Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus
Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus
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DOI：
10.1186/1471-2180-13-166
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发表时间：
2013-07-18
期刊：
影响因子：
4.2
通讯作者：
Brun, Yves V.
中科院分区：
文献类型：
--
作者：
Curtis, Patrick D.;Klein, David;Brun, Yves V.
背景：α变形菌新月柄杆菌细胞周期中的极性发育被整合到单个突变可以对极性细胞器的合成产生多效性影响的程度。编码组氨酸激酶 PleC 或其定位因子 PodJ 的基因遭到破坏，会破坏菌毛、鞭毛和粘附固着物的合成或功能。然而，这些突变影响极地发育的机制尚不清楚。本研究的目的是确定控制极细胞器发育多个方面的新调节因子。结果：为了鉴定具有多效性极细胞器合成缺陷的突变体，进行了转座子诱变，并根据对毛嗜性噬菌体 phi CbK 的抗性选择了突变体。然后筛选突变体的运动性和固着力生产方面的缺陷。仅分离出一个独立于 podJ/pleC 的突变体，该突变体在所有三种表型上均存在缺陷。定向噬菌体测定证实了噬菌体抗性表型，而该菌株在群琼脂中表现出与 podJ 突变体相似的扩散半径，并且用标记固着物的荧光凝集素处理表明该突变体没有染色。转座子已插入 ctrA 的启动子区域，ctrA 是编码细胞周期主转录调节因子的基因，破坏了天然转录，但仍然降低了这种必需蛋白质的转录活性和蛋白质产量。转录融合表明，由CtrA控制的必需基因在ctrA启动子突变体中表现出轻微到中度的表达变化，而编码菌毛丝亚基的pilA基因的基因表达急剧下降。在其天然启动子下引入 ctrA 的质粒生成副本，可以补充噬菌体抗性和固定缺陷，以及中度细胞形态缺陷，但不能弥补集群缺陷。结论：鉴定出一种突变，该突变会导致极细胞器合成中的多效性缺陷，并揭示了令人惊讶的结果，即一些 CtrA 依赖性启动子比其他启动子对 CtrA 浓度的变化更敏感。然而，在新的调控因子中没有发现多效性突变的事实表明，PleC/PodJ 的下游信号传导要么是必需的，要么是冗余的，要么是分支的，因此所有三种表型不会同时受到影响。
Background: Polar development during the alphaproteobacterium Caulobacter crescentus cell cycle is integrated to the point that individual mutations can have pleiotropic effects on the synthesis of polar organelles. Disruption of the genes encoding the histidine kinase PleC, or its localization factor PodJ, disrupts synthesis or functionality of pili, flagella and adhesive holdfast. However, the mechanism by which these mutations affect polar development is not well understood. The aim of this study was to identify new regulators that control multiple aspects of polar organelle development.Results: To identify mutants with pleiotropic polar organelle synthesis defects, transposon mutagenesis was performed and mutants were selected based resistance to the pili-tropic bacteriophage phi CbK. Mutants were then screened for defects in motility and holdfast production. Only a single podJ/pleC-independent mutant was isolated which had defects in all three phenotypes. Directed phage assays confirmed the phage resistance phenotype, while the strain demonstrated a similar dispersal radius as a podJ mutant in swarm agar, and treatment with a fluorescent lectin that labels the holdfast showed no staining for this mutant. The transposon had inserted into the promoter region of ctrA, a gene encoding a master transcriptional regulator of the cell cycle, disrupting native transcription but still allowing reduced transcriptional activity and protein production of this essential protein. Transcriptional fusions showed that essential genes controlled by CtrA exhibited minor to moderate changes in expression in the ctrA promoter mutant, while the pilA gene, encoding the subunit of the pilus filament, had a drastic decrease in gene expression. Introduction of a plasmid-born copy of ctrA under its native promoter complemented the phage resistance and holdfast defects, as well as a moderate cell morphology defect, but not the swarming defect.Conclusions: A mutation was identified that caused pleiotropic defects in polar organelle synthesis, and revealed the surprising result that some CtrA-dependent promoters are more sensitive to changes in CtrA concentration than others. However, the fact that no pleiotropic mutations were found in new regulators suggests that downstream signaling of PleC/PodJ is either essential, redundant, or branching such that all three phenotypes were not simultaneously affected.