Fremyella diplosiphon: A Chameleon of the Bacteria World

By: Amanda Porter, Alessandra De Franco, and Shelbie Spalding

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Like a chameleon, the cyanobacteria Fremyella diplosiphon changes color in response to stress from the surrounding environment; however, unlike a chameleon, F. diplosiphon is limited to the colors red and green.  This color change is stimulated by a change in light quality. F. diplosiphon is an aquatic bacterium that depends on photosynthesis for energy, but in water, light quality and quantity vary at different depths. To accommodate for the change in light quality, the bacteria utilizes complementary chromatic acclimation (CCA). This is a process by which the pigment of the bacteria changes in response to the change in light color. The bacteria have phycobilisomes, light harvesting antenna, that allow for absorption of light and facilitate photosynthesis. When the bacteria are exposed to green light, they construct green-absorbing phycobilisomes, and when exposed to red light, red-absorbing phycobilisomes are created. The color we perceive the bacteria to be is the opposite color it is absorbing. For example, when F. diplosiphon is red, it is in green light and has constructed phycobilisomes to absorb green light (Figure 1).

Figure 1. Complementary chromatic acclimation of F. diplosiphon under varying intensities of light.
Under red light, F. diplosiphon is green in color, whereas under green light, F. diplosiphon appears to
be red. Source

To facilitate the change in color and phycobilisomes, the cells must also alter their shape. Unlike CCA, a process that is well known, how the bacteria alter their shape is not fully understood. Singh and Montgomery, the authors of Morphogenes  bolA and mreB mediate the photoregulation of cellular morphology during complementary chromatic acclimation in Fremyella diplosiphon, set out to determine how the shape and structure of F. diplosiphon change during the adaption to changes in light quality.

Like the bacteria’s color, changes in light quality, or rather the intensity, alter the cell’s morphology. By modifying its size and shape, F. diplosiphon is able to absorb different wavelengths of light to maximize its ability to perform photosynthesis. Under the less intense green light, the cell takes on a rod or rectangular shape, which provides more surface area to absorb the available photons (Figure 2). Alternatively, under red light, which has a more increased intensity, the cell takes on a smaller, spherical shape, since an increased surface area isn’t needed.

Figure 2. In green light, F. diplosiphon forms a rod shape

and appears red in color. In red light the bacteria becomes

spherical and is green.

Prior to this experiment, it was established that CCA was controlled by RcaE. RcaE acts as a kinase, an enzyme that phosphorylates other proteins to activate or inhibit pathways within the cell. Under green light, RcaE is inactive, but under red, it indirectly represses green pigments and activates red pigments to allow for absorption, and thus, photosynthesis. Aware of this well-known process, Singh and Montgomery specifically investigated how RcaE impacted the cell morphology in response to changes in light.

Figure 3. Unfinished model indicating the cellular

morphological pathways initiated by red and green

light. These pathways currently include RcaE, bolA,

and mreB, but lack a connection between the

enzyme and morphogenesis.

Additionally, proteins involved with specific cell morphology in F. diplosiphon had also been established prior to this experiment. The protein MreB, which is similar to actin in Eukaryotic cells, is associated with rod shaped morphology (Figure 3). MreB typically collaborates with MreC and MreD proteins, which can be found on the same operon, a series of genomic DNA clustered together and influenced by a single promoter.

The other protein involved in cell morphology is BolA. This protein is encoded by the bolA gene and is found in spherical shaped cells under red light. BolA binds to the promoter region of the mreB operon, inhibiting the transcription of mreB, thus directly influencing morphology (Figure 3). This inhibits MreB production, which results in the cell transitioning from rod shaped to spherical. With the knowledge that the morphogenes mreB and bolA are responsible for directly altering cell shape, Singh and Montgomery set out to further determine the mechanism of  cell morphology  by examining the role of RcaE in wild type, rcaE and bolA mutant strains of  F. diplosiphon.

The authors began with an experiment examining transcript levels of bolA, mreB, mreC, and mreD genes in wild type, (deletion of) rcaE, and bolA strains of F. diplosiphon under red and green light.  They examined these transcript levels using polymerase chain reaction, or PCR.  This experiment was performed in order to observe how the absence of the genes rcaE and bolA affected the levels of the morpho-genes in varying light intensities. As an initial step to obtain a bolA deletion mutant, they found that complete deletion was unsuccessful in F. diplosiphon, thus concluding that the gene is essential. However, they managed to isolate a “partially segregated” strain consisting of cells with both wild type and deleted bolA genes.

Comparing the levels of protein expression for the various strains, they collected a number of results.  They observed that the expression of bolA and mreD was significantly lower under green light than red light in the wild type strain, while expression of mreB and mreC were lower under red and higher under green.  Since bolA expression is increased under red light, these results indicated its importance in determining spherical shape.  Although mreD expression was also increased under red light compared to its expression in green, the authors only noted this difference in expression and did not conclude or deduce further. Additionally, since mreB and mreC expression are increased under green light, these results indicated an importance in determining rectangular shape. They also observed that the expression of bolA was significantly lower for the rcaE strain than the wild type, even under red light, whereas the expression of all the mre morphogenes for the rcaE strain were higher under both light conditions.  These results indicated that RcaE is necessary in promoting the expression of bolA and that either RcaE or BolA help to inhibit the expression of the mre morphogenes.

