Cyanobacterial Responses to UV Radiation

Summary

The influence of ultraviolet radiation (UVR) on populations of microorganisms has been the subject of serious investigation for at least the past 20–25 years. UVR that is applicable to the Earth’s surface (past or present) is arbitrarily divided into UVA (400–320 or 315 nm), UVB (280–320 or 315 nm), UVC (∼180–280 nm). Although essentially all organisms are affected by UVR, microorganisms show more rapid, immediate and measurable effects than macro-organisms. This chapter is mainly relegated to UVR and cyanobacteria, although UV effects on other phototrophs and microorganisms, when relevant, will be included. Some ancestors of living cyanobacteria, the oldest oxygenic organisms, may have evolved in the Archean or early Proterozoic Eons, from 3.5 to 2.5 Gyr, respectively, in a time when UV radiation fluxes reaching the surface, particularly UVB and UVC, were much higher than at present. The latter wavelength region (UVC) does not reach the Earth’s surface at present. Thus, cyanobacteria and other microorganisms in that distant age had to have evolved a strategy to tolerate these greater levels of UV radiation, and at present this strategy may demonstrably involve multiple devices, even within one organism. The best understood in the past several years for numerous organisms has been the active metabolic strategies that compensate for the destruction of vital genetic components, such as the development of efficient metabolic DNA repair systems. The implementation of gliding motility system for escaping the effects of high visible and UV radiation has been better described and understood. Some of the most revealing results in the last 10 years have been an almost complete understanding of the regulation of the UV-protective compounds, scytonemin and mycosporine-like compounds, that partially or completely avoid the damage caused by UV radiation.

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1 Introduction

Because of the potential to negatively affect molecules and processes of biological importance, ultraviolet radiation (UVR) is considered a detriment to all life forms. There are a few exceptions where UVR may mediate some beneficial responses to high light intensity such as the UVA role in photoreactivation of damaged DNA, and, in some cases, the regulation of some biosynthetic pathways. Although UVR represents only a small proportion of the solar radiation reaching the earth’s surface, its effects are inordinately great. There have been several reviews in recent years on the effects of UV radiation (UVR) on microorganisms (e.g. Castenholz and Garcia-Pichel 2000; Vincent and Neale 2000; Whitehead et al. 2000; Roy 2000; Häder 2001; Cockell 2001; Day and Neale 2002; Shick and Dunlap 2002; Hockberger 2002; Castenholz 2004; Singh et al. 2010). This review follows the general pattern of the presentation of Castenholz (2004) and will be focused almost entirely on cyanobacteria. However, when relevant, UVR effects on other organisms will be included. In the natural habitats of cyanobacteria, UVR has a negative effect, particularly during the period of highest solar radiation. If these cyanobacteria are active metabolically, they have the ability to partially ameliorate these negative effects of UVR radiation by various repair mechanisms, often during periods of low light or darkness. Some cyanobacteria that produce the extracellular UV-absorbing compound, scytonemin, may be protected from UVR damage during periods of metabolic inactivity, such as during desiccation or dormancy as a result of suboptimal temperature or freezing (Castenholz and Garcia-Pichel 2000; Castenholz 2004).

Current paleobiological evidence suggests that oxygenic photosynthesis evolved during the early Proterozoic (∼2.45–2.32 Gyr, or possibly earlier; Knoll 2008). When the ancestors of cyanobacteria first appeared, the impact of UVR radiation was undoubtedly greater than at present, due to the near lack of O₂ and ozone in the atmosphere, although the luminosity of the sun was diminished during this period. Throughout the Proterozoic, particularly by 2.0 Gyr, cyanobacterial “look-a-like” micro-fossils dominated the presumed photosynthetic picture until about 0.54 Gyr, the end of the Proterozoic (Knoll 2008). Although a high temperature environment has been proposed for the period of early life during the Archean, recent evidence suggests that the Paleoarchean ocean temperatures were no higher than about 40°C (Hren et al. 2009). In this geologic period, before the advent of efficient grazing animals, shallow seas bounding the continental or island land masses were inhabited by cyanobacterial ancestors that formed a variety of microbialites, laminated or not. It is not known when cyanobacteria first invaded terrestrial habitats. However, evidence of microbes in anoxic sediments have been documented as early as 2.75 Gyr instead of the previous suggestion of 1.2 Gyr (Rasmussen et al. 2009). Today there is a much greater variety of both aquatic and terrestrial habitats than in the early Proterozoic or late Archean, and these usually harbour cyanobacteria. Some are exposed to high UVR. UVC (∼180–280 nm) was probably quite relevant during these early geologic periods, since it is absorbed by O₂/O₃ in the stratosphere, and only 10⁻⁵ PAL O₂ (present atmospheric level) was present according to Kasting (1987) and Pavlov and Kasting (2002). This level would not have been sufficient to attenuate UVC or UVB. Alternative conditions that may have resulted in the attenuation of UVR in the primitive anoxic atmosphere may have been sulphur vapor composed of S₈ and other sulfur molecules (Kasting et al. 1989). Also, a high concentration of ferrous ion (Fe II) may have been present in anoxic waters to significantly attenuate UVR (Pierson 1994; Pierson et al. 1993). If no screens of this type were present, 1–4 Wm⁻² of UVC may have reached the earth and water surface, a value similar to that of maximum intensities of UVB that presently reach the earth’s surface (although 5–6 Wm⁻² may be attained) (Kasting 1987; Cockell 2001). Garcia-Pichel (Fig. 6 in Garcia-Pichel 1998) has estimated the relative damage to a selected cyanobacterium from incoming UVC, UVB, and UVA radiation during the Archean, Proterozoic and Phanerozoic Eons. Cockell (1998) has also estimated the likely biological effects of UVR on early Earth. These estimates show that UVR must have been a very significant evolutionary force during their earliest evolutionary time. Only UVA, the deleterous effects of which are mostly due to indirect, sensitized photoxidative processes, is thought to have intensified as the partial pressure of oxygen increased in the atmosphere (Garcia-Pichel 1998).

This review will summarize the UV tolerance and avoidance strategies known in living cyanobacteria. Some of these adaptive strategies that evolved in cyanobacteria probably did so in the Archean and early Proterozoic, and may now represent relics of that time. The possible influence of UVR fluctuations on plant (and presumably algal and cyanobacterial evolution), particularly during the Cenozoic have been discussed by Willis et al. (2009). A few passive “strategies”, include burial at shallow sediments, growth at greater water depths (avoidance), and the seemingly altruistic sacrifice of surface layers in microbial mats, will not be discussed here. These are forms of relegation to refugia, that imply the loss of potential habitat. Instead, biochemical aspects of tolerance, UV shielding and behavioural escape mechanisms will be discussed here. This includes recently elaborated signal responses, and biochemical pathways of mycosporine-like amino acid (MAA) and scytonemin induction and synthesis. Although it is difficult to discuss the effects and responses to the three arbitrarily defined categories of UVR (UVC, UVB, UVA), an attempt will be made to present this review in that fashion. However, at first, we will describe the habitats in which cyanobacteria are most at risk to UV exposure. The readers are referred to Castenholz and Garcia-Pichel (2000) for topics not included here.

2 UVR Exposure in Natural Habitats

Latitude determines to a great extent the flux of UVR incident at ground level, the maximum instantaneous and yearly dosage, as well as the amplitude of seasonal variation. Large seasonal variation and low maximal values are associated with higher latitudes, whereas strong, seasonal irradiances correspond to lower latitudes. High altitude, by decreasing the semi-spherical path length for atmospheric attenuation, also results in higher solar radiation fluxes. Other meteorological factors may modify the incident UVR, such as cloud cover, air pollution, and the optical thickness of the stratosphere that is regionally altered by ozone depletion (Roy et al. 1994). Industrial air pollutants, such as sulphur dioxide, nitrogen dioxide and soot particles may reduce incoming UVR by as much as 20%, thus, offsetting the potential effect of ozone depletion (Kvalevåg et al. 2009). Direct measurements of UVR are obviously preferred, but irradiance data are available from various meteorological institutions, although UVR information is often missing (Castenholz and Garcia-Pichel 2000). Total solar radiation, and the UVA and UVB components have been monitored for over 10 years in Europe and some other locations including stations in the Southern Hemisphere (see Häder et al. 2007). The measurement of UVR reaching ground level is not entirely indicative of any organism’s exposure. Absorptive and scattering properties within the micro-environment of the organism may greatly modify the simple assessment of the scalar irradiance as measured by a biospherical photon collection instrument (Fig. 19.1). These modifications are not always intuitively evident or easy to measure.

