Kuo Mei-Shiue, Chen Kuei-Peng, Wu Whi Fin
Department of Agricultural Chemistry, Bldg 2, R311, National Taiwan University, Taipei (106), Taiwan, ROC.
Microbiology (Reading). 2004 Feb;150(Pt 2):437-446. doi: 10.1099/mic.0.26446-0.
Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB-lacZ was used to assay for beta-galactosidase activity and thus follow RcsA stability. The expression of cpsB-lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB-lacZ expression. Additionally, a P(BAD)-rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB-lacZ expression. The effect of RcsA on cpsB-lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA(Red)-lacZ translational fusion construct showed higher activity of RcsA(Red)-LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased beta-galactosidase levels of RcsA(Red)-LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.
大肠杆菌ClpYQ蛋白酶和Lon蛋白酶在降解细胞分裂抑制剂SulA方面具有冗余功能。一个实验线索表明，Lon的已知底物——荚膜合成激活剂RcsA可能是ClpYQ蛋白酶的特异性底物。本文表明，ClpQ和ClpY的过表达抑制了lon突变体的黏液样表型。由于参与荚膜合成的cpsB（wcaB）基因由RcsA激活，因此使用报告构建体cpsB-lacZ来检测β-半乳糖苷酶活性，从而追踪RcsA的稳定性。与野生型或lon单突变体相比，在lon与clpQ或/和clpY突变的双突变体中，cpsB-lacZ的表达增加。ClpYQ或ClpQ的过量产生降低了cpsB-lacZ的表达。此外，一个P（BAD）-rcsA融合构建体定量显示，可诱导的RcsA激活cpsB-lacZ的表达。结果表明，RcsA对cpsB-lacZ表达的影响受ClpYQ活性的影响。此外，一个rcsA（Red）-lacZ翻译融合构建体显示，在clpQ clpY菌株中，RcsA（Red）-LacZ的活性高于野生型。相比之下，细胞ClpYQ的过量产生导致RcsA（Red）-LacZ的β-半乳糖苷酶水平降低。综上所述，数据表明ClpYQ作为一种二级蛋白酶参与降解Lon底物RcsA。
