Laboratoire de Microbiologie et Génétique Moléculaire (LMGM), UMR5100, Centre National de la Recherche Scientifique (CNRS) and Université Paul Sabatier, Toulouse, France.
PLoS Genet. 2012;8(11):e1003037. doi: 10.1371/journal.pgen.1003037. Epub 2012 Nov 1.
The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.
普遍保守的 J 域蛋白（JDPs）是 Hsp70（DnaK）伴侣蛋白的必需共伴侣。它们刺激 Hsp70 的 ATP 酶活性，促进底物传递，并赋予 Hsp70 特定的细胞定位。在这项工作中，我们已经鉴定和表征了第一个由噬菌体编码的功能性 JDP 蛋白。具体来说，我们表明，T4 相关肠杆菌噬菌体 RB43 的 ORFan 基因 057w 编码一种真正的 JDP 蛋白，命名为 Rki，它与大肠杆菌宿主多功能 DnaK 伴侣蛋白特异性相互作用。然而，与 DnaK 编码的三种已知宿主 JDP 共伴侣形成鲜明对比的是，Rki 在体内或体外都不作为通用共伴侣发挥作用。单独表达 Rki 对野生型大肠杆菌毒性极高，但在没有内源性 DnaK 的情况下，或当 Rki 的保守 J 域发生突变时，毒性会被消除。进一步的体内分析表明，Rki 在 RB43 感染后早期表达，并且 rki 基因的缺失会显著损害 RB43 的增殖。此外，我们表明，宿主 dnaK 基因的突变有效地抑制了 RB43 rki 缺失突变体的生长表型，这表明 Rki 特异性干扰 DnaK 细胞功能。最后，我们表明，Rki 与宿主 DnaK 伴侣蛋白的相互作用迅速导致热休克因子 σ(32)的稳定，而 σ(32)通常被 DnaK 靶向降解。讨论了 Rki 依赖性稳定 σ(32)促进 RB43 噬菌体增殖的机制。
