Have you ever heard the story of The Little Red Hen? The moral of this classic children’s tale teaches us that we must work hard in order to reap the rewards of our labor. Well, sorry to burst your bubble, but as it turns out, this is not always true in the natural world. For example, sometimes birds deposit their eggs into other bird’s nests. Bees have been known to steal nectar from flowers without spreading any pollen. Believe it or not, such instances of “cheating” between species can even be found in the microbial world.
That’s right, even something as small as a bacterium is able to be that classmate who leeches off of everyone else without ever doing any work for the group project. Let me explain. It all starts with the bacterium known as Vibrio fischeri (Figure 1).
Figure 1. Fluorescence stained V. fischeri bacterial cells. Source: E Nelson & L Sycuro for the Vibrio fischeri Genome Project
V. fischeri are rod-shaped, bioluminescent bacteria that hold relevance not only for microbiologists, but for anyone living near a coastal environment. If you have ever visited the beach before, you have probably come into contact with V. fischeri. These bacteria can be found free-floating in seawater, as well as within the bodies of other marine organisms. V. fischeri play a fascinating role in the survival of the Hawaiian bobtail squid (Figures 2 and 3). To learn more about the symbiotic relationship between V. fischeri and the Hawaiian bobtail squid, click here.
Figure 2. Hawaiian Bobtail Squid. Source: Ronald R. Holcom
Figure 3. A confocal micrograph of the light organ of a Hawaiian bobtail squid. This light organ contains bioluminescent V. fischeri bacteria. Source: Margaret McFall-Ngai
V. fischeri as a species is well studied by microbiologists due to its pervasiveness in marine environments. However, there are many other strains and species of Vibrio bacteria that inhabit the ocean. This reality presents an interesting opportunity for scientists to explore how various Vibrio species interact with one another in shared spaces. Just what kinds of mechanisms have bacterial cells evolved to compete for limited resources? What happens to the bacterial cells that have less of a competitive advantage?
Well, the results from this recent study conducted at Princeton University by graduate student Michaela J. Eickhoff and instructor Bonnie L. Bassler have proven helpful in answering these questions. This research focuses on the fact that iron in particular is very limited in marine environments. This presents a problem to bacteria, since iron is an essential nutrient required for both DNA synthesis and the ability to release stored energy.
Some Vibrio species are able to obtain this essential nutrient through the production and release of small iron binding molecules called siderophores. Siderophores are produced within the bacterial cell and exported into the environment through the use of transporter proteins that span the cell membrane. Once released into the environment, they are able to bind to iron and form a siderophore-iron complex, which outer membrane receptors on the surface of the bacterium can then recognize and shuttle back through the cell membrane. Essentially you can think of the use of siderophores as a way for bacteria to “reach out” into the environment and “scoop up” iron for themselves. Bacterial species that are capable of producing siderophores possess a competitive advantage, since they are able to enhance their own iron acquisition while at the same time denying other bacterial species access to this essential resource.
Researchers Michaela J. Eickhoff and Bonnie L. Bassler determined that the strain of V. fischeri known as V. fischeri ES114 produces a siderophore with a very high affinity for iron called aerobactin. Since aerobactin has such a high affinity for iron, it allows V. fischeri ES114 to outcompete other bacterial species by denying access to a resource necessary for survival.
Eickhoff and Bassler demonstrated this phenomenon by designing an experiment in which another Vibrio species called Vibrio harveyi was suspended in an iron-limited culture along with aerobactin produced by V. fischeri ES114 (Figure 4). A control culture was created by combining V. harveyi with another strain of V. fischeri that does not produce any siderophores called V. fischeri MJ11. A photograph of the test tubes containing these cultures can be found below in Figure 4A.
As can be seen, the cultures in the test tube containing V. harveyi and the tube containing V. harveyi and V. fischeri MJ11 are clouded (Figure 4A). This cloudy appearance indicates bacterial growth. V. harveyi bacterial cells are able to grow and divide in these tubes because they are able to freely access iron. However, we can also see that the culture in the test tube containing aerobactin produced by V. fischeri ES114 is clear. V. harveyi bacterial cells are not able to grow and divide in this case, because aerobactin binds to the limited amount of available iron, rendering it inaccessible for use in cell growth. Since V. harveyi was not able to grow and divide, the culture in the test tube appears clear.
