Landman O E, Forman A
J Bacteriol. 1969 Aug;99(2):576-89. doi: 10.1128/jb.99.2.576-589.1969.
Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.
接种在SDG培养基上的枯草芽孢杆菌原生质体可定量产生L型菌落，并能无限期地以L型形式繁殖。置于25%明胶培养基中的原生质体或L型小体可形成杆菌菌落。本文报道了这些无壁体回复为有壁形式的详细情况。在基本培养基中制备的原生质体接种到明胶中3至4小时后相当同步地发生回复，但在富含酪蛋白水解物（CH）的基本培养基中预孵育的原生质体在明胶中1小时内就开始回复。为了实现良好的预孵育效果，需要在0.44%的CH中预孵育1.5小时。细胞必须在无壁状态下进行这种预孵育（步骤1）；这对L型小体和原生质体同样有效。预孵育被氯霉素、嘌呤霉素和放线菌素D阻断，但不受青霉素、溶菌酶或脱氧核糖核酸（DNA）合成抑制的影响。得出的结论是，步骤1需要蛋白质和核糖核酸（RNA）合成，不需要DNA合成，也不合成细胞壁粘肽。在明胶中预孵育良好的原生质体的回复（步骤2）在胸腺嘧啶饥饿且染色体停滞在末端的细胞中不受干扰地进行。在明胶中，氯霉素几乎不使其回复过程减慢，但嘌呤霉素和放线菌素D都使其延迟约3小时。在抑制剂仍在积极阻断生长时就出现了对抑制的逃逸。青霉素和环丝氨酸抑制回复，溶菌酶则使回复逆转。明胶的短暂融化会延迟回复。得出的结论是，步骤2发生粘肽合成，同时进行的RNA、DNA或蛋白质合成并非必需，但在步骤2早期，排出的细胞产物在原生质体表面的物理固定是必要的。新回复的细胞形状异常且对渗透压敏感。在氯霉素或放线菌素D存在的情况下，回复后赋予渗透压稳定性的过程（步骤3）不会发生。
