BIOCHEMICAL CHARACTERIZATION OF THE BACILLUS SUBTILIS MACROFIBER CELL SURFACE.

Abstract

Cell walls of Bacillus subtilis macrofibers have been biochemically analyzed to determine the contribution of various surface polymers in the twist regulation. Helix hand inversion was induced by a variation in either the growth temperature or the nutritional composition of the culture medium. Initial experiments had demonstrated a fivefold difference in the sensitivity of right- and left-handed forms to muramidases indicating modifications of peptidoglycan as a possible mechanism underlaying inversion. An examination of lysozyme susceptibility of purified cell walls and whole cells derived from the two structural forms, however, exhibited no significant difference suggesting loss of the relevant component(s), perhaps biomechanical in nature, during disintegration of macrofibers. The effect of various twist modulators such as trypsin, ammonium sulfate and D-alanine on the development of helical twist in both switchable and "fixed" mutants were studied. The interaction matrices have established D-alanine as the most potent of right-factors. Intestinal alkaline phosphatase is reported as a newly discovered antagonist to the development of leftward twist. Heat inactivation and protein purification experiments strongly indicated that twist modulation was due to the phosphatase activity rather than minor protease contaminants. The chemical composition of cell walls purified from right- and left-handed structures was determined. No twist correlated differences in the overall content of peptidoglycan, teichoic acid and teichuronic acid were detected. Evidence is presented for the absence of correlation between the extent of ester-linked alanine substitution and twist state. These findings suggest that gross changes in wall composition is perhaps not the mechanism for hand inversion. From the profiles of the wall associated proteins, a 200 Kdal band has been identified whose presence is strongly correlated with the development of leftward twist. This polypeptide was found to be highly sensitive to trypsin; a property it shares with a previously proposed left-twist protein. Preliminary evidence for isolation of left-hand specific polyclonal antibodies is also presented. FJ7, a switchable mutant, was successfully transformed with a plasmid containing the Streptococcus transposon Tn917. A small bank of insertional mutants has been constructed for the isolation of mutants impaired in helix hand inversion.