Why do I make EPS if I can’t run?

By: Fangliang Guo, Nisha Malik, and Ye Tian

Figure 1. Mating dance of performed by a bright-plumed male in

front of a plain-looking female, both of which are from the family

of birds of paradise. Source

People in the 21st century enjoy the convenience and fashion brought by advanced technology: it is very difficult to find someone without a social media account like facebook, twitter, instagram. However, by spending more time with these new technologies, we sacrifice time that should be spent with family and friends. This is a trade-off. Trade-offs occur in other animal kingdoms as well. Most of us are pretty familiar with the concept of sexual selection, which is best illustrated by the 39 species in the family of birds of paradise living in New Guinea, Australia. If you think that a guy wearing a formal suit for his first date is extravagant, you would be absolutely amazed by this BBC video on the mating dance of birds of paradise. However, the potential danger of their colorful feathers is that in addition to attracting potential female mates, they can also attract potential predators. Knowing that trade-offs exist in both humans and birds, would you be surprised if I tell you that trade-offs also occur in microbes? This blog’s protagonist will introduce the benefits as well as the costs of producing extracellular polymeric substances (EPS), a material essential in the formation of biofilm.

Figure 2. A schematic diagram of biofilm. Source

The example we will show you today is the formation of biofilm in Vibrio Cholerae. Biofilm is a defense strategy that microbes form at the surface to protect cell population (a gallery of different biofilms). In general,microbial cells exist in two phenotypically distinct conditions: planktonic cells and biofilm cells. Planktonic cells are the ones that float in liquids while biofilms cells are ones that attach to surfaces, secreting a matrix of EPS and growing together in matrix-rich colonies called biofilm. The EPS matrix contains polysaccharides, enzymes, lipids and extracellular DNA, which can protect the cells from disturbance and enhance quorum sensing, the system that microorganisms use to detect communication signals within colonies. V. cholerae, the notorious cholera pathogen, utilizes quorum sensing to detect intracellular messengers between cells, and further determines the timing and strength of biofilm formation. Now that we have mentioned cholera, we can’t resist to tell you more about this epidemic disease.

Figure 3. A magnified view of typical

V. cholerae bacteria - scan microscopy.

Source   

Cholera causes severe vomiting and diarrhea, which eventually leads to dehydration and death if treatment is unavailable.The culprit of this disease, V. cholerae, is a gram-negative crescent shaped organism that utilizes cholera toxin to infect hosts. The injected toxin overrides normal regulation of ion channel on cell membrane, thus causing electrolyte imbalance and severe diarrhea. Even worse, it is a frequent user of plasmid-mediated conjugation, a mechanism by which donor cell exchanges genetic material with a recipient cell through physical contact. This type of horizontal gene transfer among individuals and populations in bacterial organisms makes them antibiotic resistance. In the nineteenth century, this disease was widespread in the U.S. due to a lack of well-functioning water and sewage treatment systems. It is still a serious disease in many parts of the world where famine or poor sanitation is prevalent. Unfortunately, 3 to 5 million people are infected with the disease, causing nearly 100, 000 deaths annually worldwide.  The astonishing casualties of cholera draw the attention of scientists to focus on the biofilm production in V. cholerae.

V. cholerae is pathogenic in both planktonic and biofilm forms. Previous study conducted by Heithoff and Mahan (click here for the paper) shows that V. cholerae uses pathways that involve flagella to control EPS production and biofilm formation. When flagella is present, the cell is mostly going to become a planktonic cell and disperse away to infect more human cells while no EPS is produced. When the flagella is absent, the cell attaches to a surface. At the same time, its EPS production gene is turned on and biofilm is formed to protect the cell population from antibiotics. This striking connection between flagella and EPS production sheds light for scientists on exploring the relationship between dispersal ability and biofilm formation. By doing so, researchers may be able to discover feasible points to tackle the production and dispersal of V. cholerae, lessening the burden of the disease caused by cholera.

With the costs and benefits of producing biofilm and dispersal as the general study question, Nadell and Bassler evaluated the relationship between EPS+ and EPS- cells using two V. cholerae mutant strains. The EPS+ strain are cells that constitutively produces EPS. On the other hand, the EPS- strain cannot produce EPS. The scientists also modified the two strain to express different color fluorescent protein for visualization purposes by tagging a single copy of the V. cholerae chromosome. They also made sure that the expression of different fluorescent proteins did not influence the magnitude of the growth rate, and therefore could be used in other comparative experiments in this study.

Figure 4. Will the EPS+ cells let EPS- cells be the free riders? Source 

They started the study by asking whether EPS+ and EPS- cells are in competition. Nadell and Bassler first measured the maximum growth rate of wild type V. cholerae, EPS+ and EPS- mutant at 37°C and at room temperature. The results showed that the growth rate of EPS+ cells was at least 20% lower than that of EPS- cells. The lower maximum growth rate of the EPS+ cells further illustrated that EPS production was an energetically expensive investment. Were the EPS+ cells “willing” to let their EPS- neighbors be the free riders, enjoying the stability created by the matrix at no cost?

