Gilois Nathalie, Ramarao Nalini, Bouillaut Laurent, Perchat Stéphane, Aymerich Stéphane, Nielsen-Leroux Christina, Lereclus Didier, Gohar Michel
Institut National de la Recherche Agronomique (INRA), Génétique microbienne et Environnement, Guyancourt, France.
Proteomics. 2007 May;7(10):1719-28. doi: 10.1002/pmic.200600502.
Using 2-DE, transcriptional gene fusions and cell cytotoxicity assays, we followed changes in the Bacillus cereus strain ATCC14579 secretome, gene expression and culture supernatant cytotoxicity from the end of the vegetative phase up to 5 h after entry into the stationary phase. The concentration of each of the 22 proteins in the culture supernatant was determined at various times. In addition, the stability of the proteins was studied. Fifteen of these proteins, including 14 members of the virulence regulon PlcR, were known or predicted to be secreted. All of the secreted proteins reached a maximum concentration during early stationary phase, but there were significant differences in the kinetics of their concentrations. The time courses of protein concentrations were in agreement with gene expression data, except for cytotoxin CytK, which was unstable, and for the metalloprotease InhA1. Supernatant cytoxicity also peaked in early stationary phase, and the kinetics of cytotoxicity paralleled the time course of concentration of the PlcR-controlled toxin, CytK. Our concomitant study of the time course of protein concentrations, gene expression and supernatant cytotoxicity reveals that the pathogenic potential of B. cereus peaks during the transition state. It also suggests that there is diversity in the regulation of gene expression within the PlcR regulon.
我们运用双向电泳、转录基因融合以及细胞毒性测定等方法，追踪了蜡样芽孢杆菌ATCC14579菌株分泌蛋白组、基因表达及培养上清液细胞毒性从对数生长期末期直至进入稳定期后5小时的变化情况。在不同时间点测定了培养上清液中22种蛋白质各自的浓度。此外，还研究了这些蛋白质的稳定性。其中15种蛋白质，包括毒力调节子PlcR的14个成员，已知或预测为分泌型蛋白质。所有分泌蛋白在稳定期早期达到最大浓度，但它们浓度的动力学变化存在显著差异。蛋白质浓度的时间进程与基因表达数据一致，但细胞毒素CytK不稳定，金属蛋白酶InhA1则不然。培养上清液的细胞毒性在稳定期早期也达到峰值，且细胞毒性的动力学变化与PlcR调控毒素CytK的浓度时间进程平行。我们对蛋白质浓度、基因表达及培养上清液细胞毒性时间进程的同步研究表明，蜡样芽孢杆菌的致病潜力在过渡状态时达到峰值。这也表明PlcR调节子内基因表达的调控存在多样性。
