Roles of SigB and SigF in the Mycobacterium tuberculosis sigma factor network
Roles of SigB and SigF in the Mycobacterium tuberculosis sigma factor network
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DOI：
10.1128/jb.01273-07
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发表时间：
2008-01-01
影响因子：
3.2
通讯作者：
Bishai, William R.
中科院分区：
文献类型：
--
作者：
Lee, Jong-Hee;Karakousis, Petros C.;Bishai, William R.
为了研究SigB和Sigf在结核分枝杆菌Sigma因子调控中的作用，我们使用化学诱导的重组菌株有条件地过表达SIGB和Sigf。利用全基因组芯片分析和定量逆转录-聚合酶链式反应，我们研究了SIGB诱导后的整体转录变化，并特异性地检测了SIGB和SIGF敲入表达后其他sigma因子基因的相对表达。SIGB的过表达导致几种早期培养滤液抗原(ESAT-6样蛋白)、核糖体蛋白、PE-PGRs蛋白、酮酰合成酶KASA以及调节蛋白WhiB2和IDER的基因显著上调。值得注意的是，SIGB的诱导并没有改变其他Sigma因子基因的表达，表明SigB可能作为至少一个分支的末端调节因子调节结核分枝杆菌Sigma因子级联。对SigB依赖转录本的5‘-非翻译区(UTR)进行分析，发现了一个推测为NGTGG-N14-18-NNGNNG的一致序列。该序列出现在SIGB(Rv2710)及其后面的基因ider(Rv2711)的上游，重组SigB重组RNA聚合酶体外转录分析证实这些启动子都有SigB依赖的转录。SIGF的敲入表达表明，只有SigC基因在SIGF诱导后6h和12h显著上调。在SigC基因的5‘非编码区中鉴定到了SIGF启动子共有序列AGTTTG-N-15-GGGTTT，并利用重组SIGF重组RNA聚合酶证实了SigF依赖的启动子在SigC上游的体外转录。这两个敲入的重组菌株在巨噬细胞感染模型中进行了测试，结果表明，SIGB和SIGF的过表达导致结核分枝杆菌细胞内生长速度下降。这些结果定义了SigB启动子的共识识别序列和SigB调节子的成员。此外，数据表明，除了在西格玛因子级联中充当末端调节因子外，SigB还可以在特定条件下自动放大自己的表达。
To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5'-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N-15-GGGTTT was identified in the 5' UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.