After determining the maximum growth rate of the mutant strains, the researchers tested whether EPS production could provide a competitive advantage to outcompete the EPS- strain in different environments.  In biofilm monoculture, EPS+ strain built up more biovolume per unit area of substratum than does the EPS- strain. Similarly, the greater biovolume accumulation by EPS+ strain in biofilm not only held under monoculture condition, but also in cocultures with EPS- cells. Although EPS+ strain increased in frequency relative to the EPS- strain in biofilm, the other experiment they did indicate that EPS+ decreased in frequency in shaken liquid environments. The results suggested that EPS production is an advantage in a biofilm environment but a disadvantage in a mixed liquid environment.

Apparently, these two strains of cells are in competition. In biofilm, EPS+ cells grow more advantageously, using the EPS matrix to increase cell-cell communication. However, this benefit is only shared within the EPS+ strain, and they recognize and inhibit the growth of EPS- cells to prevent them from being the free riders. With this initial finding of the competition between EPS+ and EPS- cells, the researchers further studied the cost and benefit nature of EPS production.

Figure 5. Two V. cholerae making the serious decision of whether they should produce EPS or not. Source

Benefit: From an evolutionary point of view, researchers hypothesized that EPS+ cells would only benefit other EPS+ cells rather than EPS- cells even though they are of the same species. To better visualize the finding, researchers took a time-lapse video of co-culture of these two species on solid substrate. Even though the initial concentration of EPS+cells was less than 5 percent, EPS+ cells rapidly took over almost the entire territory within 36 hours. Interestingly, each 3-D tower of EPS+ cluster had derived from a single cell lineage as proven by a supplemental experiment, which monitored EPS+-only cells with two distinct lineages and observed single lineage in every cluster. These observations suggested an advantage of EPS+ cells: EPS producing cells are able to build 3-D structures with the same cell lineage and adhere to the substratum while resisting shear stress, outcompeting EPS- cells on solid substratum in competition for nutrients.

Figure 6. A time-series of EPS+ cells (red) growing in a biofilm with EPS- cells (blue). (Courtesy of Nadell and Bassler)

Cost: If there is such a huge advantage of EPS production for local competition, why aren’t all cells producing EPS? The answer lies in the fact that resources for biofilm are not always unlimited, and that when resources are used up, bacteria need to disperse to new colonies. Thus, researchers hypothesized that EPS production could affect a cell's ability to disperse.To test this hypothesis, they co-inoculated EPS- and EPS+ strain on either solid or liquid effluent and monitored the frequency of EPS+ cells over a 40-hour period.The observation that EPS+ cells gradually dominate the solid substratum but remain a minority in the liquid effluent suggested that EPS+ cells were more likely to adhere to substrate rather than flow in liquid to be dispersed to a new location, therefore supporting the hypothesis that inherent EPS producing affects bacteria dispersal ability.

Furthermore, Nadell and Bassler hypothesized that EPS+ strain’s disadvantaged dispersal ability has led to their poor colonization in new environments. To test this hypothesis, researchers connected the effluent of the biofilm-containing chamber to a new chamber to monitor the colonization of both EPS+ and EPS- cells. The results indicated that EPS- cells colonized at higher bacterial biovolume compared to EPS+ cells. Interestingly, even when they increased the inflow of chamber by a factor of 1000, EPS+ cells were still rarely seen in the new chamber. On one hand, the results confirmed the strong resistance of EPS+ cells to shear stress, while on the other hand, they revealed the tremendous cost of EPS production when dispersal is necessary.

Clearly, altruism does not exist between EPS+ and EPS- cells: they are in competition. Just as the famous peppered moth evolution entails, whether a trait is a beneficial phenotype depends on the environment that the organism dwells in. EPS+ cells benefit on patches where resources are long-lasting, whereas EPS- cells thrive in environments where resources are short-lasting and thus search for new resource patches is required. Furthermore, this study has important implication in treatment and prevention of cholera. While EPS producing cells form biofilm and are more likely to be antibiotic resistant, EPS non-producing cells could aid in the dispersal of  microorganisms to new colonies and facilitate the spread of cholera disease. This finding suggests that we could target EPS production and cell dispersal ability as a possible treatment for cholera by affecting the cells’ ability to form biofilms and disperse.

Nisha, Ye, and Fangliang are biology majors at Mount Holyoke College.

References

Nadell, CD, Bassler, BL (2011). A fitness trade-off between local competition and dispersal in Vibrio cholerae biofilms. PNAS, 108 (34): 14181-5. PMID: 1111147108