Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 05 Dec 2018 20:17:47 The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic Meenakshi Singh, 1 Naveen K. Sharma, 2 Shyam Babu Prasad, 3 Suresh Singh Yadav, 3 Gopeshwar Narayan 3 and Ashwani K. Rai 1 Correspondence Ashwani K. Rai akrai.bhu@gmail.com Received 20 November 2012 Revised 2 January 2013 Accepted 15 January 2013 1 Department of Botany, Banaras Hindu University, Varanasi 221 005, India 2 Department of Botany, Indira Gandhi National Tribal University, Amarkantak, (M.P.) 484886, India 3 Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi 221 005, India Glycine betaine (GB) is an important osmolyte synthesized in response to different abiotic stresses, including salinity. The two known pathways of GB synthesis involve: 1) two step oxidation of choline (choline A betaine aldehyde A GB), generally found in plants, microbes and animals; and 2) three step methylation of glycine (glycine A sarcosine A dimethylglycine A GB), mainly found in halophilic archaea, sulphur bacteria and the cyanobacterium Aphanothece (Ap.) halophytica. Here, we transformed a salt-sensitive freshwater diazotrophic filamentous cyanobacterium Anabaena (An.) doliolum with N-methyltransferase genes (ApGSMT-DMT) from Ap. halophytica using the triparental conjugation method. The transformed An. doliolum synthesized and accumulated GB in cells, and showed increased salt tolerance and protection to nitrogenase activity. The salt responsiveness of the transformant was also apparent as GB synthesis increased with increasing concentrations of NaCl in the nutrient solution, and maximal [12.92 mmol (g dry weight) ”1 ] in cells growing at 0.5 M NaCl. Therefore, the transformed cyanobacterium has changed its behaviour from preferring freshwater to halophily. This study may have important biotechnological implications for the development of stress tolerant nitrogen-fixing cyanobacteria as biofertilizers for sustainable agriculture. INTRODUCTION Salinization of soil and water is one of the major factors adversely affecting the global productivity and biodiversity (Epstein & Bloom, 2005). Low rainfall, poor irrigation water quality, excessive use of fertilizers and rising global temperatures are major factors responsible for increasing the salinity. High salinity causes both ionic and osmotic stresses resulting in growth retardation and ultimately death of the organism. A great diversity of organisms, mostly micro-organisms, survive and grow well in high salt environments (Oren, 2011). To do so, they have to balance the osmolarity of their cytoplasm with that of their surrounding medium. There are two fundamental cellular responses used by micro-organisms against salt stress. One is accumulation of K + ions and counter extrusion of Na + . Alternatively, they synthesize de novo and/or accumulate compatible solutes such as polyols (glycerol), sugars (sucrose, trehalose) and amino acid derivatives (glycine betaine, ectoine), which are highly soluble uncharged or zwitterionic low molecular mass molecules with no adverse effect on cellular enzymic machinery. If these compounds are available in the external medium, cells can take them up through membrane bound transporters and accumulate them. The strategy adapted by organisms under a particular saline condition depends upon the level of salinity and energetic cost of a reaction to complete minus energy available through various meta- bolic processes (i.e. energetic balance) (Oren, 2011). Glycine betaine (N,N,N-trimethylglycine, GB) is one of the most studied compatible solutes (Chen & Murata, 2011) that organisms synthesize and accumulate under different abiotic (salt, drought, cold), oxidative and organic (abscisic acid, polymixin B, salicylic acid, etc.) stresses (Gorham et al., 1985; Reed et al., 1986; Lai et al., 1991; Kla ¨hn & Hagemann, 2011). In bacteria, plants and animals, GB is synthesized by a two step oxidation of choline to betaine aldehyde, and then to GB. However, a different pathway Abbreviations: ApGSMT, Ap. halophytica glycine sarcosine methyltrans- ferase; ApDMT, Ap. halophytica dimethylglycine methyltransferase; GB, glycine betaine; WT, wild-type. A supplementary figure is available with the online version of this paper. Microbiology (2013), 159, 641–648 DOI 10.1099/mic.0.065078-0 065078 G 2013 SGM Printed in Great Britain 641