Lo Hsueh-Hsia, Chang Hsiao-Ching, Liao Chao-Tsai, Hsiao Yi-Min
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, 40601, Taiwan.
Antonie Van Leeuwenhoek. 2022 May;115(5):589-607. doi: 10.1007/s10482-022-01725-9. Epub 2022 Mar 23.
ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.
ATP 依赖性蛋白酶（FtsH、Lon 和 Clp 家族蛋白）在细菌中普遍存在，在众多细胞调节过程中发挥着重要作用。野油菜黄单胞菌野油菜致病变种是一种革兰氏阴性病原菌，可在十字花科植物中引起黑腐病。野油菜黄单胞菌野油菜致病变种的基因组中有多个 clp 基因，即 clpS、clpA、clpX、clpP、clpQ 和 clpY。在这些基因中，已知只有 clpX 和 clpP 是致病性所必需的。在此，我们聚焦于两个未被表征的 clp 基因（clpS 和 clpA），它们分别编码 ClpAP 蛋白酶复合体的衔接蛋白（ClpS）和 ATP 酶亚基（ClpA）。转录分析表明，clpS 和 clpA 的表达呈生长阶段依赖性，并受生长温度影响。clpA 的失活而非 clpS 的失活导致对高温敏感且在寄主植物中致病力减弱。clpA 突变体的表型改变可通过反式互补得到恢复。定点诱变显示，K223 和 K504 是 ClpA 在耐热性方面发挥功能的关键氨基酸残基。clpA 突变体在热应激下的蛋白质表达谱与野生型不同。总之，我们通过研究它们在包括胁迫耐受性和致病性等不同过程中的表达谱和功能，对两个 clp 基因（clpS 和 clpA）进行了表征。我们证明了 clpS 和 clpA 以温度依赖性方式表达，并且 clpA 是野油菜黄单胞菌野油菜致病变种在高温下存活和完全致病力所必需的。这项工作首次对野油菜黄单胞菌中的 clpS 和 clpA 进行了表征。
