Pedraza-Reyes M, Gutiérrez-Corona F, Nicholson W L
Institute of Investigation in Experimental Biology, Faculty of Chemistry, University of Guanajuato, Mexico.
J Bacteriol. 1994 Jul;176(13):3983-91. doi: 10.1128/jb.176.13.3983-3991.1994.
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination
细菌芽孢对紫外线辐射具有高度抗性，并表现出独特的DNA光化学性质。紫外线照射芽孢DNA会导致芽孢光产物（SP）的形成，即胸腺嘧啶二聚体5-胸腺嘧啶基-5,6-二氢胸腺嘧啶。枯草芽孢杆菌芽孢萌发过程中，SP的修复通过两条不同途径进行，要么通过一般核苷酸切除修复（uvr）途径，要么通过由spl基因编码的SP裂解酶介导的新型SP特异性单体化反应。SP的修复在芽孢萌发早期发生，且不依赖于从头合成蛋白质，这表明SP修复酶是在芽孢形成过程中合成并包装在休眠芽孢中的。为了验证这一假设，在枯草芽孢杆菌生长和芽孢形成过程中监测了整合在spl位点的翻译型spl-lacZ融合基因的表达。在营养生长期间，spl-lacZ融合基因的β-半乳糖苷酶表达处于沉默状态，且不受DNA损伤诱导，但在芽孢形成的形态学阶段III，特别是在前芽孢区室中被激活，这与阶段III标记酶葡萄糖脱氢酶表达的激活同时发生。结果表明，spl-lacZ融合基因的表达依赖于含有σ-G因子（EσG）的芽孢形成特异性RNA聚合酶，因为在sigG基因缺失的突变体中，spl-lacZ表达被消除，而通过反式表达sigG基因可恢复其表达。对spl mRNA的引物延伸分析显示，一个主要延伸产物起始于spl之前一个功能未知的小开放阅读框上游，还显示出另外两个较短的次要延伸产物。在芽孢形成期间和sigG诱导后，所有这三种延伸产物的量都更高。这三种推定的转录本之前都有与共有σ-G因子型启动子序列具有同源性的序列，但仅从与主要延伸产物对应的启动子检测到纯化的σ-G RNA聚合酶的体外转录。因此，开放阅读框-spl操纵子似乎是σ-G调控子的另一个成员，该调控子还包括小的、酸溶性芽孢蛋白，这些蛋白在很大程度上负责芽孢DNA光化学性质。因此，正在形成芽孢的细菌似乎协同调节其产物不仅改变芽孢DNA光化学性质，而且在萌发过程中修复主要的芽孢特异性光产物的基因。
