Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili
Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili
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DOI：
10.1128/jb.182.9.2498-2506.2000
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发表时间：
2000-05-01
影响因子：
3.2
通讯作者：
Donnenberg, MS
中科院分区：
文献类型：
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作者：
Anantha, TP;Stone, KD;Donnenberg, MS
致病性大肠杆菌表达一种IV型菌毛，称为菌毛形成菌毛（BFP），它是宿主细胞自我聚集和局部粘附（LA）所必需的。14个基因的簇足以在实验室E.杆菌我们已经进行了个别基因的系统诱变，以确定每个突变对BFP生物合成和LA的影响。在这里，我们报告的结构和分析的非极性突变的sis基因的bfp簇，bfPG，bfpB，bfpC，bfpD，bfpP，和bfpH，以及进一步分析的先前描述的bfpA突变株，是无法表达的pilin，菌毛蛋白。我们发现，bfpB，它编码外膜蛋白的突变; bfpD，它编码一个假定的核苷酸结合蛋白;和bfpG和bfpC，这在其他IV型菌毛系统中没有序列同源物，不影响prefabullin的表达或加工，但阻止BFP生物合成和LA。前菌毛蛋白肽酶基因bfpP的突变不影响前菌毛蛋白的表达，但阻断了前菌毛蛋白、BFP生物合成和LA的信号序列切割。预测编码溶解性转糖基酶的bfpH突变对prebundlin表达、prebundlin加工、BFP生物发生或LA没有影响。对于检测到表型改变的每个突变体，与含有相应野生型的质粒互补。型等位基因恢复了野生型表型。我们还发现，协会的premartlin或remartlin与蔗糖密度浮选梯度级分含有浴内外膜蛋白不需要任何辅助蛋白。这些研究表明，许多bfp基因产物的功能IV型皮利的生物发生所需的，但在个别基因的突变不会导致菌毛组装的新阶段的识别。
Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in sis genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing bath inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.