Fig. 19.1

Effects of habitat on the exposure of cyanobacteria to solar radiation: (I) Spectral scalar irradiance (sun and sky) incident at ground level at noon during a clear midsummer day at 41°N. Lat. (II) “Strong shade” habitat (N. facing surface illuminated by very diffuse sky radiation only). Although scalar irradiance is very low, the relative importance of UVR is enhanced. (III) Open ocean habitat (under 1 m of clear water) where all fluxes remain fairly high and UVB and visible light are more strongly attenuated than UVA. (IV) Surface of beach sand (quartz and feldspar) where all UVB, UVA and visible are higher than incident (by 120%, 150% and 205%, respectively) due to light trapping effects. (V) 300 μm deep in a wet topsoil where UVB and UVA have been attenuated below 5% of incident, but ca. 20% of visible light remains. (VI) Scalar irradiance within the thallus of the terrestrial cyanobacterial lichen Collema sp. (From Fig. 1 in Castenholz and Garcia-Pichel 2000, with references. With kind permission from Springer Science + Business Media B.V.)

Three types of “optical” habitats are first considered: planktonic, benthic, and terrestrial. Exposure in planktonic habitats is determined by the attenuation rate within the water column and by the mixing regime of the water mass. Typically, cells are subjected to high and low exposure periods as they are brought up and down by turbulence (Cullen and Neale 1994). A time integration of UV exposure per cell is very difficult to calculate in these circumstances because of complex hydrodynamic mixing processes (Castenholz and Garcia-Pichel 2000). A comparison of several natural marine waters showed that the UV doses received by cells in the euphotic zone ranged between 3% and 9% of the incident doses that would be received if they remained at the surface (Garcia-Pichel and Bebout 1996). Ultra-oligotrophic lake and most oceanic seawater are quite transparent to UVR. A detailed re-evaluation of visible and UV radiation penetration in the clearest oceanic waters was presented by Morel et al. (2007). UVA can penetrate to nearly the maximum depth of the euphotic zone (Smith and Baker 1981; Helbling et al. 1994; Prezelin et al. 1994). UVB is attenuated more steeply, but still a low intensity may reach the limit of the euphotic depth. Turbidity, of course, will greatly reduce UVR penetration, but more selectively dissolved organic matter (DOM), particularly humic acids, a general term for coloured organic solutes that are generally derived from vegetation, especially from littoral and terrestrial sources in lakes, ponds, and river backwaters, and from near shore macroalgae in the marine environment, will have the greatest impact. (Vincent and Roy 1993; Scully et al. 1996; Karlsson et al. 2009). In many lakes, particularly during summer stratification when cyanobacterial and algal populations may be vertically stabilized in the metalimnion or upper portion of the hypolimnion, dense populations of cyanobacteria may accumulate. This is conspicuous in eutrophic and mesotrophic lakes in which dim green light zones are dominated by gas-vesiculate filamentous cyanobacteria, red in colour due to a high cell content of phycoerythrin. However, little UVR is likely to reach such depths in these waters. In terrestrial habitats, including shallow water springs and streams, ephemeral slacks, and moist surfaces, two factors locally influence the incident UVR, the orientation of the surface with respect to the solar vertical, and the albedo of the surface. Instantaneous exposure and dose are highest on surfaces oriented orthogonally to the sun’s vertical, and lowest in those in the optical shadow of solids (Fig. 19.1). However, due to the atmospheric (Rayleigh) preferential scatter of the shorter wavelengths, the spectral composition of the diffuse light that penetrates the shade behind solids is highly enriched in UVR (Robinson 1966; Frederick et al. 1989). At the wavelength of 300 nm (UVB), approximately half of the incident radiation is diffuse and half comes directly from the solar disc. Thus, populations thriving on shaded surfaces will receive higher UVR doses relative to the visible than populations on sunny surfaces, but with lower absolute doses of UVR. The implication then is that UVR may play an important role for cyanobacteria exposed to diffuse light. The radiation reflected from the surface may change the absolute and spectral characteristics of the absorbed radiation with respect to the incident radiation. For example, substrates such as white sandstone and carbonaceous materials (e.g. travertine, other limestones, coral sand, and concrete) reflect UVR strongly (Koller 1965; Diffey et al. 1995). In contrast, sedimentary environments (including microbial mats) are characterized by the strong attenuation of both visible and UVR due to absorption and multiple scattering by the matrix and the organisms, so that phototrophic metabolism is restricted to thin (millimeter to centimeter) surface layers (e.g. Castenholz and Garcia-Pichel 2000; Kruschel and Castenholz 1998; Garcia-Pichel et al. 1994). When close to the surface, light-trapping phenomena result in localized irradiance radiation maxima that are higher than the incidence radiation. Typically, then, there is an onset of quasi-exponential attenuation of light below the light-trapping. Thus, in sedimentary habitats, extremely steep gradients of exposure ranging from zones of increased exposure to zones that are refuges from UVR. This condition also applies to endolithic populations in many habitats. Because light-trapping effects are greater at longer wavelengths, and absorption by particulates is most pronounced at shorter wavelengths, the ratio of UVR to visible invariably decreases with depth. A comparison of data from several sedimentary micro-environments showed that the space-averaged UVR dosage rates within the euphotic depth ranged from 15% to 33% of the incident value, a much higher UVR exposure than that in the euphotic zones of water columns (Garcia-Pichel and Bebout 1996).

A recent experiment that placed endo- and epilithic intertidal microorganisms into low orbit space at a height of about 300 km for 10 days that included exposure to vacuum, desiccation, and high intensity UVR resulted in the survival of a coccoid, aggregated cyanobacterium (Olsson-Francis et al. 2010). However, there was no information on the intensity of UVR that actually reached the endolithic cyanobacteria.

Other aspects of biologically effective UVR exposure, such as biological weighing functions are discussed in Castenholz and Garcia-Pichel (2000).

3 Metabolically – Timed Exposure

Cyanobacteria are often the predominant phototrophs in habitats where metabolic activity is restricted due to lack of water or nutrients, freezing or sub-optimal temperatures. Under conditions of restricted or interrupted active metabolism, the impact of exposure to solar radiation may seriously exceed that predicted by simply expressing exposure in terms of total time. Dose modification factors based on the fraction of total time that the organisms are active, can be used to obtain a more relevant metabolically-timed exposure. This may be relatively easy where the time partition is clear but quite complicated if metabolism slows rather than halts. An extreme example may be the surface-dwelling soil cyanobacteria of the Colorado Plateau (e.g. Nostoc, Scytonema, Microcoleus). These populations may experience about 100 h of wetness (active metabolism) during the winter season and additional scattered periods during and after summer rains (Garcia-Pichel and Belnap 1996). Approximately one half of the periods of wetness occur during daytime. However, the populations are exposed to UVR throughout the year. Thus, the UVR damage accumulated during a year’s exposure needs to be rectified by repair and other metabolic processes within the possible 120 h when cells are potentially active. This represents a metabolically-timed dosage of about 70 times the actual incident dose. This metabolically-timed irradiance is likely to be a better predictive factor in the terrestrial habitat of desert crusts, in polar mats, and in alpine situations, where desiccation and/or cold are important factors. However, all of these types of theoretical calculations are nullified if the organisms have an extracellular UVR-protectant, such as the sheath “pigment”, scytonemin, or layers of dust/soil that would prevent much of the UVR from reaching the cell interior.

