Expression of yeeK during Bacillus subtilis Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy
Expression of yeeK during Bacillus subtilis Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy
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DOI：
10.1128/jb.01269-08
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发表时间：
2008-12
影响因子：
3.2
通讯作者：
H. Takamatsu;D. Imamura;Ritsuko Kuwana;K. Watabe
中科院分区：
文献类型：
--
作者：
H. Takamatsu;D. Imamura;Ritsuko Kuwana;K. Watabe
枯草芽孢杆菌yeek基因编码145个氨基酸，由28%甘氨酸、23%组氨酸和12%酪氨酸残基组成。以前的研究无法在野生型孢子中检测到YEK；然而，已经在YabG突变孢子中发现了18 kDa的YEK多肽。在本研究中，我们分析了yeek的表达和定位，以探讨yeek与YabG之间的关系。对野生型RNA的Northern杂交分析表明，yeek基因的转录在孢子形成5h后启动，依赖于含有SigK的RNA聚合酶和Gere蛋白。YEK基因的破坏不会损害营养生长、抗性孢子的发育或萌发。荧光显微镜观察YEKK与绿色荧光蛋白(YEK-GFP)和红色荧光蛋白(YEK-RFP)的框内融合，证实YEKK组装成孢子层。正确定位YEK-GFP需要COTE、SAFA和SpoVID。YEKK-RFP和YabG的框内GFP融合的比较分析表明，YEKK与YabG在孢子被层中共定位。这是第一次使用荧光蛋白来显示孢子被的不同层的定位。用抗GFP抗血清进行免疫印迹实验表明，YEK-GFP是以44 kDa的分子形式合成的，并被消化成29 kDa的片段，与野生型孢子中GFP的分子大小一致。相反，在yabG突变孢子中只有极少量的44 kDa YEEK-GFP被消化。我们的发现表明，YEK是由Cote、Safa和SpoVID引导进入孢子壳的。我们的结论是YabG直接或间接参与了YEK的消化。
ABSTRACT The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.