To determine the environmental effect of stratospheric ozone depletion, research has focused on the responses of marine microorganisms to ultraviolet radiation (UVR). The majority of this research has focused on the base of marine food chains, phytoplankton, while substantially fewer studies have examined bacteria. Bacteria mediate geochemical cycles that support the activity of the entire marine food chain. An evaluation of the effect of UVR on bacteria will complement work that has been conducted on phytoplankton and significantly contribute to our understanding of the control UVR exerts on plankton community dynamics. Our studies have focused on two processes; the formation of DNA photoproducts (cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers) and induction of damage repair mechanisms determined by measurements of recA transcription and translation. UVB (280-320 nm) survival curves and photoproduct dose response curves have been determined for two marine bacteria, Vibrio natriegens and Pseudomonas stutzeri. V. natriegens is significantly more sensitive to UVB than P. stutzeri  and higher photoproduct formation per UV dose was observed in V. natriegens. Gulf of Mexico waters have been used for studies of indigenous bacterioplankton populations. Diel patterns have been observed in RecA expression and levels of RecA have been found to be higher in surface waters than in deeper waters. Preliminary data suggests that solar radiation does cause photoproduct formation in surface water bacterial populations. We are currently examining diel formation and repair of photoproducts in surface waters and photoproduct formation in waters collected at various depths. Future experiments will investigate the relationships between photodamage and RecA expression in indigenous bacterioplankton populations.

Key words: Ultraviolet radiation, DNA damage, cyclobutane dimers, marine bacteria

There is now strong evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern has been the annual pattern of ozone depletion over Antarctica and the Southern Ocean where ozone levels have declined as much as 74% compared to pre-ozone hole events. Reduction of ozone concentration selectively limits stratospheric adsorption of UV-B (290-320 nm) radiation, resulting in higher irradiance on the earth's surface. As a result, the impact of increased UV-B due to ozone depletion upon phytoplankton and primary production has received extensive interest. The potential effect of UV penetration in the marine environment has been demonstrated using biologic ultraviolet dosimeters designed to indicate the amount of biologically relevant UV reaching either surface or specific oceanic depths (Karentz and Lutze, 1990; Regan et al., 1992) or by in situ incubations for primary production (Smith et al., 1992). Depending on location, potential UVR effects have been measured to 5 to 10 meters depths.

During the past fifteen years, the importance of bacteria in oceanic processes has become widely recognized. Bacteria have been found to account for up to 90% of the cellular DNA in oceanic environments (Paul et al., 1985; Boehme et al., 1993; Fuhrman et al., 1989) and their role in elemental and nutrient cycling has received extensive study (e.g. Falkowski and Woodham, 1992). Bacteria have been found to play vital roles in carbon "repackaging" providing significant amount of organic material to higher organisms. This "microbial loop" has been estimated to cycle up to 50% or more of primary production (Azam et al., 1983). The effects of UVR on bacterioplankton in aquatic ecosystems, however, has received much less attention than has UVR effects on primary producers. The available evidence suggests that UVR may impose a chronic stress on marine bacteria (Sieracki and Sieburth, 1986; Herndl et al., 1993).