4 Examples of Cyanobacterial Habitats with High Exposure

The Proterozoic Eon (2.5–0.54 Gyr) was populated by cyanobacteria or their oxygenic ancestors, as demonstrated by micro-fossil and relic chemical evidence as well as indirectly by the early oxygenation of the waters and atmosphere (Knoll 2008). In this geologic period, before the advent of efficient grazing animals, shallow seas bounding the continental or island land masses were inhabited by cyanobacterial ancestors that resulted in the formation of a variety of stromatolites. Today there is a much greater variety of both aquatic and terrestrial habitats and niches than in the early Proterozoic or late Archean, and these usually harbour cyanobacteria. Some are exposed to high UVR.

4.1 Hot Springs

Although hot springs occur today in many locations associated either with volcanic activity (residual or current) or tectonic activity, most terrestrial springs are exposed to full sun because vegetation near them is usually low or absent due to high soil temperatures, and possibly toxic compounds such as arsenite and arsenate. High elevation is another factor that exposes such habitats to high levels of solar irradiance. This was pointed out by Phoenix et al. (2006) who studied hot spring cyanobacterial habitats at over 5,000 m elevation in Bolivia. However, the UVR fluxes measured (perhaps incorrectly) were no higher than those at the major thermal areas of Yellowstone National Park, most of which are close to 2,500 m. The measurements in Bolivia were also quite comparable to midday values in Argentina, some at sea level, but during a low ozone period (Orce and Helbling 1997). In Iceland, many geothermal springs occur only slightly above sea level, but no trees or shading vegetation occurs, and high levels of UVR should be expected. However, the solar angle in summer at over 65°N. reduces total solar intensity, but not necessarily 24 h dosage during the summer months. Many or most hot springs produce shallow runoffs of clear water that flows over microbial mats that are topped by cyanobacteria or simply biofilms of cyanobacteria at the upper end of their thermal range for growth (72–73°C), the highest known temperature for photosynthesis (Fig. 19.2) This limit applies to some areas in the western US and parts of southeastern Asia. However, the upper temperature limit is considerably lower (63–64°C) in other regions (including New Zealand, Iceland and Europe) (see Ward and Castenholz 2000) due to the absence of the highest temperature form of Synechococcus in these and other regions. Metabolic and carotenoid mechanisms for UVR damage repair occur in probably all cyanobacteria (Fig. 19.3) When hot spring runoff reaches average temperatures below about 50–55°C many cyano­bacteria also produce scytonemin, an extracellular sheath UVR-shielding pigment that may actually prevent damage even when the cells are metabolically inactive (Fig. 19.4). The scytonemin-producing cyanobacterial species in Yellowstone (e.g. Calothrix, Scytonema and Nostoc) are those that grow only below ∼45–50°C (Fig. 19.5). It has also been shown that acidic hot springs (pH 0.5–4.0, ∼35–56°C) harbour only one type of phototroph, namely the unicellular members of the Cyanidiales (Rhodophyta). In their natural shallow stream habitats they are significantly inhibited by high solar radiation, particularly UVR (Lehr et al. 2007).

Fig. 19.2

An alkaline hot spring in the Lower Geyser Basin, YNP (“Five Sisters”) in summer showing upper temperature limit (70–73°C) of Synechococcus sp.: yellow cover on left and foreground, rich in carotenoids; Synechococcus sp. also in deep shaded basin, rich in chlorophyll and phycocyanin. Gray area is covered with water (>74°C), originating from spring source in upper left. Dark moist crust along left and right edge consists of various cyanobacteria containing scytonemin (Photo by RWC)

Fig. 19.3

Alkaline White Creek, Lower Geyser Basin, YNP, with a mean temperature at this point of 50–55°C, with green trailing wisps of Mastigocladus (=Fischerella), Leptolyngbya and Synechococcus, the last attached mainly to streamers of Chloroflexus-like anoxygenic bacteria. The rich green of the cyanobacteria in the fast-flowing stream is probably a result of continuous waving motion of the streamers, thus lessening constant exposure to high solar radiation. The orange static film of cyanobacteria fanning down the embankment is rich in carotenoids, probably because of suboptimal temperature and high solar irradiance that results in a high carotenoid to chlorophyll  +  phycocyanin ratio (Photo by RWC)

Fig. 19.4

Absorption spectrum of scytonemin in tetrahydrofuran. The in vivo maximum absorption peak is at 372 nm. The spectrum of a generalized MAA has been added, as well as colour guides to nm ranges (Modified from Proteau et al. 1993. With kind permission from Springer Science + Business Media B.V.)

Fig. 19.5

Alkaline Grand Prismatic Spring in Midway Geyser Basin, YNP. High temperature Synechococcus sp. in distance (greenish at edge of pool). Orange flow in shallow water from ∼60°C to ∼50°C consisting mainly of Leptolyngbya spp. (=Phormidium) and Synechococcus spp. The cells are rich in carotenoids and low in chlorophyll and phycocyanin content. The brown band (<50°C) consists mainly of Calothrix sp. with a high content of scytonemin in the filament sheaths (Photo by RWC)

4.2 Intertidal Marine and Hypersaline Habitats

Intertidal marine habitats close to “normal” seawater salinity (∼31–35 ppt), such as mud flats and salt-water marshes are usually devoid of perennial cyanobacterial mats, probably because of an abundance of invertebrate herbivores and algal and plant competitors. Nevertheless, there are some tidal flats covered by water mainly during spring tides, and these exclude most grazing invertebrates because of desiccation and high salt intolerance as the flats dry out. Some of these flats (e.g. Laguna Guerrero Negro, Baja California Sur, Mexico) are dominated by scytonemin-producing cyanobacteria (Lyngbya cf. aestuarii and Calothrix sp.). These almost monospecific mats inhabit the mid-intertidal (Lyngbya), and uppermost intertidal (Calothrix) and cover vast areas in this location (Javor and Castenholz 1981, 1984). Both these cyanobacteria in nature contain scytonemin in their sheaths (Fig. 19.6). Other intertidal sedimentary habitats, such as the Great Sippewissett Marsh, Massachusetts, contain so much biogenic sulfide in the sediments that many grazers are absent or restricted, and highly exposed and cyanobacterial mats develop mainly in the summer. The benthic mats of shallow, natural or man-made, hypersaline pools and lagoons also provide habitats with high insolation where cyanobacteria usually dominate, at least at salinities above 50–60 ppt, a level that usually excludes grazing herbivores except for harpactipoid copepods, nematodes, and various “protozoa” (Des Marais 1995; Garcia-Pichel et al. 1994; Kruschel and Castenholz 1998). In warm, clear tropical waters, several types of cyanobacteria, including living stromatolite-like cushions, and both filamentous and gelatinous cyanobacterial tufts and epilithic biofilms attached to sediment, rocks, or macro-algae are exposed to high levels of UVR (Castenholz and Garcia-Pichel 2000).

Fig. 19.6

Intertidal flat at Laguna Ojo de Liebre, Baja California Sur, Mexico. The dark area is covered by the scytonemin-rich cyanobacterium, Lyngbya cf. aestuarii during neap tide. This area is covered by seawater of 35–50‰ salinity during periods of spring tides (∼every 2 weeks) (From Castenholz 2009, Mats/microbial, in encyclopedia of microbiology, Fig. 9, p 287. With kind permission of Elsevier, Inc.)