For their next experiment, Singh and Montgomery studied the cellular morphology of wild type and bolA strains with confocal microscopy in order to observe how the bolA gene affects cell shape (Figure 4). Through this analysis, they confirmed that under green light, the wild type cells take on a more rectangular shape, whereas under red light, they take on a more rounded shape.  However, they also discovered that the morphology of the bolA strain undergoes a few dramatic changes.  Under all light conditions, the bolA strain generated larger cells, both in length and width, a different point of attachment of cells to one another, and exhibited plasmolysis, all irrespective of light quality (Figure 2). These changes in the bolA strain indicate that BolA is necessary for the maintenance of proper size, attachment, and water retention of the cells for all light conditions.

Figure 4. Confocal microscopy of wild type and bolA strains, followed by quantification
of the cellular morphology for the two strains (Singh and Montgomery, 2014).

The researchers performed their final experiment in order to determine which mreB promoter a specific BolA protein, FdBolA, binds to.  They first identified three different promoter regions for mreB, and determined promoter region 1 as the binding site for FdBolA.  This result allowed them to conclude that BolA directly regulates mreB gene expression by binding to an mreB promoter (Figure 5).

Figure 5. Model indicating the cellular morphological
pathways initiated by red and green light (Singh and
Montgomery, 2014).

From these experiments Singh and Montgomery determined that RcaE and its ability to photoregulate bolA is crucial for F. diplosiphon to be able to change shape and color in response to different wavelengths of light. The RcaE kinase activity, triggered by red light, indirectly induces the transcription of bolA (Figure 5). Since BolA was found to regulate the transcription of both MreB and MreC by binding to the mreB promoter, RcaE indirectly controls levels of MreB and MreC. Red light promotes RcaE kinase activity, and in turn bolA expression; therefore mreBC transcription is inhibited in the presence of red light, which causes the bacterium to adopt a spherical shape. RcaE kinase activity diminishes in the presence of green light, leading to decreased levels of BolA within the cell and allowing for transcription of the mreB gene; therefore, F. diplosiphon a takes on a rod shape in the presence of green light.

In addition to discovering the details of the regulatory cellular morphology pathways initiated by light in F. diplosiphon, Singh and Montgomery determined that the bolA gene is essential for the cell’s survival.  Since the cells were not viable when the entire bolA gene was deleted, it was clear that the role of bolA extended beyond chromatic acclimation. This role included the maintenance of proper size, attachment, and water retention of the cells under all light conditions. Although the results from this experiment provide valuable knowledge regarding the function of BolA and its effect on cellular morphology, the results would have been far more concrete if Singh and Montgomery were able to delete the bolA gene entirely.  Since they were unsuccessful in this endeavor, the exact role that BolA plays within the cell remains somewhat of a mystery.

Despite the fact that the knowledge gained from this experiment does not encompass all there is to know about the bolA gene and BolA protein, the information that Singh and Montgomery discovered is still extremely valuable. Homologues of bolA are expressed in many plant, animal, and bacterial cells. The importance of BolA in the regulation of the shape of a cell can therefore be applicable to a multitude of species of life. In particular, a human bolA homologue has been known to influence mitochondrial shape similarly to the way it can influence cell shape in F. diplosiphon. Neuron function relies heavily on proper function of the mitochondria and as a result, many neurodegenerative diseases are influenced by mitochondrial shape and plasticity (Chen & Chan, 2009). Due to the parallels between bolA and the human homologue, this new information regarding bolA and its influence on mitochondrial shape could be extended to study new treatments for diseases like Parkinson’s and Alzheimer’s.

Fremyella diplosiphon is only one of many bacterial species that acts as a microbial chameleon. Although the mechanisms through which many bacteria can change pigmentation have been well studied, the ways in which other bacterial, animal, and plant cells are able to transform under different environmental conditions are still somewhat unknown. Since homologues of the BolA protein are found in many species of bacteria, it is possible that other bacteria that undergo CCA utilize a similar RcaE/bolA- dependent mechanism for shape shifting.

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References

Chen, H., & Chan, D. (2009). Mitochondrial dynamics-fusion, fission, movement, and mitophagy-in neurodegenerative diseases. Human Molecular Genetics, 18 (R2), 169-176.

Grossman, A., & Bhaya, D. (n.d.). Phytochrome Photoreceptors in Cyanobacteria: The Impact of Light Quality on Phycobilisome Biosynthesis. Retrieved November 19, 2014, from http://www.photobiology.info/Grossman-Bahaya.html

Singh, S., & Montgomery, B. (2014). Morphogenes bolA and mreB mediate the photoregulation of cellular morphology during complementary chromatic acclimation in Fremyella diplosiphon. Molecular Microbiology. 93(1), 167-182