The Responses of Cyanobacteria to UV-B Irradiation

Introduction

Cyanobacteria are a deep branching phylum of prokaryotes that are of incredible importance to the biosphere. These ancient organisms perform photosynthesis through the use of photosystems (PS) I and II, a design unique among photosynthesizing bacteria and archaea. Pigments such as phycocyanin and chlorophylls a and b are embedded in the cyanobacterial thylakoid membrane. In particular, phycocyanin is associated with cyanobacteria, as it is responsible for the blue-green appearance for which these organisms are named. Pigments absorb wavelengths of light from the visible spectrum of 400 to 700 nm (Allen, 2001). When energy from light is captured, H2O or H2S is split. When water is the substrate, this reaction releases oxygen gas, protons (H+) and donates an excited electron to PS II. As this initial electron relaxes back to its ground state, it passes its energy to electrons in adjacent atoms until the reaction center of PS II is reached. From this point, electrons reduce various proteins in PS II, allowing H+ to be pumped across the thylakoid membrane to produce a chemical gradient. The potential energy stored in this gradient is then harnessed to make a usable energy source for the cell in the form of ATP. Even more energy is stored when a second electron from PS I is excited. The protein ferredoxin can then produce the energy carrier NADPH.

It is important to note the production of oxygen gas (O2) that occurs during photosynthesis in cyanobacteria. As it turns out, cyanobacteria are the only known bacteria that produce O2 (Slonczewski). The evolution of this type of metabolism probably caused the increase of oxygen in Earth’s early atmosphere. Thus, without the presence of cyanobacteria or a similarly photosynthesizing predecessor, aerobic life as we know it could not have evolved.

Today, cyanobacteria continue to act as important contributors to the air we breathe and provide a number of other important services to ecosystems. The diverse forms seen amongst species of cyanobacteria allow this phylum to fill key roles in a range of ecosystems including the most extreme aquatic and terrestrial environments on Earth. Cyanobacteria have been found to act as a primary source of usable nitrogen to living organisms. Most species of cyanobacteria are capable of fixing nitrogen, converting atmospheric nitrogen into nitrate or ammonia. In habitats like those found in arctic regions where nitrogen is the most common limiting element, the presence of cyanobacteria accounts for up to 80% of the total annual nitrogen input (Solheim et al., 2006). The growth of organisms in these regions would be severely retarded without the nitrogen contributions of cyanobacteria. Similarly, cyanobacteria compose a large portion of microbial biomass and productivity in many ecosystems on which a multitude of higher organisms depend (Vincent, 2000).

Given these essential functions of cyanobacteria in ecosystems, it is important to understand how these organisms are adapted to their environment and how global change might impact them. The level of ozone-depleting gases in our atmosphere has increased dramatically in Earth’s recent history due to anthropogenic emissions. With a reduction of the ozone layer, organisms on Earth are being exposed to an increased level of ultraviolet (UV) radiation. UV light has shorter wavelengths (290-400 nm) than visible light, meaning that it has higher energy (Allen, 2001). Exposure to this high-energy irradiation can lead to the damage of proteins and DNA, as well as changes in growth, cellular differentiation, photo orientation, and the motility of cells (Ehling-Schulz & Scherer, 1999). UV-A (320-400 nm) radiation is not impacted by the presence of the ozone layer, and thus exposure to this form of UV light will not change due to ozone depletion (Ehling-Schulz & Scherer, 1999). However, UV-B (290-320 nm) photons are typically absorbed by the protective ozone layer, so a decrease in ozone will lead to an increased amount of UV-B hitting Earth’s surface (Ehling-Schulz & Scherer, 1999). This will be of particular importance to cyanobacteria, as these organisms depend on near-constant exposure to sunlight for survival. Accordingly, cyanobacteria have been found to poses mechanisms to avoid UV-B rays, defend against them, and repair any damage that they might cause to cellular components (Ehling-Schulz & Scherer, 1999).

