#MicroTwJC: Bacteria in SPAAAAACE !!

In 2011, Stephen Hawking declared that humanity may not survive to see the next millenium "without escaping beyond our fragile planet." That may seem like an overly dramatic statement, but there is some truth to it. As long as we confine ourselves to earth, we tie our fate to the fate of this world. It is a big uncaring universe containing unstoppable threats that can any time blast us from the surface of this world.

If we want to ensure that our species survives, we need to colonise space. This is a challenge that no other life form (as far as we know) has achieved. There are no environments on earth that can possibly prepare us for the fatigues of space. We evolved without having to worry about radiation, within the constant grasp of gravity. If we are to make space our home, we need to figure out how to adapt our physiology to make it hospitable.

The paper being reviewed in this weeks Microbiology Twitter Journal Club aims to investigate how our bacteria will be affected when we go into space. Our gut encompasses an entire bacterial ecosystem that is essential to our health, and that we still barely understand. We know that when bacteria adhere to the surfaces within the body, they aggregate together into convoluted structures known as biofilms. These colonies allow the bacteria to communicate with each other, and survive within the harsh environments of the body. A biofilms are critical to bacterial survival within the body, and by proxy, could be crucial to our survival. But what happens to these films when they are grown in space ? 

How do we  even find out ?

It's simple. We go there.

These are the experiments of the Space Shuttle Atlantis on its final missions, to study biofilms where no biofilms have been studied before*.

Performing any biological experiment without the aid of gravity presents a number of challenges. Simple techniques, like pouring one liquid into another become more challenging under zero gravity. Making sure that the bacteria can survive the trip, and that the massive acceleration occurring during take off doesn't break any equipment essential to the experiment.

The authors of this paper solved these problems by using a special piece of equipment known as the Fluid Processing Apparatus (FPA).

The FPA is a sealed tube containing everything needed to perform these experiments. It contains multiple compartments which are mixed together as an experiment progresses.
Pseudomonas aeruginosa was used in this experiment, as it's ability to form biofilms has been well documented.  The Pseudomonas was housed in a dormant state within the yellow compartment at 8'C during launch and spaceflight.

The adjacent compartment contained Artificial Urine medium, which is like urine, but mixed from a standard recipe of its constituent chemicals. This way, they could control the presence of certain nutrients and sugars. At five days before the end of spaceflight, the compartment with the bacterium was mixed with the compartment containing the AU medium using a Group Activation Pack, which allowed compartments to be mixed together simultaneously.

The FPA's slot into the Group Activation Pack as shown below 

These packs were then placed into an incubator set to body temperature, 37'C. The change in temperature woke the bacteria up. Upon finding themselves in a medium rich in nutrients, the bacteria did what came naturally. They began to grow.

Nestled within the medium was a membrane disc to which the Pseudomonas could attach and build biofilms.
Some of the FPA's had an air space separated from the biofilm by a special membrane. This membrane let oxygen enter the liquid.
The bacteria were allowed to grow and build biofilms within this environment for around three to four days.

On the final day of the experiment, the Pseudomonas were cooled to 8'C , returning them to a dormant state for the trip home. Scientists on the ground could simply shake these bacteria off the membrane, and count them.
Some of the FPA's had an additional compartment containing Paraformaldehyde. When this chemical is mixed in with bacteria in a biofilm, it acts like a glue on a molecular level. It causes bacteria within a biofilm are "fixed" in place. This allowed the structures of the biofilms grown in space to be preserved so that scientists on the ground could observe them in detail under a microscope.

While these experiments occurred in space, scientists at ground control were replicating them exactly, so that they could compare the biofilms built by bacteria in space with the biofilms built by bacteria under earths gravity.

So what did they find out ?

Bacteria build bigger Biofilms in Space.

The biofilms were measured to ascertain the total bacterial numbers within the biofilm (Figure 1 A). This is taken through shaking out bacteria from the biofilm, and counting them. The black bars represent the cultures grown on earth, the grey bars indicate those cultures grown in space. Across the bottom bars are the different growth conditions, such as the presence of phosphates (Pi) in the medium and the presence of glucose sugar as a carbon source (mAUMg).

In Figure 1 B, we are introduced to a new measurement, which the authors have called biomass, but this measurement can more truthfully be described as bio-volume. This is measured through three dimensional microscopy. A computer controlled microscope takes images of a biofilm at different depths in order to build a 3D model out of it. It can then measure the volume of all of the bacteria it images (um^3).  This value is then divided by the surface area (um^2) on which the bacteria are growing to get an estimate of the volume of bacteria growing on each unit of area.

