Structural differentiation of the Bacillus subtilis 168 cell wall

Abstract

Exponential-growth-phase cultures of BaciUus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between 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 the cell surface is indicative of regions of extreme hydrolytic activity in which ot-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.The cell wall of Bacillus subtilis 168 is a two-polymer structure comprised virtually of teichoic acids (54%) and peptidoglycan (46%) on a per dry weight basis when cultured in Spizizen minimal medium (4,30,31). Despite this chemical simplicity, the exact mechanisms by which the wall grows, divides, and turns over have not been adequately explained (2).Numerous studies indicate that the predominant mode of wall growth involves insertion of newly synthesized wall polymers in an inside-to-outside fashion at many sites randomly distributed along the cytoplasmic membrane (21, 22). According to the surface stress theory of Koch (18), this newly inserted material is cross-linked to the existing wall fabric at the inner wall surface but is unstressed. It assumes tension only after it is pushed outwards by more recently acquired wall material. Select bonds in this "stretched" wall eventually undergo hydrolysis, turning over or releasing polymers from the cell...

In Bacillus subtilis 168, teichoic acid of the cross-wall may be different from that of the cylinder: a hypothesis based on transcription analysis of tag genes

“…For instance, autoradiography and fluorescence microscopy (Mobley e t al., 1984), electron microscopy (Clarke-Sturman et al, 1989), freeze-substitution (Graham & Beveridge, 1994) and endolysin susceptibility of the cell wall (Fan et al, 1972) have all suggested a metabolic and/or a qualitative difference between cylinder and septum cell wall which, among other factors, may be due to a difference at the level of teichoic acid.…”

Survival of freeze-dried bacteria

“…Serological studies of Lactobacillus have shown that these species can be assigned to several groups on the basis of specific antigenic characteristics, including production of cell wall polysaccharides, cell wall teichoic acids, and membrane teichoic acids (Knox and Wicken, 1973;Sharpe, 1981). Teichoic acids are components of the outer layers of Gram-positive bacteria; they function like extracellular polysaccharides in processes such as antigen activity, adhesion, and biofilm formation (Birdsell et al, 1975;Doyle et al, 1975;Graham and Beveridge, 1994;Knox and Wicken, 1973;Neuhaus and Baddiley, 2003).…”

The response of selected members of the archaea to the Gram stain

“…bacteria (such as Bacillzls snbtilis) possessed thick amorphous cell walls overlying the plasma membrane (Beveridge,198l), although more recent work using cryo-electron microscopy has now shown these envelopes to be more complex (Beveridge & Graham, 1991 ;Graham & Beveridge, 1994). The staining response for eubacteria has usually been clear-cut with few exceptions (Beveridge & Davies, 1983 ;Beveridge, 1990).…”