Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis
Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis
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DOI：
10.1046/j.1365-2958.2003.03779.x
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发表时间：
2003-11-01
影响因子：
3.6
通讯作者：
Hill, TM
中科院分区：
文献类型：
--
作者：
Korch, SB;Henderson, TA;Hill, TM
高频率（10（-2））的大肠杆菌在长时间暴露于青霉素类抗生素中存活的能力，称为高持久性，与hipA基因突变有关。hip操纵子位于dif附近的染色体末端，由两个基因hipA和hipB组成。野生型hipA基因编码毒素，而hipB编码DNA结合蛋白，其自动调节hip操纵子的表达并与HipA结合以消除其毒性作用。我们已经表征了赋予高持久性的hipA 7等位基因，并确定HipA 7是无毒的，包含两个突变（G22 S和D291 A），并且这两个突变是与hip突变体相关的全范围表型所需的。此外，在没有hipB的情况下hipA 7的表达足以建立高持久性表型，表明不需要hipB。hipA 7菌株产生的持留细胞频率与细胞密度之间存在很强的相关性，随着培养物接近稳定期，hipA 7菌株产生持留细胞的频率高20倍。还证明了relA敲除减少了hipA 7突变体中的高持久性表型，并且relA spoT敲除完全消除了高持久性，这表明hipA 7通过诱导（p）ppGpp合成促进持久状态的建立。与该提议一致，显示relA'从质粒的异位表达使由hipA 7 relA双突变体产生的持久细胞的数量增加100倍或更多。一个模型提出，假设hipA 7增加的基础水平（p）ppGpp合成，允许一个显着更大的百分比的细胞在人口中承担一个持久的，抗肿瘤不敏感的状态，通过加强快速过渡到休眠状态后，应用程序的压力。
The ability of a high frequency (10(-2)) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB. The wild-type hipA gene encodes a toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is non-toxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20-fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock-outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock-outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA' from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100-fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibiotic-insensitive state by potentiating a rapid transition to a dormant state upon application of stress.