Institut für Mikrobiologie der Georg-August-Universität, D-3400 Göttingen, and Technische Universität Hamburg-Harburg, Arbeitsbereich Biotechnologie I, Technische Mikrobiologie, 2100 Hamburg 90, Federal Republic of Germany.
Appl Environ Microbiol. 1991 Apr;57(4):1062-9. doi: 10.1128/aem.57.4.1062-1069.1991.
Clostridium thermosulfurogenes EM1 formed blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by phosphate limitation or high dilution rates. Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded. Bleb and vesicle formation was also observed in the absence of substrates that induce alpha-amylase and pullulanase synthesis. No correlations existed between bleb formation and the presence of active enzyme. Similar blebs could also be observed in a number of other gram-positive bacteria not producing these enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of alpha-amylase and pullulanase in C. thermosulfurogenes EM1, two different antisera were applied. One was raised against the enzymes isolated from the culture fluid; the other was produced against a peptide synthesized, as a defined epitope, in analogy to the N-terminal amino acid sequence (21 amino acids) of the native extracellular alpha-amylase. By using these antisera, alpha-amylase and pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning, blebs could be seen, and the immunolabel pinpointing alpha-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the blebs. Cells exhibiting high or virtually no enzyme activity were labeled similarly with both antisera. This finding strongly suggests that alpha-amylase and pullulanase may occur in both active and inactive forms, depending on growth conditions.
热脱硫肠状菌 EM1 形成了泡囊，即在分批培养和连续培养过程中，从细胞质膜向外突出的部分，仍然与细胞质膜接触。在细胞壁层（表面层和肽聚糖层）似乎完整的细胞中，以及在细胞壁层由于磷酸盐限制或高稀释率而处于不同降解状态的细胞中，可以观察到泡囊被挤压在细胞壁和细胞质膜之间。当细胞壁严重降解时，泡囊会通过收缩变成膜泡。在没有诱导α-淀粉酶和普鲁兰酶合成的底物的情况下，也观察到泡囊和囊泡的形成。泡囊形成与活性酶的存在之间没有相关性。在许多不产生这些酶的其他革兰氏阳性菌中也可以观察到类似的泡囊，但在革兰氏阴性菌中没有观察到。为了对热脱硫肠状菌 EM1 中的α-淀粉酶和普鲁兰酶进行免疫电子显微镜定位，应用了两种不同的抗血清。一种是针对从培养液中分离的酶制备的，另一种是针对根据天然细胞外α-淀粉酶的 N 端氨基酸序列（21 个氨基酸）合成的肽制备的。使用这些抗血清，在连续培养或分批培养中采集的样品中，α-淀粉酶和普鲁兰酶被定位在细胞周围。在通过冷冻置换随后进行超薄切片制备的电子显微镜样品中，可以看到泡囊，并且免疫标记定位α-淀粉酶酶颗粒不仅随机分布在细胞周围，而且还沿着细胞质膜和泡囊的表面排列。用两种抗血清标记的具有高或几乎没有酶活性的细胞相似。这一发现强烈表明，α-淀粉酶和普鲁兰酶可能存在于活性和非活性两种形式，这取决于生长条件。
