Bayer M H, Keck W, Bayer M E
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
J Bacteriol. 1990 Jan;172(1):125-35. doi: 10.1128/jb.172.1.125-135.1990.
We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.
我们报道了青霉素结合蛋白1b（PBP 1b）在大肠杆菌KN126及含有质粒pHK231的过量表达构建体中的定位。我们使用PBP 1b特异性抗血清对全细胞超薄切片进行免疫电子显微镜观察，并对细胞质和分离的膜组分进行免疫电泳分析。我们研究了逐步降温后包埋的戊二醛固定细胞以及在低温包埋剂K4M和HM20中进行冷冻置换的冷冻固定细胞的超薄切片。大部分PBP 1b特异性标记见于内膜（IM）和相邻的细胞质，在外膜（OM）中观察到的较少；在大量细胞质中也可见相当数量的标记。标记的分布和强度均与温度有关：温度升至37℃，导致构建体中PBP 1b过量表达，结果显示IM的标记有统计学上高度显著的增加，包括与IM相邻的一个细胞质区域（深度至少30nm），我们将该区域称为膜相关区域。伴随温度升高，在大量细胞质中观察到标记密度降低。在IM-OM接触区域（膜粘连区域）也发现标记增加。周质未显示明显标记。蛋白质印迹法（免疫印迹法）在大多数分离的膜组分中检测到PBP 1b；然而，在中等密度的膜组分中发现标记密度最高，这支持了膜粘连区域中PBP 1b浓度增加的观点。总之，我们提出PBP 1b存在于细胞质的膜相关区域，蛋白质（如PBP 1b或硫氧还蛋白）可从该区域进入其在包膜中的特定插入位点。使用多种免疫电子显微镜方法首次明确证明了PBP 1b定位于膜粘连位点。由于这些位点被抗PBP 1b血清特异性标记，我们推测它们包含肽聚糖层组装和生长机制的部分成分。
