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Phosphorylation status of BolA affects its role in transcription and biofilm development
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-06-14 , DOI: 10.1111/febs.15447 Lisete Galego 1 , Susana Barahona 1 , Célia V Romão 1 , Cecília M Arraiano 1
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-06-14 , DOI: 10.1111/febs.15447 Lisete Galego 1 , Susana Barahona 1 , Célia V Romão 1 , Cecília M Arraiano 1
Affiliation
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BolA has been characterized as an important transcriptional regulator, which is induced in stationary phase of growth, and in response to several stresses. In Escherichia coli, its cellular function is associated with cell wall synthesis and division, morphology, permeability, motility and biofilm formation. Phosphorylation has been widely described as one of the most important events involved in the modulation of the activity of many transcription factors. In the present work, we have demonstrated in vivo and by mass spectrometry that BolA is phosphorylated in four highly conserved protein positions: S26, S45, T81 and S95. S95 is located in the C terminus unstructured region of the protein, and the other three sites are in the DNA‐binding domain. These positions were mutated to nonphosphorylated residues, and their effects were investigated on different known BolA functions. Using northern blot experiments, we showed that the regulation of the expression of these Ser/Thr BolA mutants is performed at the post‐translational level. Western blot results revealed that the stability/turnover of the mutated BolA proteins is differently affected depending on the dephosphorylated residue. Moreover, we provide evidences that phosphorylation events are crucial in the modulation of BolA activity as a transcription factor and as a regulator of cell morphology and biofilm development. Here, we propose that phosphorylation affects BolA downstream functions and discuss the possible significance of these phosphoresidues in the protein structure, stability, dimerization and function as a transcription factor.
中文翻译：
BolA的磷酸化状态影响其在转录和生物膜发育中的作用
BolA已被表征为一种重要的转录调节因子，可在生长的稳定期被诱导并响应多种压力。在大肠杆菌中，其细胞功能与细胞壁合成和分裂，形态，通透性，运动性和生物膜形成有关。磷酸化已被广泛描述为涉及许多转录因子活性调节的最重要事件之一。在目前的工作中，我们已经证明了体内通过质谱分析，BolA在四个高度保守的蛋白质位置（S26，S45，T81和S95）被磷酸化。S95位于蛋白的C末端非结构化区域，其他三个位点位于DNA结合域中。这些位置被突变为非磷酸化的残基，并研究了它们对不同的已知BolA功能的影响。使用northern blot实验，我们证明了这些Ser / Thr BolA突变体的表达调控是在翻译后水平进行的。蛋白质印迹结果表明，突变的BolA蛋白的稳定性/周转率取决于去磷酸化残基的不同。此外，我们提供的证据表明，磷酸化事件对于BolA活性作为转录因子以及细胞形态和生物膜发育的调节剂至关重要。在这里，我们建议磷酸化影响BolA下游功能，并讨论这些磷酸在蛋白质结构，稳定性，二聚作用和作为转录因子中的可能意义。
更新日期：2020-06-14
中文翻译：
BolA的磷酸化状态影响其在转录和生物膜发育中的作用
BolA已被表征为一种重要的转录调节因子，可在生长的稳定期被诱导并响应多种压力。在大肠杆菌中，其细胞功能与细胞壁合成和分裂，形态，通透性，运动性和生物膜形成有关。磷酸化已被广泛描述为涉及许多转录因子活性调节的最重要事件之一。在目前的工作中，我们已经证明了体内通过质谱分析，BolA在四个高度保守的蛋白质位置（S26，S45，T81和S95）被磷酸化。S95位于蛋白的C末端非结构化区域，其他三个位点位于DNA结合域中。这些位置被突变为非磷酸化的残基，并研究了它们对不同的已知BolA功能的影响。使用northern blot实验，我们证明了这些Ser / Thr BolA突变体的表达调控是在翻译后水平进行的。蛋白质印迹结果表明，突变的BolA蛋白的稳定性/周转率取决于去磷酸化残基的不同。此外，我们提供的证据表明，磷酸化事件对于BolA活性作为转录因子以及细胞形态和生物膜发育的调节剂至关重要。在这里，我们建议磷酸化影响BolA下游功能，并讨论这些磷酸在蛋白质结构，稳定性，二聚作用和作为转录因子中的可能意义。
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