Graupner S, Weger N, Sohni M, Wackernagel W
Genetik, Fachbereich Biologie, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg, Germany.
J Bacteriol. 2001 Aug;183(16):4694-701. doi: 10.1128/JB.183.16.4694-4701.2001.
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. [(3)H]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 P. 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 transformability. 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.
无处不在的施氏假单胞菌具有IV型菌毛，这些菌毛对于细胞的自然转化至关重要。通过转座子诱变获得的一个完全缺乏转化能力的突变体，在一个被称为pilT的基因中存在插入。推导的氨基酸序列与铜绿假单胞菌的PilT有94%的同一性，与淋病奈瑟菌的有67%的同一性，以及与其他革兰氏阴性菌的有同一性，并且它包含一个核苷酸结合基序。该突变体菌毛过多，但在其他与菌毛相关的特性方面存在缺陷，如颤动运动和菌毛特异性噬菌体PO4的平板接种。突变体结合了用[³H]胸腺嘧啶标记的DNA，但未摄取。在pilT下游鉴定出一个被称为pilU的基因，其编码的推定蛋白质与铜绿假单胞菌的PilU有88%的氨基酸同一性。插入失活不影响菌毛形成、颤动运动或PO4感染，但将转化率降低到约10%。不可转化的铜绿假单胞菌的PilU完全互补了该缺陷。当操纵pilAI基因（编码IV型菌毛前导肽）编码一种蛋白质，其中六个C末端氨基酸被六个组氨酸残基取代，然后从质粒表达时，它在pilAI::Gm(r)细胞中产生了无菌毛和颤动运动缺陷的表型，但允许转化能力。此外，突变等位基因抑制了由pilT突变引起的完全转化缺陷。考虑到pilT(+)在菌毛回缩中的假设作用以及DNA摄取对回缩的推测需求，有人提出，不依赖pilT的转化是由PilA突变蛋白促进的，该蛋白以单分子形式或作为周质中可能类似于解聚菌毛的最小菌毛组装结构存在，并且这些结构导致外膜孔打开以允许DNA进入。