Direct biological effects of UVR result from absorption of specific wavelengths by specific macromolecules and the dissipation of the absorbed energy via photochemical reactions (Vincent and Roy, 1993). The major lethal and mutagenic effects of UV radiation to marine organisms results from damage to DNA. The type and extent of damage to DNA is a function of the wavelength and intensity of the exposure. UV-A generally causes indirect damage to DNA through the formation of chemical intermediates such as oxygen and hydroxyl radicals which interact with DNA to form strand breaks, alkali labile sites and DNA protein crosslinks (Peak and Peak, 1989). Conversely, adsorption of energy from UV-B induces direct damage to DNA. The two major lesions induced by UV-B radiation are the cyclobutane pyrimidine dimer and the pyrimidine-pyrimidone (6-4) photoproduct. These photoproducts may be detected and quantified using specific radioimmunoassays (Mitchell et al., 1985). A lesion that inhibits replication or expression of essential genes is cytotoxic. A lesion may also cause mutations which may have no net effect because the mutation is in a non-transcribed region of DNA or does not alter protein function. Finally, mutations may also be either harmful (i.e. reduced fitness) or may actually be benefitial (i.e. improved fitness). Tolerance to DNA damage is determined, in large part, by two primary repair mechanisms. Photoenzymatic repair (photoreactivation) reverses cyclobutane dimers in DNA by the combined enzyme activity of photolyase and visible light. Nucleotide excision repair is more complex, requiring damage recognition, incision of the DNA backbone at or near the site of the lesion and the concomitant excision and resynthesis of the DNA around the damaged site, and finally, ligation of the single strand nick after DNA polymerase detaches.

While it is now apparent that UV-B inhibits primary production (Weiler and Penhale, 1994), the need to address UVR impact by means other than the effect on phytoplankton has been recently discussed (Bothwell et al., 1994; Culotta, 1994). Because of the interactions between bacteria and phytoplankton it might be expected that a decrease in phytoplankton production may result in a decline in bacterial production which may be compounded by direct UV-B effects on bacterioplankton (e.g. photodamage). This relationship demonstrates the need to assess UV-B impacts on both trophic groups. To our knowledge, the natural accumulation and repair of DNA photoproducts (e.g cyclobutane dimers) has not been documented for indigenous bacterioplankton populations. Rather than extrapolate the potential for UVR effects based on dosimeters or in situ incubations, cyclobutane dimer accumulation is a true measure of DNA damaging effects in situ.

Photobiology of marine bacterial cultures. UVB survival curves have been determined for Vibrio natriegens and Pseudomonas stutzeri. Cells were grown to stationary phase, harvested, and resuspended in nutrient-free medium. Percent survival was determined after exposure to up to 4000 J/m² of UVB. The experiment was repeated and survival determined after a 2 hr period of photoreactivation was allowed.

Diel patterns of photodamage in bacterioplankton vs. eukaryotic plankton. It should be expected that DNA damage and repair follow daily solar cycles with greatest damage being observed at peak sunlight and that damage would be repaired prior to sunrise the following day. The extent of damage to indigenous bacterioplankton , however, had not been determined. We examined diel patterns of damage and repair in bacterioplankton during shipboard incubations of surface waters collected from the Northern Gulf of Mexico. Surface waters were collected prior to sunrise on March 18, 1994 at 28 27' N, 88 47' W and prefiltered through a 0.8 m cartridge filter (Nuclepore, Pleasanton, CA) to remove eukaryotic microorganisms and held in shallow (15 cm) containers at ambient seawater temperatures. These containers were left exposed to unfiltered solar radiation while a control sample was incubated in a covered 200 L high density polyethylene Nalgene tank at ambient seawater temperature. At 3 or 6 hr intervals, the contents from one container (approximately 12 L) were collected onto a 0.22 m pore-size 142 mm diameter polysulfone (Supor, Gelman) filter. A similar volume was collected from the control tank. The filter samples were frozen on dry ice and stored at -70° C until DNA was extracted using a boiling SDS lysis and chloroform extraction. Cyclobutane dimers were then quantified in the extracted DNA using the radioimmunoassay methodology developed in one of our labs (Mitchell et al., 1985).