For the final part of this figure, we are shown the total thickness of the biofilms o the ground, and up in space.

In all of these cases, the bacteria that were grown in space seemed to form bigger biofilms with more bacteria in them. These differences appeared to be most prominent when high levels of phosphate or glucose was present.

Bacteria build bizarre Biofilms in space

Figure 2 A shows the clear differences between biofilms formed by wild-type (WT) Pseudomonas on earth, and those formed in space. Not only are the space biofilms bigger, but they had  a strange "Column and canopy" structure. The biofilm appeared to be tethered to the surface  by only a few columns whilst in spaceflight. You can see this difference most clearly on the figure 2B which shows slices of the biofilm taken at different heights when viewed from above.

However, you may notice that there spaceflight does not have an effect on a strain marked motABCDE.
 The motABCDE gene is the blueprint of the molecular motor which drives the flagella.  Flagella are the microscopic propellers that allow bacteria to swim through their medium, and they play a key role in forming the structure of biofilms. The motABCDE strain bacteria do not have this gene, and cannot work their flagella. Since this strain cannot produce the "Column and Canopy" structure, thus we can deduce that the strains of bacteria that do produce this structure can only do it because of their flagella.

Oxygen fixes all of the differences

The previous experiments were all performed when the bacteria were starved of oxygen. But what happens when we re-introduce oxygen into the mix. Will that change the way that bacteria make their biofilms ?

They looked at both wild type Pseudomonas, and the mutant Pseudomonas with the bad flagella, because these grew differently from each other during space-flight. So what happened ?

The differences disappeared. There were no columns or canopies to be seen when oxygen was present. Oxygen allowed the bacteria to build the biggest biofilms they could irrespective of the environment. It seems that the column and canopy structure is only important when bacteria don't have the help of oxygen to release energy from their food.

Conclusions

Under normal gravity, biofilms form mushroom-like structures if they are exposed to "hydrodynamic" conditions. That is, if water is moving over and around them, the biofilms are formed into these structures.

 Microgravity appears to cause biofilms to form a somewhat similar, albeit accentuated structure that we've seen above.

So why is this useful ?

We don't often get the opportunity to look at how gravity affects the way bacteria form biofilms. These are dynamic structures that respond to forces in different ways. Each individual bacterium is a self contained unit with the potential to form a biofilm. We don't currently understand all of these rules. Observing how bacteria form these communities is not just important to astronauts, it is relevant to those of us on the ground. We are all affected by gravity, including our bacteria. If we really want to understand how gravity affects bacterial behaviour, we need to look at what they do when it is removed. Only then can we understand the internal rules that each individual bacterium follows when it helps to build a biofilm. 

Comments

•  The statistical analysis appears to be completely missing from this paper. They list p values, but these statistical values of significance are effectively meaningless if we don't know what statistical method the researchers used to obtain them. 
• The paper appears to treat gravity as if it has no direction. A biofilm is either being grown with the action of gravity upon it, or it is not. The question I would ask is whether biofilms grown in normal gravity grow differently if they are placed upside down, or balanced on their sides. If gravity has an effect on the way biofilms structure themselves, then the orientation of a biofilm relative to the earth's gravitational field should also change the structure of the biofilm. In what orientation were the ground control cultures grown ?

• .

Future Work

There are a few things that would have been cool if they could have investigated them

• It would have been cool if thay had terminated their experiments at different time points, so that we could see how these "Column and Canopy" structures develop over time.
• It would have been fascinating if they could simulate the effects of different strengths of gravity on biofilm formation using some form of centrifuge

All we need is to book some space on the next Space Shuttle mission, and we can these experiments sorted out..... Oh wait.

What do you mean cancelled ?

Final Verdict

The greatest thing about actively reading a paper is putting yourself in the shoes of the scientists who write it. The ability to see some of the problems they overcame to produce their work. That is why this is an exciting paper to read. Most of us will never do science in space. But reading this paper can get us a taste of what that experience would be like.

Microbiology Twitter Journal Club Starts at 8pm BST tomorrow, Join in the Discussion. Just follow  #microtwjc

Reference

Kim W., Tengra F.K., Young Z., Shong J., Marchand N., Chan H.K., Pangule R.C., Parra M., Dordick J.S. & Plawsky J.L. & (2013). Spaceflight Promotes Biofilm Formation by Pseudomonas aeruginosa, PLoS ONE, 8 (4) e62437. DOI: 10.1371/journal.pone.0062437.s008

*Except on a previous space shuttle missions when astronauts growing bacteria in vats noticed that the bacteria appeared to clump together more often, and formed biofilms. But I'm ignoring this so I can squeeze in a Star Trek reference.