Spo0A Mutants of Bacillus subtilis with Sigma Factor-Specific Defects in Transcription Activation

The transcription factor Spo0A of Bacillus subtilis has the unique ability to activate transcription from promoters that require different forms of RNA polymerase holoenzyme. One class of Spo0A-activated promoter, which includes spoIIEp, is recognized by RNA polymerase associated with the primary sigma factor, sigma A (ς(A)); the second, which includes spoIIAp, is recognized by RNA polymerase associated with an early-sporulation sigma factor, sigma H (ς(H)). Evidence suggests that Spo0A probably interacts directly with RNA polymerase to activate transcription from these promoters. To identify residues of Spo0A that may be involved in transcriptional activation, we used PCR mutagenesis of the entire spo0A gene and designed a screen using two distinguishable reporter fusions, spoIIE-gus and spoIIA-lacZ. Here we report the identification and characterization of five mutants of Spo0A that are specifically defective in activation of ς(A)-dependent promoters while maintaining activation of ς(H)-dependent promoters. These five mutants identify a 14-amino-acid segment of Spo0A, from residue 227 to residue 240, that is required for transcriptional activation of ς(A)-dependent promoters. This region may define a surface or domain of Spo0A that makes direct contacts with ς(A)-associated holoenzyme.

枯草芽孢杆菌（Bacillus subtilis）的转录因子Spo0A具备独特能力，可激活依赖不同形式RNA聚合酶全酶（RNA polymerase holoenzyme）的启动子的转录。Spo0A激活的启动子可分为两类：一类包含spoIIEp，可被结合了主要σ因子（primary sigma factor）σ^A（ς(A)）的RNA聚合酶识别；另一类包含spoIIAp，则可被结合了早期芽孢形成σ因子（early-sporulation sigma factor）σ^H（ς(H)）的RNA聚合酶识别。现有证据表明，Spo0A可能通过直接与RNA聚合酶相互作用，来激活上述两类启动子的转录。为了鉴定Spo0A中参与转录激活过程的氨基酸残基，我们对完整的spo0A基因开展了PCR诱变，并基于两种可区分的报告基因融合体——spoIIE-gus与spoIIA-lacZ——设计了筛选方案。本研究成功鉴定并表征了5个Spo0A突变体，这些突变体仅特异性丧失对σ^A依赖型启动子的激活能力，却仍可正常激活σ^H依赖型启动子。通过这5个突变体，我们定位到Spo0A上一段由227号至240号氨基酸残基组成的14肽区段，该区段是激活σ^A依赖型启动子转录所必需的。该区域或许构成了Spo0A与结合了σ^A的RNA聚合酶全酶发生直接接触的表面或结构域。