Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
J Bacteriol. 2012 Dec;194(23):6479-89. doi: 10.1128/JB.01135-12. Epub 2012 Sep 21.
Five genes (cps2E, cps2T, cps2F, cps2G, and cps2I) are predicted to encode the glycosyltransferases responsible for synthesis of the Streptococcus pneumoniae serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant S. pneumoniae enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In in vitro assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose. cps2T, cps2F, and cps2I deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2G mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2I mutants contained different secondary suppressor mutations in cps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2T mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.
五个基因（cps2E、cps2T、cps2F、cps2G 和 cps2I）被预测编码负责合成肺炎链球菌 2 型荚膜重复单位的糖基转移酶，该重复单位聚合生成具有分支表面结构的物质，包含连接到葡萄糖-鼠李糖-鼠李糖-鼠李糖主链的葡萄糖-葡萄糖醛酸。Cps2E 是起始糖基转移酶，但缺乏支持其余糖基转移酶功能的实验证据。为了对糖基转移酶进行生化表征，首先使用重组肺炎链球菌酶 Cps2L、Cps2M、Cps2N 和 Cps2O 合成供体底物 dTDP-鼠李糖。在与每种糖基转移酶的体外测定中，只有包含重组 Cps2T、dTDP-鼠李糖和 Cps2E 产物（十一烯基焦磷酸葡萄糖）的反应混合物才会产生新产物，该产物与脂连接的葡萄糖-鼠李糖一致。cps2T、cps2F 和 cps2I 缺失突变体未产生可检测到的荚膜，但在Δcps2G 突变体中可检测到痕量荚膜，表明 Cps2G 添加非主链糖。所有Δcps2F、Δcps2G 和Δcps2I 突变体在 cps2E 中都含有不同的次要抑制突变，表明在没有重复单位合成减少的情况下，初始突变是致命的。Δcps2T 突变体不含影响荚膜合成的次要突变。在缺乏 Cps2F、Cps2G 和 Cps2I 的突变体中需要次要突变表明这些活性发生在荚膜合成的关键步骤之后，并且表明 Cps2T 催化该步骤。因此，Cps2T 是将第二个糖添加到重复单元的β1-4 鼠李糖基转移酶，并且作为 2 型重复单元合成的关键步骤，预计是荚膜调节的重要靶点。
