Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

Abstract

When the cell wall of B. subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). If protoplasts or L bodies are conditioned by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60-100% of the formerly naked cells give rise to bacillary colonies. The present experiments largely explain the mechanism responsible for the heritable persistence of the wall-less state in B. subtilis. Protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 .times. 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat and detergent. Efficient reversion at 2 .times. 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 .mu.g of trypsin/ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by .beta.-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 .times. 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several of the properties of the 2 activities coincide: both are inhibited by high concentrations of gelatin, are activated by .beta.-mercaptoethanol and have high affinity for cell wall. Assuming that RIF is an autolysin, models for protoplast reversion and L-form stability are proposed. A role of teichoic acid in reversion is suggested since mutants with altered teichoic acid show altered reversion behavior.