PROTOPLASTS AND L-TYPE GROWTH OF ESCHERICHIA COLI

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Abstract
E. coli growing in the presence of penicillin forms localized swellings which enlarge to yield special protoplasts which lyse in dilute media, but may be maintained in protective media containing 0.33 [image] sucrose plus 0.01 [image] Mg++. In penicillin sucrose broth, the protoplasts continue to enlarge but do not proliferate. In the absence of penicillin they revert to form normal rods. An agar medium is described which permits the further growth of protoplasts in the presence of penicillin to produce L colonies. Growth, which depends on the formation of blebs which enlarge and pinch off, occurs only in agar medium. Indefinite serial passages were made of certain such strains of E. coli K-12 as L growth in penicillin sucrose agar; these reverted to normal bacillary form in the absence of penicillin. Optimal L growth requires high concentrations of penicillin, neither L nor bacilliform colonies developing well at intermediate levels. This phenomenon has been applied to the selection of fixed L forms, which grow as L colonies in conventional media. So far, these isolates from E. coli are all auxotrophic mutants requiring diaminopimelic acid, a characteristic constituent of bacterial cell walls. These mutants grow as L colonies in agar lacking diaminopimelic acid. They may be passed in series as such, and regenerate bacilliform growth when this metabolite is restored. However, other fixed L forms of Proteus responded neither to diaminopimelic acid nor to crude bacterial hydrolysate. Penicillin-produced L colonies of E. coli become resistant to certain phages and colicines, but not others; protoplasts of genetically compatible strains retain their ability to undergo sexual recombination. Some but not all the surface receptors of the bacteria are therefore believed to have been lost. Protoplasts suspended in sucrose broth show a transparent capsule, which may represent remains of the cell wall. Lysed protoplasts, though expanded, are not fully dispersed through the solvent but occupy definite spaces. Attempts to convey genetic markers to protoplasts by means of extracts containing deoxyribonucleic acid were unsuccessful, as were efforts to promote the fusion of protoplasts of otherwise incompatible F- X F- genotypes. These observations support the proposals that: (1) the mechanism of action of penicillin is to inhibit the synthesis of the bacterial cell wall; (2) that the L forms of Dienes and Klieneberger are colonies of protoplasts, whose aberrant mode of growth is conditioned by the loss of the wall and the failure of the normal division mechanism; (3) the partial or complete defect of the wall may be brought about either by external inhibition (penicillin) or by internal genetic blocks affecting any of various aspects of wall formation. So far, two types of wall-defect mutants are known: those repaired by diaminopimelic acid, and auxotrophic for it, and those not reparable even by crude bacterial hydrolysates. The evolution of fixed growths in the presence of penicillin may be accounted for by a selective advantage under these conditions of spontaneous mutants with wall defects.