The vast majority of previous research has examined the impact of UV-B upon phytoplankton community dynamics. During a series of research cruises in the Northern Gulf of Mexico we examined the relative proportion of DNA damage in the bacterioplankton size fraction (<0.8m) and what we are operationally defining as the eukayotic planktonic size fraction (>0.8 m). We have examined the formation of cyclobutane dimers in different size fractions collected from surface waters in the Gulf of Mexico. A buoy was deployed from the RV BELLOWS at 30 05' N, 86 50' W at 6 am on April 27, 1994. Surface waters (depth <0.2 m) were collected within 100 meters of the buoy at three hour intervals. Water was pumped aboard ship using a teflon lined pump (Wilden, Colton, CA) and silicone tubing. All samples were pre-filtered through 120 m mesh Nytex screen. Water was filtered onto 0.8 m pore-size polysulfone filters (> 0.8 m fraction or eukaryotic plankton) and this sample filter was immediately frozen in dry ice. The <0.8 m filtrate was also collected onto a 0.22 m pore-size filter (bacterioplankton fraction). Cyclobutane dimers were determined in each DNA extract via radio-immuno assay (Mitchell et al., 1985).

Depth profiles of photodamage. Water samples were collected in late afternoon from four locations in the Gulf of Mexico in March, 1994. Discreet depths between the surface and 30 meters were sampled using Niskin bottles and cyclobutane dimer accumulation determined at each sampled depth.

Photobiology of marine bacterial cultures. V. natriegens was found to be more sensitive to UVB than P. stutzeri (Fig. 1). In contrast, V. natriegens appears to have more efficient photoreactivation such that percent survival is greater than for P. stutzeri after photoreactivation (Fig.1)

Diel patterns of photodamage in bacterioplankton vs. eukaryotic plankton. Diel patterns of damage and repair were observed for the duration of the 48 hr experiment (Fig. 2). Maximal damage (approximately 60 cyclobutane dimers/Kbp DNA) was observed in late afternoon each day while the majority of damage was repaired at night prior to sunrise. No damage was observed in control samples shielded from UV radiation (Fig. 2).

Diel patterns of solar induced DNA photodamage were observed in both the bacterioplankton and phytoplankton size fractions in surface waters (depth <0.2 m) collected at three hour intervals. At each time point during daylight hours, the proportional amount of DNA damage in the bacterioplankton fraction was greater than in the larger size fraction (Fig. 3). This proportional damage has been observed in greater than 90% of surface water samples collected (other data not shown). The data clearly demonstrate a naturally occurring diel pattern of damage and repair in both size fractions (Fig. 3).

Bacterioplankton were observed to have greater net accumulated photodamage at sunset than eukaryotic plankton. Greater damage may be the results of higher rates of photodamage induction owing to smaller cell size and greater surface area to volume ratios in bacterioplankton (Karentz et al., 1991). Lower rates of repair may also determine greater damage accumulation in bacteria. Damage removal is dependent upon quantitative processes such as excision, and qualitative processes such as the amount of excision repair and the presence or absence of photoreactivation. Greater damage in bacteria, therefore, could result from both greater rates of damage induction as well as lower rates of repair.

Bacterioplankton have been found to suffer up to twice as much DNA damage per unit DNA than larger planktonic organisms. The importance of this is emphasized by the realization that bacterioplankton often account for the majority of cellular DNA in oligotrophic waters (Paul et al., 1985; Boehme et al., 1993; Fuhrman et al., 1989). The impact of UV-B on bacterioplankton, therefore, may be much greater than the impact on larger organisms or other trophic levels.

Depth profiles. Although UVR attenuates exponentially in the water column, no consistant pattern of photodamage with depth was observed (Figure 4). The results suggest that mixing in the water column may have a significant effect on net photodamage accumulation in the water column. The results demonstrate the complexity of extrapolating UVR effects to indigenous planktonic populations from measures of UVR and fixed depth dosimeters or in situ incubations (as have been done inother studies), especially in high energy environments such as the Southern ocean. In contrast, low energy environments such as some lakes may be less effected by daily mixing. In addition, coral reefs,in which UVR has been implicated in bleaching, (Gleason and Wellington, 1993) are at fixed depths so the net effects would be determined less by wave driven mixing and more by water clarity and solar radiation intensity.

Future research will focus on evaluating the role of vertical mixing and net UVR effects in the water column. In addition, mechanisms and kinetics of photodamage repair will be evaluated in both bacterial cultures and natural populations of bacterioplankton.

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