4.3 Benthic Freshwater Habitats

Clear water lakes and streams are common in many areas distant from human populations. These include a large percentage of oligotrophic alpine/subalpine and polar lakes and ponds (Vinebrooke and Leavitt 1996; Vincent 2000). Although many of these habitats are not dominated by cyanobacteria, there are various algae that are typical. However, in ultra-oligotrophic lakes and ponds in polar regions cyanobacterial mats again become predominant, possibly because of the lesser ability of many or most eukaryotic algae to tolerate long- or short-term and frequent freezing and thawing (Vincent et al. 1993, 2004; Tang et al. 1997; Bonilla et al. 2005) and also, in some cases, because of the low herbivore diversity and density. One ultra-oligotrophic lake in the Oregon Cascades (Waldo Lake) that is essentially equivalent to distilled water in terms of solutes, is dominated by benthic populations of cyanobacterial populations of the genera, Stigonema and Scytonema, that have sheaths rich in scytonemin to depths of at least 15 m (Johnson and Castenholz 2000) (Figs. 19.4 and 19.7). These oligotrophic waters are dominated by heterocystous cyanobacteria by virtue of an extreme deficiency of combined nitrogen. Another example of this is the slow-growing giant Nostoc cf. pruniforme, forming scytonemin-containing colonies that form an almost monospecific population in a few cold water spring-fed ponds in southern Oregon that never exceed ∼5°C (Dodds and Castenholz 1988; photo in Castenholz and Garcia-Pichel 2000). Although dominance by cyanobacteria in cold waters would have been unexpected some years ago, Antarctic and Arctic freshwater and saline ponds and lakes are nevertheless dominated by cyanobacterial mats (Vincent 1988; Tang et al. 1997). It was long thought that cyanobacteria were more adapted to warmer waters. It appears, however, that most of the cyanobacterial species in these habitats are simply more tolerant of cold and freezing than most eukaryotic algae, although their optimal temperature for photosynthesis and growth may be considerably higher than in their ambient cold water habitats (Nadeau and Castenholz 2000; Castenholz and Schneider 1993). Heterocystous Nostoc with scytonemin is very common in glacial meltwater streams and ponds in the Antarctic dry valleys (Vincent 1988; Castenholz, unpublished observations).

Fig. 19.7

Stigonema sp. from 1 m depth in ultra-oligotrophic Waldo Lake, Oregon Cascade Mountains: Yellow colour of sheath is caused by scytonemin. Diameter of main axis is ∼7 μm (Photo by RWC)

4.4 Marine and Freshwater Plankton

Although much information is available on the effects of UVR on marine, planktonic diatoms, and various photosynthetic flagellates (Gieskes and Buma 1997; Bouchard et al. 2005; Sobrino et al. 2008; Laurion and Roy 2009), there is little information on the specific effects of UVR on marine cyano-plankton. The filamentous gas-vesiculate Trichodesmium, the principal N₂-fixer of warmer, nitrate-depleted oceanic waters, is subject to the production of high amounts of reactive oxygen species, including oxygen radicals due to the impact of high visible and UV radiation. It produces the highly potent antioxidant, all-trans-ß-carotene that may counter the detrimental effects of reactive oxygen (Kelman et al. 2009). Trichodesmium and some other marine and freshwater planktonic cyanobacteria are known to accumulate large quantities of MAAs (mycosporine-like amino acids) that are, in these cases, intracellular UVR absorbers (Fig. 19.8). However, MAAs apparently do not occur in small unicellular planktonic cyanobacteria (Garcia-Pichel and Appel, unpublished data). Meador et al. (2009) demonstrated UVR-DNA damage in surface dwelling microorganisms over a long latitudinal range (70°N–68°S in the Pacific with some consideration of the picoplanktonic cyanobacteria, Synechococcus and Prochlorococcus. UVR may penetrate to considerable depth in clear oceanic waters; in some cases UVB to over 30 m and UVA in excess of 60 m (Holm-Hansen et al. 1993; Jeffrey et al. 1996; Booth and Morrow 1997; Morel et al. 2007). However, in coastal waters UVR (and especially UVB) may show a rapid attenuation to <1% in less than 0.5 m depth (Nielsen and Ekelund 1995).

Fig. 19.8

Chemical structure of some compounds in cyanobacteria involved in UVR protection, including scytonemin and 5 types of mycosporine-like amino acids. OS-MAA is found in the EPS of Nostoc cf. commune (Ehling-Schulz et al. 1997) (From Fig. 2 in Castenholz and Garcia-Pichel 2000, with other references. With kind permission from Springer Science + Business Media B.V.)

In general, high-altitude lakes are not dominated by cyanobacteria, but although cyanobacteria (Synechococcus sp.) predominated in several lakes of the Tibetan Plateau (∼3,200–4,700 m elevation), a low taxon richness was evident (Xing et al. 2009). In any case, most subalpine and alpine lakes and ponds contain clear water and thus, have high exposures to solar irradiance. Freshwater unicellular cyanobacteria of pico-planktonic size also occur in many of these waters (Weisse 1993; Eguchi et al. 1996; Postius et al. 1996). In many temperate lakes, especially eutrophic types, gas-vacuolate cyanobacteria often predominate (e.g. Aphanizomenon, Anabaena, Planktothrix, Limnothrix, Microcystis) and may rise to the surface during periods of low wind and are destroyed by high solar irradiance. During such a calm period in sunlit Upper Klamath Lake, Oregon , there was there was an almost complete extermination (only phycocyanin remained) of dense Aphanizomenon populations that floated to the surface (Castenholz, unpublished observations).

4.5 Terrestrial Habitats

In exposed terrestrial habitats that are often extreme by virtue of long-term or periodic desiccation, many species of cyanobacteria thrive because of their tolerance to desiccation, but also because of a high tolerance, in many cases, to UVR (Potts 1994). The terrestrial cyanobacterial mats or crusts (often known as biological soil crusts) occur in many of the warm and cold deserts of the earth, where these may form an extensive ground cover when undisturbed by human practices, and sometimes surrounding protective desert shrubs (Garcia-Pichel and Belnap 1996; Mazor et al. 1996). The harder substrates, such as rocks and cliff faces are often covered with thin dark covers of epilithic cyanobacteria (“tintenstriche”) that by virtue of their unprotected location, obviously receive much UVR whether wet or dry. Most of these cyanobacteria are dark in colour because of sheaths or EPS that contain scytonemin. These generally do not occur on the most sun-exposed faces which are often too desiccated to allow any free-living phototrophs, except for lichens, some of which are inhabited by cyanobacterial photobionts that also contain scytonemin (Büdel et al. 1997). The well-known Nostoc flagelliforme of semi-desert regions appears to be insensitive to UVR under both desiccated and rehydrated conditions (Gao and Ye 2007). There is a large literature on endolithic and chasmolithic cyanobacteria within limestone (including travertine), dolomite, sandstone, and other somewhat porous rocks (Norris and Castenholz 2006). Most of these situations involve a narrow band (1–2 mm thick) 1–3 mm below the rock surface. Although only one publication has measured light penetration to the level of the phototroph band (Matthes et al. 2001), it is assumed that the specific depth of the chlorophyll band represents many strategies, such as avoidance of high light and UVR, as well as optimization of the light intensity, moisture retention, and prevention of erosion by wind. Such endolithic populations are known world-wide in hot and cold, deserts (e.g. Nienow and Friedmann 1993) as well as in rocks exposed to seasonal desiccation, wetting, and freezing, such as in travertine in Yellowstone National Park and the Rocky Mountains (Norris and Castenholz 2006; Walker and Pace 2007). Some isolates of the desiccation tolerant Chroococcidiopsis (a unicellular cyanobacterium) are also capable of tolerating up to 5 kGy of ionizing radiation (X-rays), while desiccated, and may be comparable to the tolerance and DNA repair mechanisms of Deinococcus, a heterotrophic bacterium well-known for this ability (Billi et al. 2000).

