Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis F. Kamoun 1 , H. Mejdoub 2 , H. Aouissaoui 1 , J. Reinbolt 3 , A. Hammami 4 and S. Jaoua 1 1 Laboratoire des Biopesticides, Centre de Biotechnologie de Sfax, Sfax, Tunisia, 2 Faculte´des Sciences de Sfax, Route de soukra km 4, Sfax, Tunisia, 3 UPR 9002 du CNRS, Institut de Biologie Mole´culaire et Cellulaire, rue Rene´ Descartes, Strasbourg, France, and 4 Faculte´ de Me´decine de Sfax, Avenue Majida Boulila, Sfax, Tunisia 2004/0615: received 28 May 2004, revised 4 August 2004 and accepted 2 October 2004 ABSTRACT F. KAMOUN, H. MEJDOUB, H. AOUISSAOUI, J. REINBOLT, A. HAMMAMI AND S. JAOUA. 2005. Aims: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. Methods and Results: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70°C and resisted up to pH 3Æ0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160Æ05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. Conclusions: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160Æ05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. Significance and Impact of the Study: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products. Keywords: Bacillus thuringiensis, Bacthuricin F4, purification. INTRODUCTION The field of antimicrobial compounds has since its discov- ery, been subject of intense research demonstrating its evident usefulness in several areas. Amongst these antimi- crobial compounds, the bacteriocins are defined as proteins or peptides antibiotics which do not harm the producer strain but have the ability to kill closely related bacteria (Tagg et al. 1976). Bacteriocins have received increasing attention because of the surprisingly high levels of their diversity. Bacteriocins produced by Gram-positive bacteria have attracted great interest because of their potential use as food preservatives as they can be successfully used to inhibit undesirable micro-organisms in food. In fact, the most widely studied bacteriocins are those produced by lactic acid bacteria such as nisin (Delves-Broughton 1990). In agriculture, bacteriocins are used for the retardation of spoilage by plant pathogens and for grain preservation (Jack Correspondence to: Samir Jaoua, Laboratoire des Biopesticides, Centre de Biotechnologie de Sfax, PO Box ÔKÕ, 3038, Sfax, Tunisia (e-mail: samir.jaoua@cbs.rnrt.tn). ª 2005 The Society for Applied Microbiology Journal of Applied Microbiology 2005, 98, 881–888 doi:10.1111/j.1365-2672.2004.02513.x