Ultrastructure of a Temperature-sensitive Rod- Mutant of Bacillus Subtilis

Mutant 168ts-200B, resulting from nitrosoguanidine treatment of Bacillus subtilis 168 (trp(-) C2), exhibits a rod-to-sphere morphogenetic interconversion when the incubation temperature is 30 or 45 C, respectively. Ultrathin sections of rods grown at 30 C, after glutaraldehyde-osmium uranium-lead fixation and staining, show trilaminar cell walls with a well-developed underlying periplasm as in wild-type cells. However, the outer wall layer is irregular, and abnormal protrusions of wall material occur at the cross-walls. In contrast, cells growing at 45 C become rounded and are intersected randomly by irregular cross-walls which fail to split normally, resulting in large spherical masses. In these, the outer and inner wall layers and periplasm are lost, and the wall consists only of irregularly thickened and loosely organized middle layer. Wall ultrastructure is reversible in either direction as cell shape changes during temperature shifts. Mesosomes are rare and atypical at either temperature. It thus appears that cell wall ultrastructure is altered by the conditional (temperature-sensitive) mutation, and that loss of normal wall and submural organization is correlated with changes in cell size and shape as well as with inability to complete cell division. Preliminary studies after transformation of the mutant locus to another strain and growth at 45 C showed an increase in mucopeptide, loss of wall teichoic acid, failure of phage adsorption, and identical ultrastructural changes. The site of expression of the basic defect-be it in wall, submural region, or membrane-is undetermined.