A second prepilin peptidase gene in Escherichia coli K-12.
A second prepilin peptidase gene in Escherichia coli K-12.
复制标题
大肠杆菌 K-12 中的第二个前菌毛蛋白肽酶基因。
DOI：
10.1046/j.1365-2958.1998.00723.x
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发表时间：
1998
影响因子：
3.6
通讯作者：
Pugsley,AP
中科院分区：
文献类型：
--
作者：
Francetić,O;Lory,S;Pugsley,AP
在 37°C 或 42°C（但不是在 30°C）下生长的大肠杆菌 K-12 菌株会加工淋病奈瑟菌 IV 型菌毛蛋白 PilE 和产酸克雷伯菌 IV 型假菌毛蛋白 PulG 的前体，其方式让人想起这些细菌中发生的这些蛋白质的前菌毛蛋白肽酶依赖性加工。大肠杆菌中 prePulG 的加工需要在 -1 位有甘氨酸，同源前菌毛蛋白肽酶 (PulO) 的加工也是如此，并且不受使几种非特异性蛋白酶失活的突变的影响。这些数据表明大肠杆菌K-12具有功能性的前菌毛蛋白肽酶，尽管事实上它本身在允许prePilE和prePulG加工的条件下似乎不表达IV型菌毛蛋白或假菌毛蛋白基因。大肠杆菌 K-12 基因组包含两个编码与前菌毛蛋白肽酶具有显着序列相似性的蛋白质的基因：基因图谱上第 74.5 分钟的 gspO 和第 67 分钟的 pppA (f310c)。我们之前已经获得证据表明gspO编码活性酶但不被转录。pppA被克隆并显示编码能够处理典型的前菌毛蛋白肽酶底物的功能性前菌毛蛋白肽酶。 pppA 的失活消除了内源性热诱导的前菌毛蛋白肽酶活性。当从高拷贝数质粒表达时，PppA 能够取代在大肠杆菌中重建的支链淀粉酶分泌系统中的 PulO 前菌毛蛋白肽酶，但当存在于单个染色体拷贝中时则不能。 pppA-lacZ融合体的分析表明pppA表达非常低并且在翻译水平上受到生长温度的调节，这与观察到的PppA活性的温度依赖性一致。聚合酶链反应和Southern杂交分析揭示了15个大肠杆菌分离株中的12个中存在pppA基因。
Escherichia coliK‐12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of theNeisseria gonorrhoeaetype IV pilin PilE and theKlebsiella oxytocatype IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase‐dependent processing of these proteins that occurs in these bacteria. Processing of prePulG inEscherichia colirequires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non‐specific proteases. These data suggested thatE.coliK‐12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. TheE.coliK‐12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases:gspOat minute 74.5 andpppA (f310c)at minute 67 on the genetic map. We have previously obtained evidence thatgspOencodes an active enzyme but is not transcribed.pppAwas cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation ofpppAeliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted inE.coliwhen expressed from high‐copy‐number plasmids but not when present in a single chromosomal copy. The analysis ofpppA–lacZfusions indicated thatpppAexpression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of thepppAgene in 12 out of 15E.coliisolates.