Endospore Degradation in an Oligosporogenic, Crystalliferous Mutant of Bacillus thuringiensis Pa ´vel Sierra-Martı ´nez, 1 Jorge E. Ibarra, 2 Mayra de la Torre, 1 Gabriela Olmedo 3 1 Departamento de Biotecnologı ´a y Bioingenierı ´a, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Irapuato, Gto. 36500, Me ´xico 2 Departamento de Biotecnologı ´a y Bioquı ´mica, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Irapuato, Gto. 36500, Mexico 3 Departamento de Ingenierı ´a Gene ´tica, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Apartado Postal 629, Irapuato, Gto. 36500, Me ´xico Received: 9 May 2003 / Accepted: 16 June 2003 Abstract. We isolated a new oligosporogenic mutant from Bacillus thuringiensis var. kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions. Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them). At a late stage, the coat surrounding the forespore seems to lack shape and to be empty. Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore. The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter. Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV. Bacillus thuringiensis is able to produce insecticidal crystal proteins. The expression of most of these Cry proteins occurs concomitantly with the sporulation pro- cess, resulting in the formation of the insecticidal crys- talline inclusions within the sporangium. The temporal and spatial regulation of cry gene expression is con- trolled at the transcriptional level by the Sigma factors E and K (also known as Sig 35 and Sig 28), which share homology with, and are functionally equivalent to, SigE and SigK from Bacillus subtilis [1]. The cry1A genes are sequentially transcribed during sporulation from BtI and BtII promoters. Transcription from BtI is dependent on the SigE, and BtII is recognized by SigK. The overlap- ping promoters specific for each holoenzyme ensure the transcription of cry genes throughout most of the sporu- lation process [10], which explains in part the high expression level underlying the production of these abundant proteins. The Cry proteins may reach up to 25% of the cell’s dry weight [3]. Because, in most cases, expression of the cry genes is associated with sporulation, spore-deficient mutants (Spo - ) are also frequently crystal deficient (Cry - ). De- spite this, Bravo et al. showed that cry1Aa is expressed in a sigK mutant of B. thuringiensis, with its expression controlled solely by sigE [9]. Cry + oligosporogenics mutants of B. thuringiensis var. kurstaki, arrested at stage II of sporulation, have also been described [13]. The commercial product Foil BFC is produced by using a spontaneous oligosporogeneous mutant of B. thuringien- sis var. kurstaki transformed with cry3A gene. The Cry3A protein overproduction phenotype of this strain appears to be caused in part by the sporulation-indepen- dent nature of cry3A expression, the prolonged synthesis of Cry3A protein during the terminal stationary phase, and the absence of sporulation-dependent proteases [7]. Information about the stage of sporulation at which the mutant is blocked has not been published. This report details the isolation and characterization of a new oligosporogenic mutant of B. thuringiensis var. kurstaki that retains the ability to form an insecticidal crys- tal and forespores; however, maturation of the endospores does not take place. One of the most striking features is that sporulation in this mutant follows what seems a normal pathway, and forespores are evident under electron micros- copy. At later stages, however, the forespores cannot be observed any longer, and only bacilli ghosts containing the crystal are observed. Interestingly, in this mutant SigK is Correspondence to: G. Olmedo; email: golmedo@ira.cinvestav.mx CURRENT MICROBIOLOGY Vol. 48 (2004), pp. 153–158 DOI: 10.1007/s00284-003-4148-8 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2004