involved in matrix production in biofilms are LadS, RetS and GacS. These three things are membrane associated proteins (proteins that span the membrane) and therefore search the environment for signals.

See those red things? Those are proteins embedded in the membrane.

What do the proteins search for? We don’t know. But it’s something, and when they find that special something it triggers the histadine kinase to phosphorelate the response regulators. In turn, the response regulators turn on groups of genes which do all manner of things, including producing the matrix.

Look at it go!

Once the matrix is in place, the cells are immobile and go on living as a community.

This formation of stalks and caps is characteristic of

3. Maturation stage 1

At this point, the layers of cells become greater than 10 micrometers thick.

The formation of mushroom caps involves the presence of some of the same things as initial attachment: eDNA, flagella, and type IV pili. The type IV pili may bind to the eDNA, while the flagellum allows the cell to get to its required place on the stalk.

During this stage, a second quorum-sensing system is switched on: the Rhl system.

This differentiation continues on until the thickness of the biofilm reaches a maximum of 100 micrometers, which introduces:

4. Maturation 2

This stage is characterized by the cell layers at their thickest points, the cells are nonmotile, and most of the cells are differentiated. At this point, the biofilm is at its most mature, and involves water channels, a highly organized system of cell colonies, and the ability to share genes among individual cells.

5. Dispersal

After several days of maturation, cells at the center of many of these clusters become motile and begin to disperse into the surrounding liquid.

Of course, the problem with attempting to characterize stages of development is that a mature biofilm may exhibit all 5 stages of development at any given time.

Well, that’s it in a nutshell. Biofilms are becoming more and more important in all manner of things, from medicine to engineering. I’m betting that this is the direction that microbiology will go in the future (and is probably why I need to know everything I can about the glory that is biofilm).


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