Inactivation of Cyanobacterial Nitrogenase After Exposure to Ultraviolet-B Radiation Ashok Kumar, 1 Madhu Bala Tyagi, 2 Prabhat Nath Jha, 1 Gannavarapu Srinivas, 1 Abha Singh 3 1 Microbial Biotechnology Unit, School of Biotechnology, Banaras Hindu University, Varanasi–221 005, India 2 Department of Botany, MMV, Banaras Hindu University, Varanasi–221 005, India 3 Department of Botany, Patna Women’s College, Patna– 800 001, India Received: 13 June 2002 / Accepted: 22 July 2002 Abstract. Exposure of the N 2 -fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m -2 ) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure. Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation. The damaging effects of solar ultraviolet (UV-B, 280 – 320 nm) radiation have been studied in a variety of biological systems [2, 8, 17, 24]. Depletion of strato- spheric ozone leads to an increased incidence of UV-B on the earth’s surface, causing worldwide environmental effects, leading to imbalances in natural ecosystems [4, 21]. Most studies on UV-B have been confined to only a few species of higher plants, algae, and animals, and hence no general conclusions pertaining to the deleteri- ous effects of UV-B have been put forward [2, 17]. Furthermore, the intensity of UV-B reaching the earth surface varies in different ecosystems, and in certain ecosystems only particular types of organisms are ex- posed to UV-B radiation [11, 21]. There is a great need for studying the effects of UV-B on a much wider spectrum of organisms from a diversity of habitats than hitherto explored. Cyanobacteria, the oldest oxygenic-photosyn- thetic prokaryotes [1], occur widely in nature, in soil and water and in association with a variety of plant and marine life [12, 14]. Many species can tolerate various climatic conditions and are found in diverse habitats including hot springs, arctic regions, desert soils, and rocky surfaces [1, 3, 22]. Members show considerable metabolic plasticity and, in addition to CO 2 and O 2 assimilation, some also assimilate N 2 and H 2 [22]. Because of efficient N 2 fixation potential, certain members of cyanobacteria are known to play an important role in the fertility of soil, especially rice fields in the tropics. In spite of these added features in cyanobacteria, no systematic investigations related to UV-B effects on a particular metabolic event have been made [17]. Studies so far conducted reveal that UV-B affects the growth, survival, pigmentation, ori- entation, development, general metabolism, photosyn- thesis, nitrogen fixation, and assimilation of nitrogen in diverse types of cyanobacterial species [5, 10, 13, 16, 18]. It has been suggested that these effects are in part due to direct effects of UV-B radiation on mem- brane proteins, photosystem-II, DNA, enzymes, pig- Correspondence to: A. Kumar; email: kasok@epatra.com CURRENT MICROBIOLOGY Vol. 46 (2003), pp. 380 –384 DOI: 10.1007/s00284-001-3894-3 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003