Simpson W, Wang C Y, Mikolajczyk-Pawlinska J, Potempa J, Travis J, Bond V C, Genco C A
Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Infect Immun. 1999 Oct;67(10):5012-20. doi: 10.1128/IAI.67.10.5012-5020.1999.
We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS1126(1), indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS1126(2), was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis: (i) the transposition of the endogenous insertion sequence element IS1126 and (ii) the modulation of gingipain transcription and translation as a result of IS1126 transposition.
我们之前报道过牙龈卟啉单胞菌的一个由Tn4351产生的突变体（MSM-3），该突变体表达增强的精氨酸特异性蛋白酶活性，且在生长过程中不利用血红素或血红蛋白（C.A. 根科等人，《感染与免疫》63:2459 - 2466，1995）。在对牙龈卟啉单胞菌MSM-3中的基因损伤进行表征的过程中，我们确定牙龈卟啉单胞菌内源性插入序列元件IS1126能够在牙龈卟啉单胞菌内发生转座。我们还确定IS1126转座调节编码赖氨酸特异性蛋白酶牙龈蛋白酶K（kgp）和精氨酸特异性蛋白酶牙龈蛋白酶R2（rgpB）的基因的转录。牙龈卟啉单胞菌MSM-3的序列分析表明，Tn4351插入到牙龈卟啉单胞菌内源性IS元件IS1126上游60 bp处。此外，与亲本菌株A7436相比，牙龈卟啉单胞菌MSM-3表现出另外两个IS1126拷贝。对第一个额外的IS1126元件IS1126(1)的检测表明，它已插入到牙龈卟啉单胞菌kgp基因的假定启动子区域。对从牙龈卟啉单胞菌MSM-3中提取的总RNA的分析表明未检测到kgp转录本；同样，牙龈卟啉单胞菌MSM-3缺乏赖氨酸特异性蛋白酶活性。牙龈卟啉单胞菌MSM-3所表现出的增强的精氨酸特异性蛋白酶活性被证明与rgpA和rgpB转录本的增加相关。发现第二个额外的IS1126元件IS1126(2)插入到一个新鉴定的基因hmuR上游，该基因与许多参与血红素和铁摄取的依赖TonB的基因具有同源性。对牙龈卟啉单胞菌MSM-3的总RNA的分析表明hmuR被转录，这表明IS1126的插入未对hmuR转录产生极性效应。牙龈卟啉单胞菌MSM-3中的血红素 - 血红蛋白缺陷被认为是由于Kgp失活导致的，最近已证明Kgp在血红蛋白结合中起作用。综上所述，此处呈现的结果表明，将Tn4351引入牙龈卟啉单胞菌染色体导致了牙龈卟啉单胞菌中两个以前未记录的现象：（i）内源性插入序列元件IS1126的转座，以及（ii）由于IS1126转座导致牙龈蛋白酶转录和翻译的调节。
