Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisboa, Portugal.
BMC Microbiol. 2010 Jun 23;10:180. doi: 10.1186/1471-2180-10-180.
The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell.
Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport.
This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology.
先前已经证明，苜蓿中华根瘤菌中的 TolC 蛋白对于与紫花苜蓿建立成功的生物固氮共生关系是必需的。它还需要在蛋白质和胞外多糖分泌以及抵御渗透和氧化应激方面发挥作用。在这里，描述了自由生活的苜蓿中华根瘤菌 1021tolC 突变体的转录谱，作为理解其在细胞生理学中作用的一步。
tolC 突变体和野生型菌株转录组的比较表明，有 1177 个基因的表达显著增加，而 325 个基因的表达水平显著降低。表达增加的基因表明细胞质和细胞外应激反应的激活，可能分别由 sigma 因子 RpoH1 和肠杆菌的 CpxRA 双组分调节系统的蛋白质同源物介导。应激条件可能是由细胞包膜的扰动引起的。与基因表达数据一致，生化分析表明 tolC 突变体受到氧化应激的影响。这可以通过检测到的过氧化氢酶、超氧化物歧化酶和谷胱甘肽还原酶的酶活性水平升高来证明。观察到参与中心代谢和营养物质摄取转运体的基因表达增加，表明 tolC 突变体的代谢率更高。我们还证明了 tolC 突变菌株的 swarm 运动增加。功能缺失的 TolC 导致参与氮代谢和运输的基因表达降低。
这项工作表明，外膜蛋白 TolC 的突变，常见于许多细菌运输系统，如何影响许多协同作用以恢复细胞内稳态的基因的表达。这一发现进一步强调了该蛋白在苜蓿中华根瘤菌生物学中的基本作用。
