Comparison of gene expression in spo0J mutant and wild-type B. subtilis. Bacillus subtilis

We investigated the genome-wide DNA binding of the chromosome partitioning and sporulation protein and ParB family member Spo0J in Bacillus subtilis using chromatin immunoprecipitation and DNA microarrays. We identified ten parS loci to which Spo0J binds, two of which were unexpectedly distant (>1 Mb) from the origin of replication. We used all ten sites to refine the consensus sequence for parS. We found that Spo0J spreads along the DNA around each site. Binding was near maximal levels up to 1.6 kb away from parS, and significantly above background as far away as 18 kb. Deletion of soj (parA) had no detectable effect on spreading. In contrast, the spo0J93 allele appeared to cause a significant decrease in spreading in vivo, without significantly affecting the DNA binding affinity in vitro. spo0J93 causes a phenotype similar to that of a spo0J null mutant and alters the region thought to be involved in interaction between Spo0J dimers. Our findings indicate that spreading is important for in vivo function of Spo0J. Gene expression in areas near parS sites was similar in wild-type and a spo0J null mutant, indicating that binding and spreading of Spo0J on DNA does not silence transcription of nearby genes. Keywords: genetic modification Overall design: Gene expression was compared in spo0J mutant and wild-type B. subtilis by isolating total RNA from three independent cultures of each strain and comparing them on microarrays. One of the arrays (GSM152506) used the reverse dye assignments relative to the other two.

本研究采用染色质免疫沉淀（chromatin immunoprecipitation, ChIP）与DNA微阵列（DNA microarray）技术，对枯草芽孢杆菌（Bacillus subtilis）中参与染色体分离与孢子形成的ParB家族蛋白Spo0J的全基因组DNA结合特征展开分析。研究共鉴定出10个Spo0J结合的parS位点，其中2个距离复制起始位点（origin of replication）超过1 Mb，该结果超出预期。我们利用全部10个位点优化了parS的保守序列（consensus sequence）。研究发现，Spo0J会沿结合位点周围的DNA发生扩散：在距离parS位点1.6 kb以内的区域，其结合水平接近最大值；而在最远18 kb的区域内，结合水平仍显著高于背景信号。敲除soj（parA）基因对该扩散过程无显著可检测影响。与之相反，spo0J93等位基因（allele）可在体内显著降低Spo0J的扩散能力，但并未显著改变其体外DNA结合亲和力。spo0J93等位基因所引发的表型与spo0J基因完全敲除突变体一致，且会改变被认为参与Spo0J二聚体（dimer）相互作用的区域。本研究结果表明，DNA扩散过程对于Spo0J的体内功能至关重要。在野生型与spo0J完全敲除突变体中，parS位点邻近区域的基因表达水平无显著差异，这说明Spo0J在DNA上的结合与扩散并不会沉默邻近基因的转录。关键词：基因修饰 整体实验设计：通过分别提取spo0J突变体与野生型枯草芽孢杆菌各3份独立培养物的总RNA，利用DNA微阵列比较二者的基因表达水平；其中1张芯片（GSM152506）采用了与另外2张芯片相反的染料标记方案。