Anaerobe 13 (2007) 6–13 Ecology/environmental microbiology Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea Tatyana G. Yudina a , Andrei L. Brioukhanov a,Ã , Igor A. Zalunin b , Ludmila P. Revina b , Andrei I. Shestakov a , Nina E. Voyushina b , Galina G. Chestukhina b , Alexander I. Netrusov a a Department of Microbiology, Biological Faculty, M.V. Lomonosov Moscow State University, Leninskie Gory 1/12, 119992 Moscow, Russian Federation b Scientific Research Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhny proezd 1, 117545 Moscow, Russian Federation Received 15 July 2006; received in revised form 26 September 2006; accepted 27 September 2006 Available online 27 November 2006 Abstract Proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium Bacillus thuringiensis (subspecies kurstaki, galleriae, tenebrionis) as well as some fragments of these proteins, obtained by limited proteolysis, are capable of antimicrobial action against anaerobic bacteria and archaea—Clostridium butyricum, Clostridium acetobutylicum and Methanosarcina barkeri. The MICs are 45–150 mg/mL. Electron microscopy showed that lysis of M. barkeri cells in the presence of 49 kDa fragment of Cry3Aa toxin is generally similar to the bacterial cell lysis, which has been previously detected in the presence of Cry11A, Cry1Ab and other Cry proteins. The Cry1D-like toxin from crystals of B. thuringiensis subsp. galleriae has been put forward as an example of the supposition that cell wall and some of its components like teichoic acid and N-acetylgalactosamine have possible influence on Cry toxins, enhancing their antimicrobial activity. The possible ecological role of the antimicrobial activity of Cry proteins is also discussed. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cry proteins; Clostridium butyricum; Methanosarcina barkeri; Antimicrobial activity; Cell walls 1. Introduction The entomopathogenic bacterium Bacillus thuringiensis produces protein parasporal crystals. Cry proteins, com- posing crystals of more than 80 subspecies of the bacterium, are receptor-specific toxins for some insects, nematodes and ticks larvae. More than 300 Cry proteins, subdivided into 49 classes, depending on their amino acid sequences, are known at the moment. Many of the Cry proteins (e.g., the endotoxins Cry1A) are protoxins and their proteolytic activation yields the activated toxins [1,2]. Another family of endotoxins represents Cyt proteins. These are particularly found in subspecies of B. thur- ingiensis that are toxic for Diptera. Cyt proteins cause the lysis of a broad range of cells via direct binding to membrane phospholipids and act highly synergistically with Cry toxins [3]. Previous research showed the polyfunctionality of Cry proteins that affect not only the sensitive invertebrates, but also some microorganisms, with various specificity and, possibly, by different mechanisms. This was shown, first of all, for the aerobic bacteria. Certain fragments of Cry proteins, either with or without insecticidal activity, have the antibacterial activity as well. The antibacterial action of Cry proteins is less specific than the insecticidal one [4–6]. Cyt toxins and some of their fragments also have the antibacterial activity [3,4]. The bactericidal effect of Cyt1A against Micrococcus luteus is five and 10 times higher than the activity of Cry11A and Cry4B, correspondingly [4]. Cyt toxins and some of their fragments cause distortion of cell morphology and loss of the colony-forming ability of Escherichia coli and they lyse a bacterial cytoplasmatic membranes (CM) [3]. Previously, we also found antibiotic action of parasporal inclusion toxins of three subspecies of B. thuringiensis against the following anaerobic methanogenic archaea: Methanobrevibacter arboriphilus (symbiotic strain for termites) and M. barkeri (free-living microorganism) [7]. ARTICLE IN PRESS www.elsevier.com/locate/anaerobe 1075-9964/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2006.09.006 Ã Corresponding author. Tel./fax: +7 495 9392763. E-mail address: brjuchanov@mail.ru (A.L. Brioukhanov).