5 Negative Effects of UVR and Physiological and Biochemical Strategies of Counterbalance

The targets of damage by UVR with respect to metabolism, DNA, development, and behaviour for cyanobacteria were summarized in Table 1 in Castenholz and Garcia-Pichel (2000), while studies on the effects of UVR on the morphology of filamentous cyanobacteria have been reported by Wu et al. (2005) and Gao et al. (2008). Summaries of the effects of UVR on other organisms were summarized by Jägger (1985), Häder (2001), and Hockberger (2002). The number of targets and detrimental effects mount exponentially with shortening wavelengths (Jägger 1985; Whitehead et al. 2000). However, under standard conditions of environmental exposure, UVC is no longer environmentally relevant, because of complete atmospheric absorption. Since UVC and UVB radiation have the most lethal effects, and because these spectral regions reached the Earth’s surface without atmospheric interference only in the Archean and early Proterozoic, they will be discussed first.

5.1 UVC and UVB

Germicidal lamps that emit in the UVC range have been used in many experiments with bacteria, viruses, DNA and proteins (see Jägger 1985). Damage is mainly through direct absorption by DNA. DNA absorbs in three spectral regions: I (with a peak at 260–264 nm), II (with a peak at 192 nm), and III (below 125 nm). Spectral regions II and III would only have relevance in outer space (see Zalar et al. 2007a, b; Olsson-Francis et al. 2010). The effect of UVC radiation (254 nm max) negatively affected short-term photosynthetic rate and survival in Agmenellum quadruplicatum (now Synechococcus sp. PCC 73109)(Van Baalen 1968). However, the damage to DNA was cured by photoreactivation at wavelengths from ∼395 to 450 nm (Van Baalen and O’Donnell 1972).

Chloroflexus aurantiacus, a primarily photoheterotrophic anoxygenic bacterium, showed relatively high UVC-tolerance which may have had relevance during the Archean when the Chloroflexi may have evolved (Pierson et al. 1993). Results of continuous UVC radiation within the possible Archean limits, resulted in yields similar to those of controls under anoxic conditions at 0.01 Wm⁻² UVC, an intensity that severely inhibited E. coli. This exposure could not occur indefinitely without DNA damage and deleterious accumulation of mutations. Pierson et al. (1993) suggested that the Archean sediments may have carried a load of ferric and ferrous ions that blocked UV wavelengths, but still passed the longer visible wavelengths that support photosynthesis.

Cyanobacteria that inhabit exposed, near-surface habitats usually synthesize the UVR-absorbing pigment, scytonemin, in their sheaths or EPS. This compound has major absorption maxima in the UVC and UVA spectral regions, but also ample absorption in the UVB (Proteau et al. 1993) (Fig. 19.4). During the early Precambrian an extracellular compound that absorbed UVC may have been of considerable benefit. O₂ is a requirement for scytonemin synthesis, but internal O₂ release as a product of oxygenic photosynthesis must have occurred long before the general oxygenation of the waters. Although scytonemin functions primarily today as a UVA and UVB screen, Dillon and Castenholz (1999) found that it afforded considerable protection against UVC photosynthetic damage in scytonemin-containing Calothrix and Chroococcidiopsis cultures when 0.5–1.0 Wm⁻² UVC radiation was added to natural solar irradiance. Many intracellular and extracellular compounds absorb in the UVC region, however, the presence of scytonemin in the external sheath provided protection that the glycan sheath without scytonemin did not.

UVB is less potent energetically than UVC, but the type of damage is similar. UVB includes the wavelengths that are most harmful to humans by causing sunburn and skin cancer (e.g. melanoma). The recent increase in UVB in some regions is due to periodic stratospheric losses of ozone. The UVB (280–315/320 nm) intensity reaching the earth’s surface is sufficient to cause damage to many cellular components in micro-and macro-organisms, including DNA as a direct target. The result is the formation of various photoproducts (e.g., cyclobutane-pyrimidine dimers, pyrimidine (6–4) pyrimidone products, and cytosine products) (Ravanat et al. 2001). Purine bases are also photoreactive. Other impairments include accelerated degradation of photosystem II proteins (e.g. D1/D2) and destruction of the light-harvesting phycobiliproteins, since these have a small absorbance peak in the UVB (Lao and Glazer 1996; Wingard et al. 1997). Also, various activities such as synthesis of chlorophyll, energy transfer in light harvesting, nitrogen fixation, RUBISCO and ATP synthase activities, nitrate and ammonium uptake, nitrogen fixation, motility and photoorientation, and cell differentiation are negatively affected by UVB radiation (Häder 1984; Sass et al. 1997; Choi et al. 1999; Castenholz and Garcia-Pichel 2000). UVB damage may be more severe under nutrient limitation. Damage to light-harvesting complexes in cyanobacteria may exceed the damage to DNA in some cases (Lao and Glazer 1996). D1/D2 proteins of PS II reaction center may be destroyed not only directly by UVR but also indirectly by reactive oxygen produced by high intensity violet/blue light or UV radiation. Rapid de novo synthesis of D1/D2 proteins is an essential part of the repair process (Sass et al. 1997). Several proteins may be photooxidized by UVB radiation since tryptophan, tyrosine, phenylalanine and histidine absorb in the 290–315 nm range (MacDonald et al. 2003). In one case, photooxidative inhibition and damage by moderate UVB was reversed after about 2 weeks of continuous exposure in a species of Anabaena (He et al. 2002). MacDonald et al. (2003) found that a naturally high ratio of visible light to UVB resulted in a greater tolerance to UVB in Synechococcus PCC 7942.

The exposure of the same unicellular cyanobacterium (Anacystis nidulans R-2  =  Synechococcus PC 7942) to UVB and UVA resulted in the production of several “UV-shock” proteins, some of which may function as enzymes that scavenge reactive oxygen molecules (Shibata et al. 1991). Earlier, the induction of the synthesis of a few specific proteins in thermophilic Phormidium cf. laminosum was reported in response to inhibitory UV radiation (Nicholson et al. 1987). Another study, again using Synechococcus PCC 7942, has demonstrated that an ATP-dependent Clp protease is essential for acclimation to UVB, and results in the rise of a modified D1 PS II protein (D1:2) that is more resistant to UVB stress (Porankiewicz et al. 1998). The psbA genes (psbA2 and psbA3) that code for a more UVB tolerant, PS II D1 protein may be activated in Synechocystis PCC 6803 under even low levels of UVB exposure (Máté et al. 1998; Campbell et al. 1998). After 2 h exposure of Synechocystis PCC 6803 to 20 μE m⁻² s⁻¹ UVB the levels of expression of 55 genes rose more than twofold and those of 44 genes fell more than twofold (Los et al. 2008). With whole genome profiling of some phototrophic prokaryotes now possible, transcripts from genes involved in various light (or UVR) intensity transitions can be probed with full genome DNA microarrays (see Gill et al. 2002; Los et al. 2008).

UVB affects shallow benthic phototrophs, (e.g. in intertidal flats, shallow lagoons and lakes), also phytoplankton, especially in clear oceanic waters where harmful intensities may reach over 20 m (much less in turbid or waters rich in coloured dissolved organic matter), as well as exposed terrestrial crusts and epi- and endo-lithic populations of cyanobacteria and microalgae. There is a particularly large literature on the effects of UVR (mainly UVB) on phytoplankton which often includes diatoms, dinoflagellates, and other micro-algae, but there is little specific information on the well described cyanobacterial picoplankton, such as Synechococcus, Prochlorococcus and the less well-known Crocosphaera. Effects of UVB in lakes and the sea are particularly difficult to assess because of vertical and horizontal mixing and, thus, like other field studies, requires extrapolation of short term daylight results to full days, weeks and seasons (Day and Neale 2002). Nevertheless, work in the Southern Ocean by various investigators have estimated that phytoplankton productivity has been reduced by ∼0.2–2.4% as a result of increased UVB that was presumably due to periodic ozone depletion. However, estimates of this type differ greatly (see Day and Neale 2002 for references). As with UVC, some of the negative effects on DNA (formation of cyclobutane pyrimidine dimers and 6–4 photoproducts that block DNA and RNA polymerases) may be repaired by photoreactivation, utilizing UVA and/or violet/blue wavelengths that are absorbed by chromophores of photolyase, a DNA-bound enzyme that restores the intact bases, and also by nucleotide excision repair (Jägger 1985; Sancar 1994, 1996). Increases in temperature have been shown to enhance the DNA photoreactivation repair rates in algae (Pakker et al. 2000).

