Electron Microscope Study of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

Abstract

A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis . The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect “commitment” to sporulation.

Comparison of inclusions in different Bacillus thuringiensis strains. An electron microscope study

“…Comparative studies between two lepidopteranpathogenic and three mosquito-pathogenic strains of B. thuringiensis reveal that in all strains the sequence of sporulation is very similar to that found for bacilli including the various B. thuringiensis serotypes [4][5][6][7]28]. In all strains the sporulation went through the seven defined stages described by Ryter [29] and Murrel [28] for spore-forming bacteria.…”

“…Strains were stored, lyophilized and subcultured routinely on TYG-agar slants (Difco tryptone 5.0, Difco yeast extract 2.5, glucose 1.0 g/l) or on GYS-agar slants [26]. For sporulation studies the bacterial strains were cultured in liquid GYSmedium at 28°C and synchronized using the active culture technique of Bechtel and Bulla [7] with slight modifications. For negative staining GYS or mGYS-medium [27] were used.…”

“…The sequence of sporulation and the formation of the crystal have been studied for different B. thuringiensis serotypes. The crystal is initiated during stages II-III of sporulation [4][5][6][7], and progressively assembled from protein subunits with dimensions of approx. 5.0.15.5 nm [8].…”

“…5.0.15.5 nm [8]. Reports concerning the site of inclusion synthesis in the cell have been contradictory [5][6][7][9][10][11].…”

“…The size and the form of the crystals is also affected by growth conditions [16][17][18][19]. In addition to the typical bipyramidal crystal found in B. thuringiensis isolates, there may also exist smaller inclusions which are oval or cubic in shape [7,20,21]. * To whom correspondence should be addressed.…”