Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system:: Point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype
Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system:: Point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype
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DOI：
10.1128/jb.187.21.7317-7324.2005
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发表时间：
2005-11-01
影响因子：
3.2
通讯作者：
Melefors, Ö
中科院分区：
文献类型：
--
作者：
Tomenius, H;Pernestig, AK;Melefors, Ö
BarA-UvrY双组分系统家族与毒力密切相关，但在分子水平上了解甚少。在我们尝试补充barA缺失突变体的过程中，我们一致地产生了各种突变的BarA蛋白。我们推断，突变体的表征将有助于我们更好地理解三方传感器中的信号转导机制。这得益于所证明的独立于BarA传感器用乙酰磷酸激活UvrY调节剂的能力。许多突变的BarA蛋白具有较差的互补活性，但可以以显性负性方式抵消野生型传感器的活性。这些蛋白在最近鉴定的HAMP接头中或附近携带点突变，该接头先前涉及周质和细胞质之间的信号转导。这产生了具有受损的激酶活性和净去磷酸化活性的传感器蛋白。使用进一步的HAMP接头突变的蛋白质的定点诱变，我们可以证明，磷酸接受天冬氨酸718和组氨酸861是至关重要的去磷酸化活性。HAMP接头突变的BarA传感器的额外分析表明，脱磷酸化活性可以通过体内三重传感器二聚体内的磷酸转移来操作。这也意味着即使在去磷酸化模式下，三部分传感器也可以被布置为二聚体。
The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.