In an extensive review, Bailey and Grossman (2008) have discussed the evidence that a photoprotective method of dissipating excess photon (quantum) energy by the light-harvesting complex is by down-regulating the transfer of this excitation to the photosynthetic reaction centres. This is termed non-photochemical quenching (NPQ), and the authors have concluded that this process also occurs in cyanobacteria although only previously known in algae and plants. NPQ in cyanobacteria appears to involve the absorption of blue light by a carotenoid-binding protein and probably involves quenching in the phycobilisome core. Although these reactions are known only for the short wavelength visible spectrum, it is possible that excess UVR absorption may be dissipated by a NPQ quenching reaction as well.

Sytonemin has substantial absorbance in the UVB spectral region. However, the degree of protection afforded specifically by scytonemin has not been tested in this spectral region (Fig. 19.4). It may, however, be insufficient, as most scytonemin synthesizers complement their sunscreen capabilities with mycosporine-like amino acids (MAAs), Mycosporine-like amino acid derivatives (MAAs) are known in very many cyanobacteria (Garcia-Pichel and Castenholz 1993), numerous microalgae, and marine zooplankton (Castenholz and Garcia-Pichel 2000; Shick and Dunlap 2002). MAAs are low molecular weight, water soluble compounds that are condensation derivatives of a cyclohexenone ring and amino acid (or imino alcohol residues), but are acquired secondarily by invertebrates through consumption of primary producers (Fig. 19.8) Radiotracer experiments have shown that in cyanobacteria the core cyclohexenone is derived from the shikimate pathway, and amino acids are condensed onto it directly as precursors (Portwich and Garcia-Pichel 2003). Gadusols, that are putative intermediates in the shikimate pathway of MAA synthesis, may have been early absorbers of UVC/B radiation and may also have served as strong antioxidants (Shick and Dunlap 2002). The absorption peaks of MAAs range from 310 to 360 nm (i.e. from UVB into the UVA) (Fig. 19.4). Short wavelength MAAs are monosubstituted and direct precursors of more derived, bi-substituted, long wavelength MAAs (Portwich and Garcia-Pichel 2003) In most cyanobacteria, they are synthesized when exposed to UVB. However, in all but one of the cyanobacteria examined (Nostoc), these compounds occur in the cytoplasm and could serve as alternative targets for perhaps only about 10–30% of UVB photons penetrating a cell (Garcia-Pichel and Castenholz 1993, Garcia-Pichel et al. 1993). In cells of small diameter (<10 μm), the beneficial effect would be negligible (Garcia-Pichel 1994). MAAs appear to be important in some way in UVB protection, since they are so tightly associated with UVB exposure. Perhaps, MAAs have an unknown function that is merely associated with UVB exposure (Shick and Dunlap 2002). It is apparent that intracellular MAAs cannot provide complete protection from UVB/A radiation. Some species of cyanobacteria apparently do not synthesize MAAs or scytonemin, and therefore utilize only metabolic repair mechanisms for UVB damage (Quesada and Vincent 1997). However, the employment of migratory escape motility in some may avoid UVR exposure altogether (see behavioral strategies later). In terrestrial Nostoc cf. commune, however, MAAs are bound to oligosaccharides in the inner external glycan sheath (EPS), with scytonemin in the outer portion (Ehling-Schulz et al. 1997; Ehling-Schulz and Scherer 1999). In this case, then, there is no doubt that MAAs, together with scytonemin, provide a nearly perfect spectral shield against UVB as well as UVA (Fig. 19.8). The UV energy absorbed must be dissipated, which is no problem within the sheath, but intracellular absorption may result in transfer of energy to sensitive molecules. Many cyanobacteria synthesize one or more MAAs even if they have no extracellular sheath or EPS. However, it is probable that most or all sheathed scytonemin producers are capable of making MAAs either constitutively or by UVB induction (Garcia-Pichel and Castenholz 1993). Various MAAs are induced through the absorption of UVB radiation (Ehling-Schulz et al. 1997; Sinha et al. 2001). However, Portwich and Garcia-Pichel (1999) have shown that either UVB or osmotic stress induced MAA synthesis in a strain of Chlorogloeopsis. The photoreceptor involved in the induction of MAA synthesis is a pterin with a distinct absorption peak at 310 nm in one cyanobacterial strain (Portwich and Garcia-Pichel 2000). Biosynthetic pathways of MAA synthesis have now been reported (Balskus and Walsh 2010; Gao and Garcia-Pichel 2011).

High levels of MAAs accumulate in cyanobacterial cells of hypersaline environments (Oren 1997), suggesting they could function as compatible solutes in osmoprotection; but this role, while suggested by the author, can only be minor in comparison to that of known compatible solutes in the same cells, the concentration of which may be very much higher.

MAAs and scytonemin are absent in many cyanobacteria (e.g. Fischerella [=  Mastigocladus], Synechococcus spp. and many Leptolyngbya [=  Phormidium]) types from hot springs, as well as in vertically migrating types in soft microbial mats, and from many others in the Culture Collection of Microorganisms from Extreme Environments (CCMEE) at the University of Oregon (Castenholz and Garcia-Pichel 2000 and Castenholz, unpublished data) and in the Pasteur Culture Collection. It is these that must have other means of tolerating UVR or avoiding it. In the case of exposure to full solar irradiance, without any possibility of escape because of various features of the habitat, it is apparent that only efficient repair mechanisms can make up for damage done during daylight hours of clear skies with summer insolation at temperate latitudes (Miller et al. 1998). Most repairs probably take place during darkness or during periods of low light as in morning or before dusk. Cyanobacteria are known to have a very efficient daytime photoreactivation system as compared to Escherichia coli (Castenholz and Garcia-Pichel 2000). There is, however, little known of UVR effects and tolerance capability outside of the laboratory, with the exception of papers on cyanobacteria in hot springs (Miller et al. 1998; Dillon et al. 2003; Norris et al. 2002). Although some dinoflagellates have the ability to swim vertically out of the damaging intensities of UVB, this is not true of the marine planktonic cyanobacteria that depend on vertical circulation. Gas-vesiculate cyanobacteria in oceanic waters of the tropics and sub-tropics (e.g. Trichodesmium) may circulate to great depths, but in very calm conditions may rise to near the surface and become exposed to detrimental UVR.

5.2 UVA

UVA may have both direct and indirect effects on cyanobacteria and algae. NAD(P)H (component cofactor in energy metabolism of anoxygenic and oxygenic phototrophs) and other essential nucleotides with absorption maxima at ∼340 nm may be regarded as direct targets of UVA. UVA also has some of the same effects as UVB but with less consequence. Pterines can be additional targets or sensitizers. UVA radiation affects many phenomena negatively, but the exact mechanism is not often known. UVA photooxidative damage primarily involves the guanine components of DNA as a result of singlet oxygen generation (Ravanat et al. 2001), which necessitates a photosensitizing compound. Photosynthesis, growth rate and chlorophyll synthesis are negatively affected by UVA. However, the main effects may be through the production of reactive oxygen that may also be produced by the violet/blue wavelengths if the intensity is sufficiently high. The products are singlet oxygen (¹O₂), peroxy radicals, and free radical reactions such as the production of OH• (see Asada and Takahashi 1987). Targets of oxidative damage include unsaturated lipids such as phospholipids (Girotti 2001).

