Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University of M. Curie-Skłodowska, Lublin, Poland.
J Bacteriol. 2013 Aug;195(15):3412-23. doi: 10.1128/JB.02213-12. Epub 2013 May 24.
Rhizobium leguminosarum bv. trifolii pssA encodes a glucosyl-isoprenylphosphate (IP)-transferase involved in the first step of exopolysaccharide (EPS) synthesis. It was found that the pssA gene is an important target for regulation of this biosynthetic pathway. The data of this study indicate that pssA transcription is a very complex and mainly positively regulated process. A detailed analysis of a 767-bp-long pssA upstream region revealed the presence of several sequence motifs recognized by regulatory proteins that are associated with phosphate-, carbon-, and iron-dependent regulation. In addition, numerous inverted repeats of different lengths have been identified in this region. pssA transcription is directed from two distal P1 and proximal P3 promoters whose sequences demonstrate a significant identity to promoters recognized by RNA polymerase sigma factor σ(70). Among rhizobial proteins, RosR seems to be a primary regulator that positively affects pssA expression. This protein binds to RosR box 1 located downstream of the P1 promoter. In addition, phosphate and the carbon source strongly affect pssA transcription. A significantly lower level of pssA expression was observed in both the wild-type strain growing under phosphate-rich conditions and the phoB mutant. In this regulation, the PhoB protein and Pho box 2 located upstream of the P3 promoter were engaged. pssA transcription is also significantly affected by glucose. Transcriptional analysis of a set of pssA-lacZ fusions expressed in Escherichia coli wild-type and cyaA and crp mutants confirmed that cyclic AMP (cAMP) receptor protein (CRP) and two cAMP-CRP boxes located upstream of the P1 are required for this upregulation. Moreover, the production of EPS was totally abolished in R. leguminosarum bv. trifolii mutant strains 4440 and 1012 containing a Tn5 insertion downstream of the P3 promoter and downstream of the P3 -35 hexamer, respectively.
根瘤菌属三叶草亚种 psaA 基因编码一种葡萄糖异戊烯基磷酸（IP）-转移酶，参与多糖（EPS）合成的第一步。研究发现，psaA 基因是调节这一生物合成途径的重要靶标。本研究的数据表明，psaA 转录是一个非常复杂的过程，主要受正调控。对长达 767bp 的 psaA 上游区的详细分析表明，存在几个被认为与磷酸盐、碳和铁依赖调节相关的调节蛋白识别的序列基序。此外，在这个区域还鉴定出了许多不同长度的反向重复序列。psaA 转录由两个远侧 P1 和近侧 P3 启动子指导，其序列与 RNA 聚合酶 sigma 因子σ（70）识别的启动子具有显著的同一性。在根瘤菌蛋白中，RosR 似乎是一种主要的正调控因子，它能正向影响 psaA 的表达。该蛋白与位于 P1 启动子下游的 RosR 框 1 结合。此外，磷酸盐和碳源强烈影响 psaA 转录。在富磷条件下生长的野生型菌株和 phoB 突变体中，psaA 的表达水平明显降低。在这种调节中，PhoB 蛋白和位于 P3 启动子上游的 Pho 框 2 参与其中。葡萄糖也显著影响 psaA 的转录。在大肠杆菌野生型和 cyaA 和 crp 突变体中表达的一组 psaA-lacZ 融合的转录分析证实，环腺苷酸（cAMP）受体蛋白（CRP）和位于 P1 上游的两个 cAMP-CRP 框是这种上调所必需的。此外，在分别位于 P3 启动子下游和 P3-35 六聚体下游携带 Tn5 插入的根瘤菌属三叶草亚种 4440 和 1012 突变株中，EPS 的产生完全被阻断。
