Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
J Bacteriol. 2019 Jan 28;201(4). doi: 10.1128/JB.00642-18. Print 2019 Feb 15.
At a late stage in spore development in , the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafA, and localizes to the cortex-coat interface in mature spores. SafA is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafA, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafA that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores. spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafA is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVID functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.
在芽胞发育的后期阶段，母细胞指导合成一层称为皮层的肽聚糖，位于两个前芽胞膜之间，以及在芽胞外膜表面组装保护性蛋白衣。SafA 是内衣组装的关键决定因素，首先被招募到发育中的芽胞表面，然后在形态发生蛋白 SpoVID 的控制下包裹芽胞。SafA 具有 LysM 肽聚糖结合结构域，SafA，并定位于成熟芽胞中的皮层-衣界面。SafA 之后是一个区域，A，需要与 SpoVID 相互作用并包裹。我们现在表明，SafA 中的残基 D10 和 N30 虽然参与与肽聚糖的相互作用，但也需要与 SpoVID 相互作用并包裹。我们进一步表明，SafA 上残基 S11、L12 和 I39 的单个丙氨酸取代，强烈削弱与纯化皮层肽聚糖的结合，会影响 SafA 定位的后期阶段，这也依赖于 SpoVE 的活性，SpoVE 是形成皮层所必需的转糖基酶。因此，SafA 的组装涉及到由 LysM 结构域介导的顺序的蛋白-蛋白和蛋白-肽聚糖相互作用，这些相互作用首先需要被包裹，然后需要将蛋白最终定位于成熟芽胞中。芽胞被包裹在一个多蛋白衣中，该衣包围着一个底层的肽聚糖层，即皮层。这两层之间的连接是如何加强的还没有很好的建立。在这里，我们阐明了存在于两个蛋白 SafA 和 SpoVID 中的肽聚糖结合 LysM 结构域的作用，这两个蛋白控制着额外蛋白向衣的定位。我们发现 SafA 是衣组装早期阶段的蛋白-蛋白相互作用模块，也是形态发生后期阶段的皮层结合模块，皮层结合功能促进了皮层和衣之间的紧密连接。相比之下，SpoVID 仅作为一个蛋白-蛋白相互作用结构域发挥作用，在衣组装开始时将 SpoVID 靶向到芽胞表面。