UVA (with UVB excluded) resulted in decreases in photosynthetic rate in various cyanobacteria, such as thermophilic Synechococcus (Miller et al. 1998), marine Oscillatoria and Spirulina (Kruschel and Castenholz 1998), Antarctic Oscillatoria (Nadeau et al. 1999) and thermophilic Mastigocladus (Fischerella) (Castenholz, unpublished data) all in outdoor experiments in which the effects of visible irradiance alone, vis+UVA, and vis+UVA and B were compared. The specific targets of these UVA effects have not been investigated.

Photosynthetic prokaryotes may prevent or alleviate the various detrimental effects of UVA (320–400 nm) and short wavelength visible radiation (400–∼500 nm) by synthesizing or maintaining a high carotenoid content which may act as an effective antioxidant or “quencher” of reactive oxygen. This may inhibit lipid peroxidation, and thus stabilize membranes (Britton 1995; Niyogi 1999). A high photon flux in excess of what can be used in the photosynthetic reaction may still be absorbed by tetrapyrroles (e.g. chlorophylls and phycobilins) and will result at least in triplet chlorophyll (³Chl) and resultant ¹O₂ production. Specific carotenoids (e.g. myxoxanthophyll, echinenone, zeaxanthin) may be effective through the thermal dissipation of excitation energy of chlorophyll and singlet oxygen. Recent evidence shows that singlet oxygen may be quenched by carotenoid-cyclodextrin complexes (Kanofsky and Sima 2009). It is probable that a high ratio of effective carotenoids to photosensitizer (i.e. tetrapyrroles) may be extremely important, particularly at suboptimal temperatures when repair and synthetic processes are slow (see Castenholz and Garcia-Pichel 2000). The protective xanthophyll cycle occurs in eukaryotes in vivo, but not in prokaryotic phototrophs (Josue and Frank 2002). Zeaxanthin alone, however, may be sufficient for some degree of photoprotection in cyanobacteria (Niyogi 1999). One of the most conspicuous aspects of high light intensity regimes in natural communities of photosynthetic prokaryotes (e.g. hot spring mats) is the yellow or orange colouration in summer in regions of high light intensity with a very high ratio of carotenoids to chlorophyll and phycobilins (Norris et al. 2002; Vincent et al. 1993). However, the effect of carotenoids as a UVR screen is minimal, since almost all of carotenoid absorption is in the violet/blue/green region of the visible spectrum in prokaryotes. Carotenoids, will absorb these wavelengths and be of great benefit directly under high solar intensity. In some cases certain carotenoids may function mainly as major or partial light harvesting complexes, although this is the case mainly in anoxygenic phototrophic bacteria and various algae (e.g. diatoms, chrysomonads, dinoflagellates)(Castenholz and Garcia-Pichel 2000).

Superoxide radical anions are also produced with high light intensity. For example, increased synthesis of Mn SOD above constitutive levels of Fe SOD may occur in cyanobacteria (Castenholz and Garcia-Pichel 2000). H₂O₂, a product of SOD activity, is considered to be relatively harmless compared to superoxide. In Synechocystis PCC 6803, peroxide decomposition is catalyzed by catalase-peroxidase and a thiol-specific peroxidase (Tichy and Vermaas 1999). Tocopherols, ascorbates, and glutathione are also used as peroxide detoxifying compounds (Niyogi 1999).

An important preventative and passive method of protection from UVA results from the presence of scytonemin in the sheath or EPS of cyanobacteria, but this does not apply to all cyanobacteria. Only those that possess some type of EPS and are commonly exposed to UVR (even low intensity UVR) synthesize scytonemin. This includes numerous surface layers of microbial mats in flats infrequently covered by tidal waters (Cockell and Rothschild 1999; Fleming et al. 2007), lower temperature (<∼50°C) hot spring mats (Brenowitz and Castenholz 1997; Dillon et al. 2003), terrestrial mats and crusts (Garcia-Pichel and Belnap 1996), shallow and clear oligotrophic fresh waters (Johnson and Castenholz 2000), cyanolichens (Büdel et al. 1997) and many other exposed habitats (Garcia-Pichel and Castenholz 1991; Castenholz and Garcia-Pichel 2000). Scytonemin, as mentioned earlier, has major absorption peaks at ∼250 and 370–372 nm (in vivo). It is a unique dimeric indole alkaloid with a molecular weight of 544 (Proteau et al. 1993). In mature surface filaments of cyanobacteria scytonemin-rich sheaths may screen out >95% of the UVA and a substantial portion of UVB (see above). This passive method of preventing or slowing the inhibition caused by UVA radiation apparently results in great benefits in terms of fitness and survival, particularly in habitats where desiccation or suboptimal growth conditions prevail (Garcia-Pichel et al. 1992; Brenowitz and Castenholz 1997; Dillon et al. 2003; Dillon and Castenholz 2003).

The synthesis of scytonemin is initiated and sustained by exposure to UVA although violet/blue wavelengths have some effect (Garcia-Pichel and Castenholz 1991), but some rock-inhabiting cyanobacteria synthesize a small content of scytonemin constitutively (Castenholz, unpublished data). Stresses such as increased temperature and photooxidative conditions, in conjunction with UVA, caused a small increase in scytonemin production in one species of Chroococcidiopsis (Dillon et al. 2002). In addition, a moderate osmotic stress, without UVR or blue light, resulted in some scytonemin synthesis. Periodic desiccation in addition to UVA exposure also increased scytonemin content (Fleming and Castenholz 2007). It has been shown that the content of scytonemin per unit area in intertidal Lyngbya cf. aestuarii increased during the higher solar irradiance period of summer (Karsten et al. 1998). Another stress that promoted the synthesis of scytonemin is diazotrophic growth, a condition that could be considered a stress because of the high energy requirement for N₂-fixation (Fleming and Castenholz 2008). In this case, Nostoc ATCC 29133 produced scytonemin under UVA radiation only when no combined nitrogen was available.

The genetic analyses of a scytonemin-less Nostoc punctiforme ATCC 29133 mutant (Soule et al. 2007), have opened the door in recent years to the study of the molecular basis of scytonemin biology. Much has been accomplished in this respect in a short time. The mutant implicated a genomic region containing 18 contiguous genes in the synthesis of scytonemin. Similar regions have been since found by comparative genomics or by direct sequencing in a variety of other cyanobacteria (Sorrels et al. 2009; Soule et al. 2009a). Homologous regions in non-heterocystous strains (Lyngbya and Cyanothece) contain 5 additional genes in the cluster (Soule et al. 2009a). These are also conserved in Nostoc and other heterocystous cyanobacteria, where they are non-contiguous. Most of the genes in the upstream region of the Nostoc cluster, with the exception of scyA and scyB that code for novel proteins without an easily predictable function, but a transposon inserted in scyD effectively eliminated scytonemin biosynthesis under otherwise inducing conditions. Balskus and Walsh (2008) could subsequently show that the protein coded for in one of these genes (scyA) catalyzed the condensation of phenol- and indole-pyruvate moities, a fact that is in agreement with the expected scytonemin synthetic pathway, and with the pyruvate-condensing activity predicted for scyA on the basis of sequence similarity. The functions of most of the other upstream genes have not been clarified, but tentative assignments on the basis of comparative genomics have been carried out for some (Soule et al. 2009a). Downstream in the cluster, one finds redundant orthologs of genes coding for enzymes in the aromatic amino acid pathway. Those present suffice to lead to p-hydroxyphenyl-pyruvate from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found downstream as well. Thus, it seems that the downstream region is dedicated to the delivery of monomeric blocks for scytonemin synthesis. Additionally, two genes immediately upstream from the scytonemin-associated cluster likely encode a two-component sensor kinase and response regulator, respectively. These also are highly conserved in sequence and location among cyanobacterial strains that possess the scytonemin cluster, and may be involved in regulating the expression of the scytonemin-related genes. As expected the expression of all genes in the cluster is positively regulated by exposure to UVA, and transcription proceeds in an operon-like fashion as a single transcript (Soule et al. 2009b). In recent years, much has been learned about the biosynthesis of scytonemin, although it is uncertain in what form it is transported or assembled in the sheath or EPS. Since scyD, scyE, and scyF, have export signal domains, and others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell, and that monomer synthesis and initial condensation are cytoplasmic. Later reactions are predicted to be periplasmic (Soule et al. 2009a) (Fig. 19.9).

