The Formation of Protoplasts and Quasi-spheroplasts in Normal and Chloramphenicol-pretreated Bacillus subtilis

Abstract

The lytic action of lysozyme upon Bacillus subtilis walls was studied by following the disappearance of bacillary-colony-forming units and the appearance of L-colony-forming-units. The rapidity of cell wall removal by lysozyme fluctuated markedly during growth in a chemically defined medium, presumably because subtle changes in the cell wall were constantly occurring. When lysozyme-sensitive bacilli were grown with chloramphenicol 10 pg./ml. for 3 hr they showed a notable increase in lysozyme resistance; at the same time, their walls almost doubled in thickness. As lysozyme attack proceeded in a given culture, the bacilli passed first through a rod-shaped osmotically sensitive stage, and then a spherical stage characterized by incomplete removal of cell wall before finally reaching the naked protoplast stage. The spherical forms with adherent wall residues formed L colonies on a medium containing the reversion inhibitor D-methionine and bacillary colonies on the same medium without D-methionine. Under the latter conditions, the cell wall residue served as a starting point for rebuilding of complete wall, much as residual wall permits reversion of Gram-negative spheroplasts to the bacillary state. In the presence of Dmethionine, the feedback sequence required for wall formation was severed, resulting in heritable propagation of the protoplast state.

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action

“…Protein synthesis inhibitors induce cell wall thickening by stopping enlargement of the cell wall surface [188,189], possibly by inhibiting synthesis of the peptidoglycan hydrolases needed to loosen the expanding walls [189,194]. This, in the absence of any accompanying decrease in synthesis or incorporation of peptidoglycan precursor, leads to thickening of the existing cell wall [188].…”

“…aureus [150,155] and Enterococcus faecalis [157,188], but can also occur in Gram-negative cocci [125,148] and Gram-positive bacilli [69,189].…”

“…8) [125,150,155,156,160,191]. Thickening occurs following ≥4 hours treatment with supra-inhibitory concentrations (3xMIC to 40xMIC) of RNA synthesis inhibitor (actinomycin [60] and rifampicin [150,155]), or ≥1 hours treatment with inhibitory or supra-inhibitory concentrations (1xMIC to ~10xMIC) of protein synthesis inhibitor (chloramphenicol [60,189], puromycin [60], lincosamides [160,191], macrolides [156,160,191,192], and tetracyclines [156,193]; observable as long as 24 hours following initial exposure [156,160]). Amino acid starvation has a similar effect [59,189].…”

Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Morphological Changes and Macromolecular Synthesis after Germination of Spores Treated with Cyclic Polypeptide and Aminoglycoside Antibiotics

“…There have been several reports on protein synthesis inhibitory antibiotics causing abnormal thickness of the cell wall of gram-positive bacteria; e.g., tetracycline on Staphylococcus aureus (8), chloramphenicol on Streptococcus faecalis (18), S. aureus (7), B. subtilis (12), and Bacillus cereus (5), and lincomycin, clindamycin, erythromycin, and spirarnycin on S. aureus (13). We have shown in this paper that the cell wall of kanamycin-treated ( Fig.…”

Reversion of Bacillus subtilis Protoplasts to the Bacillary Form Induced by Exogenous Cell Wall, Bacteria and by Growth in Membrane Filters

“…Thus, spheroplasts of Gram-negative species and the 'reverting' L forms obtained through multiplication of such spheroplasts (these morphologically mostly exhibit ' 3~' forms; see Dienes & Weinberger, 1951) promptly synthesize more wall as soon as lysozyme, or the inhibitory antibiotic, is removed (Landman, Altenbern & Ginoza, 1958 ;Lederberg & St Clair, 1958 ;Altenbern, 1963). Similarly, ' quasi-spheroplasts' of Bacillus subtilis, produced by partial removal of the wall by lysozyme, can apparently repair wall without difficulty (Miller, Zsigray & Landman, 1967;reviewed, Landman, I 968).…”