Cellular Damage Caused by UV-B Irridation

UV-B irradiation has been found to have a large number of damaging effects on cyanobacteria and many other organisms. While a small amount of potentially toxic forms of oxygen are normally produced within cyanobacteria, uncommonly high levels of UV light can lead to the creation of an increased number of reactive oxygen species (ROS) from excited photosynthetic pigments (He & Hader, 2002). These ROS can cause oxidative damage to polyunsaturated fatty acids in the cellular membrane and breaks in DNA strands (He & Hader, 2002). Proteins and DNA absorb UV-B irradiation, which makes them common targets for damage (Ehling-Schulz & Scherer, 1999). It has been found that UV-B wavelengths often harm the D1 reaction center protein of PS II, Rubisco, phycobili proteins (like phycocyanin) and nitrogenase (Ehling-Schulz & Scherer, 1999). When DNA absorbs UV-B photons, double-stranded breaks, single-stranded breaks, DNA-protein cross links, cyclobutane dimmers, and pyrimidine-(6,4)-pyrimidone photoproducts have all been shown to occur (Peak & Peak, 1986; Mitchel & Niara, 1989 as cited in Ehling-Schulz & Scherer, 1999). The energy that must be used to protect against and repair this damage impacts the cells’ energy budget. Organisms affected by UV-B exposure have thus been shown to grow more slowly and replicate less frequently than those animals with less of a UV-B burden (Garcia-Pichel et al., 1993; Ehling-Schulz et al., 1997).

Motility in Response to UV-B Irradiation

Cyanobacteria can often evade the damaging effects of UV-B irradiation by moving. Some species form mat communities in soils by migrating down to the level at which each species can absorb the maximum amount of light and receive the minimum amount of threat from UV irradiation (Quesada & Vincent, 1997). Because even closely related species of cyanobacteria show a large degree of variability in their sensitivity to UV light (Quesada & Vincent, 1997), mat communities tend to show distinct layers of cyanobacteria. Layers closer to the surface will consist of those species that have a higher tolerance for UV-B irradiation than the species found in lower layers. In fact, it has been found that this distinct layering is lost when cyanobacteria are grown in the absence of UV-B radiation (Sheridan, 2001). Likewise, aquatic cyanobacteria can sink to lower levels in the water column to avoid intense radiation (Reynolds et al., 1987 as cited in Ehling-Schulz & Scherer, 1999). Moving up and down occurs daily in some species to avoid the higher light intensity of the afternoon (Garcia-Pichel et al., 1994), whereas other species may only respond to long-term changes. Migration, however, comes at a price. Moving to areas with less light penetration can lead to a decrease in productivity of these photosynthetic organisms (Bebout & Garcia-Pichel, 1995).

The motility of cyanobacteria is based on the little-understood method of gliding and the simple concept of floating. Gliding involves no obvious change in cell shape or external organelle, and may be related to internal rotation or contraction of some unidentified cell components (Burchard, 1980). This movement can occur over semi-solid or solid surfaces with a film of water on top (Burchard, 1980). It has been shown that cyanobacteria exposed to UV-B light will move to less intensely irradiated areas (Quesada & Vincent, 1997; Bebout & Garcia-Pichel, 1995), indicating that these organisms are photoactive (Burchard, 1980). Despite this similarity in response by different species of cyanobacteria, it has also been shown that the rate at which species react and glide away is variable (Quesada & Vincent, 1997). When in the water column, gliding becomes less useful, and cyanobacteria rely on gas-filled vesicles to maintain buoyancy. Cyanobacteria are capable of changing their buoyancy by altering the volume of their gas vesicles as a response to environmental conditions including light intensity (Brookes and Ganf, 2001). Altering gliding patterns and buoyancy based on the strength of UV-B radiation is thus an important way in which cyanobacteria can avoid UV damage.

