Klein G, Zmijewski M, Krzewska J, Czeczatka M, Lipińska B
Department of Biochemistry, University of Gdańsk, Poland.
Mol Gen Genet. 1998 Aug;259(2):179-89. doi: 10.1007/s004380050803.
We cloned the DNA region of the Vibrio harveyi chromosome containing the heat shock genes dnaK and dnaJ and sequenced them. These genes are arranged in the chromosome in the order dnaK-dnaJ, as in other proteobacteria of the alpha and gamma subdivisions. The dnaK gene is 1923 nucleotides in length and codes for a protein of 640 amino acid residues, with a predicted molecular mass of 69,076 Da and 81.2% similarity to the DnaK protein of Escherichia coli. The V. harveyi dnaJ gene has a coding sequence of 1158 nucleotides. The predicted DnaJ protein contains 385 amino acids, its calculated molecular mass is 41,619 Da and it has 74.7% similarity to the DnaJ protein of E. coli. Northern hybridization experiments with RNA from V. harveyi cells and a DNA probe carrying both the dnaK and dnaJ genes showed a single, heat-inducible transcript, indicating that these genes form an operon. Primer extension analysis revealed five heat-inducible transcriptional start sites upstream of the dnaK gene, two of which (T1 and T4) are preceded by sequences typical of the E. coli heat shock promoters recognized by the sigma 32 (sigma32) factor. Location of these promoters is highly similar to that of the E. coli dnaK promoters. No transcriptional start sites were detected upstream of the dnaJ gene. The V. harveyi dnaKJ operon cloned in a plasmid in E. coli cells was transcribed in a sigma32 dependent manner and the size of the transcript, the kinetics of transcription, and the transcriptional start sites were as in V. harveyi cells. This indicates a high conservation of the transcriptional heat shock regulatory elements between E. coli and V. harveyi, both belonging to the gamma subdivision of proteobacteria. We tested the ability of the cloned dnaKJ genes to complement E. coli dnaK and dnaJ mutants and found that V. harveyi DnaJ restored a thermoresistant phenotype to dnaJ mutants and enabled lambda phage to grow in the mutant cells. V. harveyi DnaK did not suppress the thermosensitivity of dnaK mutants but complemented the dnaK deletion mutant with respect to growth of lambda phage. V. harveyi DnaK, in contrast to DnaJ, failed to modulate the heat shock response in E. coli. Our results suggest that the DnaK chaperone may be more species specific than the DnaJ chaperone.
我们克隆了哈维氏弧菌染色体中包含热休克基因dnaK和dnaJ的DNA区域并进行了测序。这些基因在染色体上按dnaK - dnaJ的顺序排列，与α和γ亚群的其他变形菌一样。dnaK基因长度为1923个核苷酸，编码一个由640个氨基酸残基组成的蛋白质，预测分子量为69,076道尔顿，与大肠杆菌的DnaK蛋白相似度为81.2%。哈维氏弧菌的dnaJ基因有一个1158个核苷酸的编码序列。预测的DnaJ蛋白包含385个氨基酸，计算分子量为41,619道尔顿，与大肠杆菌的DnaJ蛋白相似度为74.7%。用来自哈维氏弧菌细胞的RNA和携带dnaK和dnaJ基因的DNA探针进行的Northern杂交实验显示有一个单一的、热诱导转录本，表明这些基因形成一个操纵子。引物延伸分析揭示了dnaK基因上游有五个热诱导转录起始位点，其中两个（T1和T4）之前有被σ32（sigma32）因子识别的典型大肠杆菌热休克启动子序列。这些启动子的位置与大肠杆菌的dnaK启动子高度相似。在dnaJ基因上游未检测到转录起始位点。克隆到大肠杆菌细胞质粒中的哈维氏弧菌dnaKJ操纵子以依赖σ32的方式转录，转录本大小、转录动力学和转录起始位点与哈维氏弧菌细胞中的情况相同。这表明大肠杆菌和哈维氏弧菌（均属于变形菌的γ亚群）之间转录热休克调节元件具有高度保守性。我们测试了克隆的dnaKJ基因互补大肠杆菌dnaK和dnaJ突变体的能力，发现哈维氏弧菌DnaJ使dnaJ突变体恢复了耐热表型，并使λ噬菌体能够在突变细胞中生长。哈维氏弧菌DnaK没有抑制dnaK突变体的热敏感性，但在λ噬菌体生长方面互补了dnaK缺失突变体。与DnaJ不同，哈维氏弧菌DnaK未能调节大肠杆菌中的热休克反应。我们的结果表明，DnaK伴侣蛋白可能比DnaJ伴侣蛋白具有更强的物种特异性。
