Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Campus de la UAM, 28049, Madrid, Spain.
Extremophiles. 2012 May;16(3):485-95. doi: 10.1007/s00792-012-0448-x. Epub 2012 Apr 19.
Regular surface protein layers (S-layers) from most Gram-positive bacteria and from the ancestral bacterium Thermus thermophilus attach to pyruvylated polysaccharides (SCWP) covalently bound to the peptidoglycan through their SLH domain. However, it is not known whether the synthesis of SCWP and S-layer is coordinated enough as to follow a similar pattern of incorporation to the cell wall during growth. In this work we analyse the localization of newly synthesized SCWP on the cell wall of T. thermophilus by immunoelectron microscopy. For this, we obtained mutants with a reduced amount of pyruvylated SCWP through mutation of the csaB gene encoding the SCWP-pyruvylating activity, and its upstream gene csaA, a putative sugar transporter. We hypothesized that CsaA would be required for the synthesis of the SCWP. However, we found that csaA mutants showed only a minor decrease in the amount of SCWP immunodetected on the cell walls in comparison with csaB mutants, revealing its irrelevance in the process. Complementation experiments of csaB mutants with CsaB expressed from inducible promoters revealed that newly synthesized SCWP was homogeneously distributed along the cell wall. Fusions with thermostable fluorescent protein revealed that CsaB was distributed also in homogeneous pattern associated with the membrane. These data support that synthesis of SCWP takes place in disperse and homogeneous form all over the cell surface, in contrast to the zonal incorporation at the cell centre recently demonstrated for SlpA.
大多数革兰氏阳性菌和古细菌 Thermus thermophilus 的常规表面蛋白层 (S-层) 通过其 SLH 结构域与通过共价键结合到肽聚糖上的丙酮酸化多糖 (SCWP) 结合。然而，目前尚不清楚 SCWP 和 S-层的合成是否足够协调，以至于在生长过程中以类似的方式整合到细胞壁中。在这项工作中，我们通过免疫电子显微镜分析了 T. thermophilus 细胞壁上新合成的 SCWP 的定位。为此，我们通过突变编码 SCWP-丙酮酸化活性的 csaB 基因及其上游基因 csaA（一种假定的糖转运蛋白）获得了 SCWP 量减少的突变体。我们假设 CsaA 将用于合成 SCWP。然而，我们发现与 csaB 突变体相比，csaA 突变体在细胞壁上免疫检测到的 SCWP 量仅略有减少，表明其在该过程中无关紧要。用可诱导启动子表达的 CsaB 对 csaB 突变体进行互补实验表明，新合成的 SCWP 均匀分布在细胞壁上。与热稳定荧光蛋白的融合显示 CsaB 也以与膜相关的均匀模式分布。这些数据支持 SCWP 的合成以弥散和均匀的形式发生在整个细胞表面，与最近在 SlpA 中证明的在细胞中心的区域整合形成对比。
