STRUCTURAL DIFFERENTIATION OF THE BACILLUS-SUBTILIS-168 CELL-WALL

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by th e addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respecti vely. Isolated cell walls, lysozyme-digested cell walls, and cell wall s treated with mild alkali to remove teichoic acid were also treated w ith PCF. After labelling, whole cells and walls were processed for ele ctron microscopy by freeze-substitution. Thin sections of untreated ce lls showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated tha t the wall was thicker at locations adjacent to septa and at pole-cyli nder junctions (P < 0.001). Labelling studies showed that at saturatin g concentrations the PCF probe labelled the outermost limit of the cel l wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surfa ce locations adjacent to or overlying septa and at the junction betwee n pole and cylinder. Labelling was rarely observed along the cell cyli nder or directly over the poles. Cells did not label along the cylindr ical wall until there was visible evidence of a developing septum. Ide ntical labelling patterns were observed by using concanavalin A-horser adish peroxidase-colloidal gold. Neither probe appeared to penetrate b etween the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on th e cell surface is indicative of regions of extreme hydrolytic activity in which a-glucose residues of the wall teichoic acids and electroneg ative sites (contributed by phosphate and carboxyl groups of the teich oic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially g rowing B. subtilis cells contains regions of structural differentiatio n.