Requirement of novel competence genes pilT and pilU of Pseudomonas stutzeri for natural transformation and suppression of pilT deficiency by a hexahistidine tag on the type IV pilus protein PilAI
Requirement of novel competence genes pilT and pilU of Pseudomonas stutzeri for natural transformation and suppression of pilT deficiency by a hexahistidine tag on the type IV pilus protein PilAI
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DOI：
10.1128/jb.183.16.4694-4701.2001
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发表时间：
2001-08-01
影响因子：
3.2
通讯作者：
Wackernagel, W
中科院分区：
文献类型：
--
作者：
Graupner, S;Weger, N;Wackernagel, W
无处不在的stutzeri假单胞菌具有IV型菌毛，这对细胞的自然转化至关重要。转座子诱变后获得的绝对转化缺陷突变体在称为pilT的基因中插入。推导出的氨基酸序列与铜绿假单胞菌（94%）、淋病奈瑟菌（67%）和其他革兰氏阴性菌的PilT序列一致，并含有一个核苷酸结合基序。该突变体有多毛，但在其他与菌毛相关的特性方面存在缺陷，如抽搐运动性和菌毛特异性噬菌体PO4的电镀。[H-3]胸腺嘧啶标记的DNA与突变体结合但未被吸收。在pilT的下游，被称为pilU的基因编码了一个假定的蛋白质，与铜绿假单胞菌的pilU具有88%的氨基酸一致性。插入失活不影响收缩、抽搐运动或PO4感染，但将转化减少约10%。该缺陷被不可转化的铜绿假单胞菌的PilU完全补充。当编码IV型前毛菌的pilAI基因编码一种蛋白质，其中6个c端氨基酸被6个组氨酸残基取代，然后在质粒中表达时，它在pilAI::Gm(r)细胞中产生无毛状和抽搐的运动缺陷表型，但允许转化能力。此外，突变等位基因抑制了由pilT突变引起的绝对转化缺陷。考虑到pilT（+）在菌毛缩回中的假设作用和DNA摄取的假设要求，我们提出，PilA突变蛋白以单分子形式或以周质中可能类似于解聚的菌毛的极小的菌毛组装结构的形式促进了与菌毛无关的转化，这些结构导致外膜孔打开以供DNA进入。
The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed pilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [H-3]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termed pilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable A aeruginosa. When the pilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gm(r) cells but allowed transform ability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT(+) in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.