Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64
Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64
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DOI：
10.1007/s00438-005-1143-8
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发表时间：
2005-06-01
影响因子：
3.1
通讯作者：
Komano, T
中科院分区：
文献类型：
--
作者：
Akahane, K;Sakai, D;Komano, T
在许多 IV 型菌毛中，菌毛蛋白亚基的 N 末端氨基酸是 N-甲基化苯丙氨酸。前菌毛蛋白肽酶从前体中去除前导肽，并将新形成的苯丙氨酸的氨基甲基化。 Pi IS 是质粒 R64 编码的菌毛蛋白前体，由前菌毛蛋白肽酶 PilU 加工，但成熟菌毛蛋白的 N 端氨基酸是经过其他修饰的非甲基化色氨酸。为了研究PilU结构和功能之间的关系，通过PCR和定点诱变构建了42个错义pilU突变体，并分析了这些pilU突变体在液体培养中与pilU无效突变体互补交配的能力。尽管实际上没有注意到 21 个突变体发生接合，但其余 21 个突变体支持不同水平的残留质粒转移活性。两个在保守基序中被天冬氨酸取代的突变体没有表现出 PilU 活性，这表明 pilU 基因的产物是天冬氨酸肽酶，如霍乱弧菌的前菌毛蛋白肽 TcpJ。在 21 个突变体中未检测到 PilS 加工，但其余 21 个突变体表现出不同水平的残留 PilS 加工。注意到残留的 PilS 加工活性和接合转移之间存在密切相关性，表明 pilU 基因产物具有前菌毛蛋白肽酶活性，但不能甲基化 N 末端色氨酸。基于编码 PilU 不同片段的 pilU-phoA 和 pilU-lacZ 融合基因的活性，还提出了该蛋白质的膜拓扑模型。此外，发现pilU-phoA和pilU-lacZ融合基因的pilU部分中的一些氨基酸取代改变了产物的膜拓扑结构。
In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. Pi IS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.