Aukema Kelly G, Kron Erin M, Herdendorf Timothy J, Forest Katrina T
Department of Bacteriology, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706-1521, USA.
J Bacteriol. 2005 Jan;187(2):611-8. doi: 10.1128/JB.187.2.611-618.2005.
PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic alpha helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases.
PilT是一种六聚体ATP酶，在革兰氏阴性菌IV型菌毛回缩过程中发挥作用。IV型菌毛的回缩介导细菌与宿主细胞的紧密附着和信号传递、表面运动、生物膜形成、自然转化以及噬菌体敏感性。我们研究了PilT中独特且高度保守的C末端AIRNLIRE基序中每个氨基酸在体内和体外的作用。通过表面运动性丧失和噬菌体敏感性测定，A288、I289、L292和I293氨基酸的替换以及R290和R294的双重替换消除了铜绿假单胞菌PilT在体内的功能。当将AIRNLIRE基序中的替换引入纯化的嗜热栖热菌PilT时，并未破坏ATP酶活性或寡聚化。相比之下，广泛保守的核苷酸结合基序中的K136Q替换在体内和体外均阻止了PilT的功能。我们提出，AIRNLIRE基序形成一个两亲性α螺旋，该螺旋在表面暴露的蛋白质相互作用位点和PilT的ATP酶核心之间传递信号，并且我们认识到其他II型分泌ATP酶中存在潜在的功能同源性。
