Beadle Center for Genetics, School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666, USA.
RNA. 2011 Jul;17(7):1381-92. doi: 10.1261/rna.2679911. Epub 2011 May 27.
The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.
泉古菌门包括处于动态热条件下的高温微生物。先前对 Sulfolobus solfataricus 的转录组学研究鉴定 vapBC6 为 Vap 毒素-抗毒素基因家族中的热休克（HS）诱导成员。在这项研究中，通过靶向基因破坏使 vapBC6 操纵子失活产生了与适应性相关的两个隐性表型，分别为 HS 敏感性和生长速度对热的依赖性降低。对框内 vapBC6 缺失突变体进行了分析，以检查每种蛋白质的各自作用。由于 vapB6 转录物丰度在 vapC6 缺失时升高，因此 VapC6 毒素似乎调节其同源抗毒素的丰度。相比之下，在 vapB6 缺失时 vapC6 转录物丰度降低。推定的基因间终止子可能通过协调 vapBC6 表达来解释这些观察结果。正如结构建模所预测的，使用伴侣共合成生产的重组 VapC6 对 S. solfataricus 总 RNA 表现出热依赖性核糖核酸酶活性。通过添加预加热的重组 VapB6 可以阻止这种活性。通过评估在 vapBC6 缺陷细胞中自然响应热应激的基因的相对表达来鉴定体内转录靶标。在 vapBC6 缺陷细胞中观察到 dppB-1 和 tetR 的优先增加，以及 rpoD 和 eIF2 gamma 的优先减少。在体外，也可以针对在 vapBC6 缺陷菌株中在热休克期间表达增加的 RNA 观察到特定的 VapC6 核糖核酸酶作用。这些发现为 vapBC6 介导的对微生物生长和在极端温度下生存的控制提供了生化机制，并确定了细胞靶标。
