Analele Ştiinţifice ale Universităţii „Alexandru Ioan Cuza”, Secţiunea Genetică şi Biologie Moleculară, TOM XI, 2010 ZYMOMONAS MOBILIS LEVAN PRODUCTION IN THE PRESENCE OF ANTIMETABOLIC ANGENTS CRISTINA STURZOIU 1 *, ANDREI TANASE 1 , ANCA DINISCHIOTU 1 , GHEORGHE STOIAN 1 * Keywords: levan, Zymomonas mobilis, antimetabolic angents Abstract: Our studies have focused on the screening of Zymomonas mobilis bacterial strains capable to produce levan under the action of three antimetabolic agents (sulfafurozol, sulfametaxazol, trimetoprim). The experiments were carried out using spontaneous mutant strains derived from Z.mobilis NCIB 11163 and 11163/70, obtained by supplemented media with methotrexate (600 μg/mL) and trimethoprim (1000 μg/mL). Exponential growth profiles of bacterial cells and the production of levan were assessed in the absence and presence of antimetabolites of different concentrations. The studies have shown that Z.mobilis 11163/70 strains manifest a progressive growth in the presence of trimethoprim (50 μg/mL), an inhibition growth in the presence of sulfametoxazol (100 μg/mL). and also good resistance in the presence of sulfafurazol (100 μg/mL). Sulphonamides can inhibit the production of levan (Z.mobilis CP4PRRif, Z.mobilis NCIB 11163/70). On the other hand, a stimulation of levan production has been observed in the presence of trimetoprim (50 μg/mL) (Z.mobilis CP4Rif and 10988). INTRODUCTION Levans are microbial fructose polymers whose units are linked by type β-(2,6)-fructosyl-fructose. They are produced by several levansucrase microorganisms, among them Zymomonas mobilis, a mobile Gram-negative bacterium. Their synthesis is accompanied by FOS (fructooligosacharides) production (Bekers et al.,2002). It has been reported that levans produced by Z.mobilis possess antitumour activity (Yoo et al., 2004) (up to 72% inhibition) and that the activity depends on the polysaccharide molecular weight (Calazans et al., 2000,1997). Gram-negative bacteria Z.mobilis is a single bacterium in the evolution of bacterial world whose taxonomic position is not fully established (Rogers et al, 1984). On the sucrose-based media Z.mobilis is producing ethanol and various secondary products, especially high molecular weight levan (1975-2000 kDa) sorbitol, gluconic acid and FOS (Viikari, 1988; Vına et al, 2001). Z.mobilis bacterium is able to tolerate high concentrations of sucrose than glucose (Swings and De Ley, 1977) and at high concentrations of glucose cell membranes are affected by the rate of 10-15% while the high efficiency of hydrolysis of sucrose concentration is kept constant until 46% (Viikari, 1988). MATERIALS AND METHODS Preparation of extract of sweet sorghum. In our study we used sweet sorghum (Sorghum bicolor) from the National Institute for Research and Development of Technical Plants: (a) 11100 variety (b) 11061 variety (c) LC 99 variety (d) Prut04 variety (e) Sudan grass. Sweet sorghum extract was achieved by boiling extraction of sucrose from the plant stem and chemical characterized . Selection of strains with different degrees of resistance/sensitivity to antimetabolite. As antimicrobial agents capable of producing mutant strains were used methotrexate and trimethoprim, compounds with antifolic activity. In our paper we used wild bacterial stains which came from the Laboratory of biochemistry, genetics and molecular biology of Univerităţii of Ioannina, Greece. Cultures were kept in optimal conditions (frozen at -80°C in a mixture composed of growth medium and glycerol in 1:1 ratio). Were tested the resistance of three pure strains belonging to the genus Zymomonas mobilis (ATCC 10988, NCIB 11163 and ATCC 31381-CP4RifR) to methotrexate and trimethoprim. Sugars and levan were determined by the method described by Dubois et al (1956; Kushwaha and Kates, 1981; Mukhopadhyay, 2005). The compounds with antimetabolite activity: sulfafurazol, sulfamethoxazole and trimethoprim were analyzed. Growth curves in the presence of sulfafurazole, sulfamethoxazole and trimethoprim. Analysis of levan produced. In order to study the exoenzimes involved in levan biodegradation produced by Z.mobilis, cells in exponential growth phase were used as inoculum to obtain a initial liquid culture (107 cells/ml). The increased turbidity was monitored at wavelength of 600 nm. The optical density at 600 nm (OD600nm) equal to 0.9 corresponds to a weight of 0.35 mg dry material / ml (Douka, 1999). Growth profiles were studied of the four microbial strains in the absence and presence of trimethoprim (50μg/ml, 100μg/ml respectively), sulfafurazol (100μg/ml, 500μg/ml respectively) and sulfamethoxazole (100μg/ml, 500μg/ml respectively) (Sigma Chemical Co). At the end of the experiment the levan produced was extracted and analyzed (Andersone, 2004). 7