UV-B Absorbing Compounds

Cyanobacteria can protect themselves against UV-B radiation that does reach the cell by removing the dangerous rays before they can cause damage. UV-absorbing molecules are one way in which cyanobacteria can defend against UV-B photons. Mycosporine Amino Acids (MAA) are water soluble cyclohexanones that are linked to amino acids or amino alcohols (Ehling-Schulz & Scherer, 1999). Because MAA absorb wavelengths between 310 nm and 360 nm, these compounds can absorb UV-B before it can damage other important cellular structures (Ehling-Schulz & Scherer, 1999). MAA block three out of ten photons from being absorbed by proteins or DNA (Garcia-Pichel, 1993). The higher the concentration of MAA in a cell, the more resistant to UV-B irradiation a species of cyanobacteria tends to be (Garcia-Pichel, 1993). Heightened concentrations of MAA have been found to arise in response to UV-B irradiation, but not UV-A exposure (Ehling-Schulz et al., 1997). However, there seems to be a limit on the amount of MAA a cell can contain (Oren, 1997), therefore placement of the MAA is important as well. MAA located in the cytoplasm has been found to absorb 10-26% of UV-B photons, whereas MAA located in the extracellular glycan absorbs closer to 67% of UV-B photons (Bohm et al., 1995 as cited in Ehling-Schulz & Scherer, 1999). This is presumably due to the fact that photons are absorbed before UV-B irradiation gets a chance to enter the cell. While MAA plays a significant role in defense against UV-B irradiation, it is thought that sunscreen may not be the only role of MAA. It is suspected that these molecules might serve as antioxidants to control ROS produced from UV exposure, transfer energy from UV light to PS reaction centers for photosynthesis, or assist with osmotic regulation (Dunlap and Yamamoto, 1995, as cited in Sinha et al., 2001; Oren, 1997).

Scytonemin is a pigment that absorbs a maximum wavelength of 370 nm (Ehling-Schulz & Scherer, 1999), enabling this compound to serve a similar sunscreen function as MAA. This dimeric pigment is a yellow-brown color and is lipid soluble (Ehling-Schulz & Scherer, 1999). Researchers have observed an increase in the production of scytonemin with exposure to UV-A light and weakly with UV-B irradiation (Ehling-Schulz et al., 1997). Scytonemin is commonly found in the cyanobacterial sheath where it captures approximately 90% of UV-A photons before they can enter the cell (Garcia-Pichel et al., 1992). This pigment can make up as much as 5% of cellular dry weight, and degradation within the cell is not rapid (Sinha et al., 2001). After synthesis of this pigment, the cell need not invest any more energy in order for scytonemin to screen UV photons effectively (Sinha et al., 2001). These characteristics of scytonemin suggest that the compound plays a primary role as a UV-A sunscreen (Garcia-Pichel et al., 1992; Ehling-Schulz et al., 1997). Because of the abundance of scytonemin in cells and prevalence of the pigment across species, it is thought that scytonemin may have evolved early in the history of cyanobacteria. This adaptation would have allowed this phylum to move into shallow waters and terrestrial habitats where exposure to UV light was more intense (Dillon and Castenholz, 1999 as cited in Sinha et al., 2001). A combination of scytonemin and MAA in cells appears to optimize protection against UV-B photons (Ehling-Schulz & Scherer, 1999).

In order to enhance the effectiveness of MAA and scytonemin, cyanobacteria produce bacterial extracellular polysaccharides (EPS). These EPS are located in the sheath of a cyanobacterium, and help to form a boundary between the environment and the cellular interior (Ehling-Schulz et al., 1997). UV-B absorbing compounds, including some oligosaccharides, can then attach among this matrix of EPS and prevent harmful photons from reaching the inside of the cell (Ehling-Schulz et al., 1997). It is again observed that an increase in UV-B irradiation can stimulate the production of EPS in order to defend the cyanobacterium from UV-B damage (Ehling-Schulz et al., 1997).

Defense Against Reactive Oxygen Species

When UV light penetrates a cell and ROS production increases, cyanobacteria are equipped to remove these dangerous molecules with the use of antioxidants. Some antioxidants are scavenging enzymes that include superoxide dismutase, peroxidases, reductases and catalase (He & Hader, 2002). These enzymes bind ROS, yielding them harmless or help slow or prevent oxidative reactions from occurring. There are also non-enzymatic antioxidants within the cell that include ascorbate (vitamin C) and the pigments known as carotenoids (He & Hader, 2002). Ascorbate is typically available at high concentrations within a cell to reduce ROS to less harmful products (He & Hader, 2002). Carotenoids can remove singlet oxygen, triplet chlorophyll, and can reduce lipid peroxidation by quenching (Ehling-Schulz & Scherer, 1999). It has also been found that antioxidant activity is improved with carotenoids and abscorbate working together. As carotenoids quench ROS, charged carotene radicals are formed in the pigment (Bohm et al., 1998). These radicals are repaired when abscorbate is attracted by their charge and transfers an electron to the damaged carotenoid (Bohm et al., 1998). Thus, an abundance of both carotenoids and vitamin C lead to a more effective removal of ROS from cyanobacteria.