While simply looking at the photo of the test tubes in Figure 4A can provide us with an idea about how much bacterial growth occurred, more specific measurements are necessary. The graph to the right of the photo in figure 4A measures the optical density of the cultures in the test tubes. The optical density of a culture involves measuring how quickly light is able to travel through the sample. Cloudier cultures decrease the speed at which light is able to travel through them, resulting in a higher ocular density measurement.
The graph in Figure 4A shows us that the cultures containing V. harveyi and the mixture of V. harveyi and V. fischeri MJ11 have a much higher optical density than the V. harveyi and V. fischeri ES114 culture. This again indicates that V. harveyi is able to grow on its own and in the presence of V. fischeri MJ11, but not in the presence of aerobactin produced by V. fischeri ES114.
Figure 4. A. Growth of V. harveyi alone, in the presence of aerobactin produced by V. fischeri ES114, and in the presence of V. fischeri MJ11. B. Growth of V. harveyi, P. angustum, V. cholerae, V. parahaemolyticus, and V. vulnificus in the presence of aerobactin produced by V. fischeri ES114
Figure 4B shows us the results of a second experiment conducted by Eickhoff and Bassler. This time, cultures of aerobactin produced by V. fischeri ES114 were combined with cultures containing five other bacterial species. The gray bars of the graph represent bacterial growth in the presence of aerobactin and the black bars represent bacterial growth when not in the presence of aerobactin. Interestingly enough, the results this time around indicate that the aerobactin produced by V. fischeri ES114 does not inhibit the growth of V. parahaemolyticus and V. vulnificus. Based on the results from the first experiment, we would expect that aerobactin would be able to inhibit the growth of these two bacterial species due to its ability to block access to iron.
To ensure that aerobactin was actually the cause of the inhibited growth observed in earlier results, the researchers deleted the genes responsible for the production of aerobactin in V. fischeri ES114. They recreated cultures using this new mutant strain, and as expected, V. harveyi was able to grow when no aerobactin was produced.
To be extra sure that this mutant strain was not producing aerobactin, a special dying technique was used to measure the siderophore content in cultures of wild-type V. fischeri ES114 and mutant-type V. fischeri ES114. As expected, the presence of the siderophore aerobactin was detected in cultures containing wild-type V. fischeri ES114, but not in cultures with mutant-type V. fischeri ES114.
So why would aerobactin with its high affinity for iron be unable to inhibit the growth of certain bacterial species in iron-limited environments?
Well, as it turns out, some bacterial species are cheaters! These species possess the genes required for the recognition and uptake of siderophore-iron complexes while at the same time not possessing the genes required for siderophore production. So what is really going on here, is that V. fischeri ES114 is producing aerobactin and sending it out into the environment, but the bacterial species Vibrio parahaemolyticus and Vibrio vulnificus are the ones picking it back up along with that sweet, sweet iron.
This hardly seems fair right? Well, it’s actually a bit more than unfair. This ability is a huge competitive advantage, because while V. fischeri ES114 must expend valuable energy creating and releasing aerobactin, it only reaps some of the iron necessary for survival, while V. parahaemolyticus and V. vulnificus do not have to waste any energy to obtain this resource.
So what can we learn from the results of these experiments? These discoveries drive home the reality that bacteria are just as much a part of the evolutionary arms race as eukaryotic organisms are. Bacteria must be able to overcome harsh environmental conditions in order to survive and reproduce, even at the expense of other microbial life forms. This research will hopefully inspire and emphasize the importance of research into the other forms of competitive mechanisms that bacteria must have evolved over their long existence on earth.
While the implications and impacts of this research are important, I think that the most important lesson of all for those who work hard for their rewards is that the next time you bake a loaf of bread after harvesting all that wheat, pull it out of the oven and run far, far away to where no cheaters can get to it.
About the Author:
Amelia Strimple is a graduating senior in the class year of 2022 at Mount Holyoke College. She is a Biological Sciences major and a Psychology minor. In her spare time she enjoys making delicious lattes at the Frances Perk Cafe, walking her German Shepherd around campus and spending time playing board games with her friends.
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