MIRCENJournal, 1985, 1,269-275 Isolation of a hydroxyproline-secreting pigment-deficient mutant of Nostoc sp. metronidazole selection by H. D. Kumar & A. K. Tripathi Laboratory of Algal Biology, Centre for Advanced Study in Botany, Banaras Hindu University, Varanasi - 221005, India Received 10 November 1984; accepted 11 February 1985 Introduction Fixation of atmospheric carbon dioxide and dinotrogen occurs simultaneously only in certain cyanobacteria and photosynthetic bacteria. In these organisms, reduced ferredoxin donates electrons to various ferredoxin-linked reductases such as nitrogenase and nitrate or nitrite reductases (Apte et al. 1978). Metronidazole is toxic to these diazotrophs because ferredoxin reduces its nitro groups. Nitrogenase activity, electron transport via the enzyme couple fer- redoxin + ferredoxin-NADP reductase and certain other reductases are sensitive to metro- nidazole (Eisbrenner & Bothe 1979; Tetley & Bishop 1979). Schmidt et al. (1977) used metronidazole for selective enrichment of pigment mutants of the eukaryotic green alga Chlamydomonas reinhardii. To us, the relevance of this approach appeared even more pertinent in cyanobacteria which contain additional enzyme systems such as nitrogenase. Accordingly, we have studied the effects of metronidazole on growth of a Nostoc sp. and on the activities of its nitrogenase, nitrate reductase and glutamine synthetase. Herein we also describe the characteristics of a hydroxyproline-producing mutant which was isolated by selection with metronidazole. Materials and methods Cultivation of the Nostoc sp. Axenic clonal cultures of an unidentified species of the cyanobacterium Nostoc (most prob- ably N. linckia), isolated from a Varanasi paddy field, were grown in Allen and Arnon's culture medium (Allen & Arnon 1955) lacking combined nitrogen, at 27 + 2~ illuminated with ca 1800 lux for 16 h daily. Culture growth was estimated by measuring absorbance at 663 nm. Selection of the mutant A modified method of Schmidt et al. (1977) was employed for selection of pigment mutants. Exponential phase cultures were centrifuged and the pelletized Filaments were broken into 1-2-ceUed units. This suspension of cells was washed twice and used in three sets. In the first set, 0.2 ml of homogeneous suspension was inoculated on agar plates (basal medium) to act