Fig. 19.9

Working model of scytonemin biosynthesis in Nostoc punctiforme based on genomic and enzymological analyses:UVA is absorbed and activates the genes in the scytonemin operon (b), the gene products of which catalyze the sequence of reactions depicted in (a), likely in a compartimentalized fashion. The genes in purple color constitute redundant orthologs of key shikimic acid and aromatic amino acid biosynthetic pathways. (From Gao and Garcia-Pichel 2011, Fig. 3, p 795. Courtesy of Nature Publishing Company)

As an aside, it appears that an unknown compound is present in many marine and freshwater green macro- and micro-algae that acts an UVA and UVB shield with a relatively high protective efficiency (Pescheck et al. 2010). In the case of green freshwater micro-algae there appear to be macromolecules (possibly sporopollenin-like compounds, i.e. algenans) bound to the cell walls.

6 Behavioural Methods of Escaping UVR Exposure

Although much is known about vertical movements and regulation of buoyant or semi-buoyant gas-vacuolate cyanobacteria, and anoxygenic phototrophic bacteria in the water column of lakes (Walsby 1994), work that focuses on the effects of the UV portion of the spectrum is lacking.

Gliding filamentous cyanobacteria (as well as a few species of unicellular forms) often move vertically in soft microbial mats and sediments in response to high intensity visible light and UVR (for a summary, see Castenholz and Garcia-Pichel 2000). The downward movement of filamentous cyanobacteria (0.4–>1 mm) in dense microbial mats has been shown to be a response to high solar irradiance, particularly UVR, both UVB and UVA in temperate habitats, hot springs, and cold Antarctic ponds (Garcia-Pichel et al. 1994; Ramsing and Prufert-Bebout 1994; Bebout and Garcia-Pichel 1995; Kruschel and Castenholz 1998; Richardson and Castenholz 1987; Nadeau et al. 1999) (Fig. 19.10). In general, the return towards or to the mat surface occurred in darkness or in dim daylight until UVR or high visible light intensities again appear to stop the upward movement (Garcia-Pichel et al. 1994; Kruschel and Castenholz 1998). In two cases it has been shown that failure to migrate downward from the surface during periods of high solar irradiance (including UVR) would have resulted in extreme inhibition of photosynthesis or death (Garcia-Pichel et al. 1994; Kruschel and Castenholz 1998). Filamentous and unicellular cyanobacteria have also been shown to move vertically in soft hot spring mats, also as a response to high light intensity, but UVR was not specifically taken into account (Castenholz 1968; Richardson and Castenholz 1987; Ramsing et al. 1997). In almost all of the cases cited, the cyanobacteria involved did not possess MAAs nor did they have the ability to withstand large doses of UVR. Thus, escape was essentially the only tactic for survival. Garcia-Pichel et al. (1994) have also shown in one case, by the use of micro light sensors, that the gradual descent of Spirulina and Oscillatoria to ∼0.5 mm and later the ascent in a soft hypersaline mat would result in maximum photosynthetic rates during most daylight hours. Thus, the daytime descent may not be simply an escape but a finely regulated optimization of light available for photosynthesis. These cyanobacteria also retained high cellular contents of chlorophyll a and phycocyanin. Retaining this condition would be distinctly advantageous during overcast periods of low light intensity and during early morning and late afternoon when these cyanobacteria would be at the mat surface and able to absorb the low photon flux more efficiently for photosynthetic activity. A high content of chlorophyll and/or phycocyanin could also become a detriment and act as photosensitizers if the cells were suddenly exposed to high solar irradiance. In more sedentary cyanobacteria the synthesis of these pigments are regulated downward by stopping synthesis or by regulated degradation, thus increasing the ratio of carotenoids to photosensitizers, allowing an increased tolerance to photo-oxidative stress.

Fig. 19.10

Upward migration of Oscillatoria cf. priestleyi in hypersaline “Salt Pond” (Bratina Island area, Antarctica), under the lowest light intensity with filter removed after 4 h of treatment (upper right dark area). The intensity was ∼5–8 Wm⁻² of PAR with no detectable UVR, and the temperature 6–8°C. The same result occurred under darkness, but not under ∼12–20 Wm⁻² (next filter to left) where UVA was barely measurable, or under full solar radiation (230–360 Wm⁻²) last ring to left. A more intricate series of experiments (also in “Salt Pond”) are described by Nadeau et al. (1999). The rate of movement was about 50% slower than that observed for motile cyanobacteria in temperate mats in Baja California (Kruschel and Castenholz 1998). Oscillatoria cf. priestleyi (∼7 μm trichome diameter) is shown in the lower panel (Photos by RWC)

7 Effects of Decrease of UVR on Phototrophic Communities

Bothwell et al. (1993, 1994) studied the effects of 3–5 weeks of UVR applied to a lotic community that colonized an artificial substrate. As a result of enhanced UVB a greater biomass of diatoms accumulated than in the control. However, chironomid larvae that normally graze on diatoms were more sensitive than the diatoms to UVB. Since these were indoor experiments the +UV treatment may be closer to natural field conditions than those of the controls. Epiphytic cyanobacterial mats from tropical mangrove communities were subjected to +UV and -UV treatments for 27 days with laboratory UVB+UVA+PAR, UVA+PAR, or PAR only. Large differences in species arrangement and abundance occurred (Sheridan 2001). With UVA and UVB the top layer of scytonemin-containing, diazotrophic Nostoc cf. commune and Scytonema sp. were maintained as in the natural mat. When UVR was excluded, the Nostoc was overrun or overgrown by a less UV-tolerant species of Phormidium which was not capable of diazotrophy. As a result, overall N₂-fixation decreased greatly.

Two alkaline geothermal streams in Yellowstone National Park (40–47°C) were covered for 1–3 months with filters that excluded or transmitted UVR (Norris et al. 2002). There were no apparent cyanobacterial community composition changes during the summer with or without high or low UVR, as assessed by DGGE (denaturing gradient gel electrophoresis). Although the cyanobacterial composition of these communities was apparently stable, surface layers of cyanobacteria protected by filters from UV radiation were not as competent photosynthetically as those that had been maintained under UVR. This decrease in competence was expressed as a temporary loss of the ability to perform at a maximum rate under full solar irradiance (including UVR). Recovery occurred within a week under full irradiance.

8 Conclusions

UV radiation effects on microbial populations have been studied in recent years with increasing frequency and with refined molecular methodology. One reason is the awareness of decreasing levels of ozone in the stratosphere in some regions that result in increased UVB flux. Microbial populations may exhibit a more immediate and measurable sensitivity to small increases in UVR than larger macrophytes and metazoans that may require weeks, months, or years to show UVR effects. Some cyanobacteria, representing descendants of the oldest oxygenic inhabitants of the planet, have evolved many methods (or strategies) for coping with present levels of UVR (Table 19.1).

Table 19.1 Multiple methods of tolerating UV radiation by cyanobacteria (From Castenholz 2004, Table 1, p 457. With kind permission from Springer Science + Business Media B.V.)

Since cyanobacteria have invaded (or remain as relics) in a large number of extreme environments, including shallow water and terrestrial surfaces, they currently must often cope with high solar irradiance in which UVR can be the most detrimental factor. They have done so by evolving sunscreen pigments that envelope the cells and work even when cells are at rest, and by synthesizing other compounds such as mycosporine-like amino acids (the true value of which is still not completely evaluated). Also, they have probably evolved several efficient methods of dissipating excess photon energy, and by using regulated systems for repair of damaged DNA and for replacement of UV damaged compounds, and by using directed, “phototactic” gliding motility in soft microbial mats or sediments for escaping the daytime high intensities of solar irradiance.