J P P JOURNAL OF PROTEINS AND PROTEOMICS 7(3), 2016, pp. 177-185 Corresponding Author: Babu Vamanrao Vakil E-mail: bvvakil@gmail.com Received: August 4, 2016 Accepted: September 21, 2016 Published: September 25, 2016 Research Article ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS Kavita Rajesh Pandey, Aparna Babanrao Patil, Babu Vamanrao Vakil* Guru Nanak Institute for Research and Development, G. N. Khalsa College of Arts, Science and Commerce, Nathalal Parekh Marg, King’s Circle, Matunga, Mumbai 400019, India Abstract: Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII) obtained in previous studies (Dubey and Vakil, 2010), were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell. Keywords: 2D gel electrophoresis; Isoelectric focussing; membrane proteins; probiotic; Bacillus coagulans mutants. Note: Coloured Figures available on Journal Website in “Archives” Section Introduction Probiotics are live microorganisms which when consumed in adequate amounts confer several health benefits to the hosts as evident from the literature (Harish and Varghese, 2006; Alloui et al., 2013). Probiotics are produced as biomass by the route of fermentation and are used in foods or as medicine. The resultant biomass is harvested, separated from the spent growth medium and then concentrated and preserved as bulk before formulating it as a probiotic (Brüssow, 2001; Shah, 2001; Lacroix and Yildirim, 2007; Ding and Shah, 2009). The fermentation process is prone to contamination by strain specific phages which normally lead to batch failures due to lysis of actively growing probiotic cultures resulting in huge financial losses (Pandey et al., 2015). The traditional microbiological strategies to keep phages away from the fermentation process include rotation of cultures, use of phage inhibitory media and maintaining of aseptic processing conditions from laboratory to commercial production area (Brüssow, 2001). A more effective way to prevent the phage infection is to make the cultures genetically resistant. Physical and chemical mutagens have been used effectively to induce changes in the host DNA to make them resistant to phage attacks (Marci et al., 2004; Dubey and Vakil, 2010). Classical mutation and selection programs have been used in the industries for many years and have resulted in screening and isolation of mutants with improved characteristics that have resulted in large productivity increments. Such strain improvement programs have substantially driven