Institute of Genetics, University of Bayreuth, D-95445 Bayreuth, Germany.
AMB Express. 2011 Jul 21;1(1):22. doi: 10.1186/2191-0855-1-22.
Bacillus subtilis codes for two putative sortases, YhcS and YwpE, and two surface proteins, YhcR and YfkN, harboring sorting motifs supposed to be recognized by the putative sortase(s). However, there is no experimental evidence to show a direct link between these sortases and sorting sequences. To study the role of these two putative sortases on displaying YhcR and YfkN on the cell wall, expression of yhcS and ywpE was analyzed by transcriptional fusions and by Northern blot. It turned out that yhcS gene is expressed at a higher level during the late stationary phase from both experiments, while ywpE expression is not confirmed in the Northern blot analysis. Next, we constructed yhcS and ywpE single and double knockout strains and plasmids that express one or both genes to restore the functions of the knockout strains. It could be shown that display of YhcR and YfkN on the surface depended on the presence of YhcS while YwpE seems not to play a major role if any as a sortase. Finally, the putative sorting motif together with a 123-amino-acid spacer derived from YhcR and YfkN designated YhcR123 and YfkN123, respectively, were fused to an α-amylase reporter enzyme. The fusion protein YhcR123-AmyQ could be displayed on the surface at high amounts, while YfkN123-AmyQ could be hardly detected. We conclude that the sortase YhcS can recognize and anchor YhcR on the cell wall. This result further indicates that the YhcR sorting sequence can be used to display recombinant proteins on the surface of B. subtilis cells.
枯草芽孢杆菌编码两个假定的分选酶，YhcS 和 YwpE，以及两个表面蛋白，YhcR 和 YfkN，它们含有假定被假定的分选酶识别的分选基序。然而，没有实验证据表明这些分选酶和分选序列之间存在直接联系。为了研究这两个假定的分选酶在细胞壁上展示 YhcR 和 YfkN 的作用，通过转录融合和 Northern blot 分析了 yhcS 和 ywpE 的表达。结果表明，yhcS 基因在两个实验中的晚静止期表达水平更高，而 ywpE 的表达在 Northern blot 分析中未得到证实。接下来，我们构建了 yhcS 和 ywpE 的单敲除和双敲除菌株以及表达一个或两个基因以恢复敲除菌株功能的质粒。结果表明，YhcR 和 YfkN 的表面展示依赖于 YhcS 的存在，而 YwpE 似乎不是主要的分选酶。最后，将来自 YhcR 和 YfkN 的假定分选基序和 123 个氨基酸间隔区分别融合到一个α-淀粉酶报告酶上，得到融合蛋白 YhcR123-AmyQ。该融合蛋白可以大量展示在表面上，而 YfkN123-AmyQ 则很难检测到。我们得出结论，分选酶 YhcS 可以识别并将 YhcR 锚定在细胞壁上。这一结果进一步表明，YhcR 分选序列可用于在枯草芽孢杆菌细胞表面展示重组蛋白。
