Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis
Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis
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DOI：
10.1111/omi.12122
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发表时间：
2016-06-01
影响因子：
3.7
通讯作者：
Fletcher, H. M.
中科院分区：
文献类型：
--
作者：
Dou, Y.;Aruni, W.;Fletcher, H. M.
牙龈卟啉单胞菌中的PG0162是一个胞质外功能(ECF)西格玛因子，由193个氨基酸组成。正如先前报道的那样，缺失PG0162的突变体P.gigivalis FLL350与亲本菌株相比，牙周疼痛活性显著降低。由于该ECF sigma因子可能参与牙龈假单胞菌的毒力调节，因此对其遗传特性进行了进一步的研究。5‘-RACE分析表明，PG0162基因转录起始于ATG翻译起始密码子上游69个核苷酸的鸟嘌呤(G)残基。以pG0162启动子为模板，从大肠杆菌中纯化的rPG0162和RNAP核心酶进行体外转录，证实了pG0162作为Sigma因子的功能。由于PG0162诱导环境信号的适宜性尚不清楚，因此我们建立了一株高表达PG0162基因的菌株，命名为P.gigivalis FLL391。与野生型相比，牙龈假单胞菌FLL391的转录组分析表明，大约24%的基因组显示出基因表达的变化(260个上调基因，286个下调基因)。另外两个ECF西格玛因子(PG0985和PG1660)上调了两倍以上。在凝胶迁移率改变实验中，pG0162蛋白与pG0162启动子结合，证实了pG0162的自身调节作用。此外，rPG0162蛋白还能与牙龈假单胞菌中表达最高的两个基因(PG0521和PG1167)的启动子区域结合。综上所述，我们的数据表明，PG0162是一个西格玛因子，可能在牙龈假单胞菌的毒力调控网络中发挥重要作用。
PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P.gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P.gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off invitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P.gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P.gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P.gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P.gingivalis.