Cloning and Characterization of a Novel Crystal Protein from a Native Bacillus thuringiensis Isolate Highly Active Against Aedes aegypti Corina M. Berón, Graciela L. Salerno Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas (FIBA), C.C. 1348, 7600 Mar del Plata, Argentina Received: 31 May 2006 / Accepted: 20 June 2006 Abstract. We characterized a novel Bacillus thuringiensis isolate native to Argentina (FCC 41) that exhibits a mosquitocidal activity higher than the reference B. thuringiensis subsp. israelensis. This isolate shows a rounded crystal harboring two major proteins of about 70–80 kDa. Moreover, we cloned and sequenced the encoding gene of one of the crystal proteins (Cry) consisting of an open reading frame of 2061 pb that encodes a protein of 687 amino acid residues. The deduced amino acid sequence has a predicted relative molecular mass of 78 kDa and is 52% and 45% identical to those of the reported Cry24Aa and Cry24Ba sequences, respectively. The novel Cry protein was designated as Cry24Ca, which also exhibited larvicidal activity against Aedes aegypti when its encoding gene was expressed in an Escherichia coli host strain. Blood-sucking insects such as mosquitoes and blackflies are vectors of many human and animal infectious dis- eases. One of the most important is Aedes aegypti, the primary carrier for viruses that cause dengue fever and yellow fever. According to the World Health Organi- zation, despite advances in medical science and new drugs, dengue fever is the most important arboviral disease to man in the world (http://www.who.int/topics/ dengue/en). Dengue has become a major international public health concern, spreading geographically in incidence and severity [28]. The control of this disease is based mainly on the elimination of its vector with syn- thetic products, which has been shown a rapid increase in mosquito resistance to various chemical insecticides. In addition, there has been a growing public concern regarding their potential adverse environmental effects that prompted the development of alternative methods for mosquito control that were compatible with the environment [16]. Although more than 100 bacterial species has been identified as insect pathogens, only certain Bacillus species have met success as bioinsecti- cides [24]. Particularly, formulations based on Bacillus thuringiensis subsp. israelensis (Bti) and B. sphaericus (Bs) have been shown to be highly effective against mosquito larvae and blackfly larvae. The insecticidal activity of these bacteria is mainly ascribed to proteins produced during sporulation that are included in a crystal complex, which in the case of Bti are Cry4A, Cry4B, Cry11A, and Cyt1A [10] and in Bs are a single binary protein (Bin) and other reported toxins [9]. The successful use of Bti and Bs strains in some dipterans control programs stimulated the search for more potent bacterium isolates [10]. Among other examples that demonstrate the value of searching for new mosquito- cidal strains, it was reported the isolation of B. thurin- giensis subsp. jegathesan native to Malasya, which produces a very complex parasporal body containing at least seven major polypeptides, including Cry11B. This toxin showed higher toxic activity against mosquito larvae than the previously reported Cry11A of Bti [8]. Recently, we have isolated a new mosquitocidal B. thuringiensis strain (FCC 41), native to Argentina, which might produce at least one Cry protein unrelated to those of Bti [4, 5]. This article describes the charac- terization of a novel B. thuringiensis isolate native to Argentina (FCC 41) that exhibits a mosquitocidal activity higher than the reference Bti (HD 567). More- over, we isolated and sequenced the encoding sequence Correspondence to: G.L. Salerno; email: gsalerno@fiba.org.ar CURRENT MICROBIOLOGY Vol. 54 (2007), pp. 271–276 DOI: 10.1007/s00284-006-0299-8 Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2007