Karatzas Kimon A G, Wouters Jeroen A, Gahan Cormac G M, Hill Colin, Abee Tjakko, Bennik Marjon H J
Wageningen Centre of Food Sciences, Wageningen, The Netherlands.
Mol Microbiol. 2003 Sep;49(5):1227-38. doi: 10.1046/j.1365-2958.2003.03636.x.
A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.
先前已分离出一种自发的耐高静水压（HHP）的单核细胞增生李斯特菌ScottA突变体，命名为AK01。与野生型（wt）相比，该突变体无运动能力，且对热、酸和H2O2的抗性增强（Karatzas，K.A.G.和Bennik，M.H.J. 2002年，《应用与环境微生物学》68：3183 - 3189）。在本研究中，我们最终将菌株AK01对HHP和应激耐受性的增强与ctsR（三类应激基因阻遏物）中一个密码子的缺失联系起来，该缺失位于编码高度保守甘氨酸重复序列的区域。CtsR负向调节clp基因的表达，包括clpP、clpE和clpC操纵子（包括ctsR自身），这些基因属于III类热休克基因。用突变的ctsR基因（命名为ctsRDeltaGly）在wt背景下替换ctsR基因，使突变体的表型和蛋白质表达谱与菌株AK01相同。在ctsRDeltaGly突变体中，CtsR、ClpC和ClpP蛋白的表达水平显著高于wt菌株，表明CtsRDeltaGly蛋白无活性。进一步证明CtsRDeltaGly蛋白缺乏其阻遏功能的证据来自以下发现：突变体中的Clp蛋白在热休克后未进一步诱导，且ctsR缺失菌株的HHP耐受性与ctsRDeltaGly突变体一样高。高HHP耐受性可能是由于在没有（活性）CtsR阻遏物的情况下clp基因表达增加所致。重要的是，与wt菌株相比，表达CtsRDeltaGly的菌株毒力显著减弱；然而，在突变菌株中未发现PrfA失调的迹象。我们的数据突出了CtsR富含甘氨酸区域在抗逆性和毒力中的重要调节作用。
