Escherichia coli trxA gene as a molecular marker for genome engineering of felixounoviruses,Biochimica et Biophysica Acta (BBA) - General Subjects

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Escherichia coli trxA gene as a molecular marker for genome engineering of felixounoviruses
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 2.8 ) Pub Date : 2021-07-27 , DOI: 10.1016/j.bbagen.2021.129967
Monika Šimoliūnienė ₁ , Darius Kazlauskas ₂ , Aurelija Zajančkauskaitė ₁ , Rolandas Meškys ₁ , Lidija Truncaitė ₁

Affiliation

Background

Bacterial viruses (bacteriophages or phages) have a lot of uncharacterized genes, which hinders the progress of their applied research. Functional characterization of these genes is often hampered by a lack of suitable methods for engineering of phage genomes.

Methods

Phages vB_EcoM_Alf5 (Alf5) and VB_EcoM_VpaE1 (VpaE1) were used as the model phages of Felixounovirus genus. The phage-coded properties were predicted by bioinformatics analysis. The ‘pull-down’ assay was used for detection of protein-protein interactions. Primer extension analysis was used for the DNA polymerase (DNAP) activity testing. Bacteriophage lambda Redγβα-assisted homologous recombination was used for construction of phage mutants.

Results

Bioinformatics analysis showed that felixounoviruses encode DNA polymerase, which is homologous to the T7 DNAP. We found that the Escherichia coli thioredoxin A (TrxA) in vitro interacts with the predicted DNAP of Alf5 phage (gp096) and enhances its activity. Phages Alf5 and VpaE1 do not grow on E. coli strains lacking trxA gene unless it is provided in trans. This feature was used for construction of the deletion/insertion mutants of non-essential genes of felixounoviruses.

Conclusion

DNA replication of phages from Felixonuvirus genus depends on the host trxA, which therefore may be used as a molecular marker for their genome engineering.

General significance

We present a proof-of-principle of a strategy for targeted engineering of bacteriophages of Felixounovirus genus. The method developed here will facilitate the basic and applied research of this unexplored phage group. Furthermore, detected functional interactions between the phage and host proteins will be significant for basic research of DNA replication.

中文翻译：

大肠杆菌trxA基因作为felixounoviruses基因组工程的分子标记

背景

细菌病毒（噬菌体或噬菌体）有很多未表征的基因，阻碍了其应用研究的进展。由于缺乏合适的噬菌体基因组工程方法，这些基因的功能表征常常受到阻碍。

方法

噬菌体 vB_EcoM_Alf5 (Alf5) 和 VB_EcoM_VpaE1 (VpaE1) 被用作Felixounovirus属的模型噬菌体。通过生物信息学分析预测噬菌体编码的特性。“下拉”分析用于检测蛋白质-蛋白质相互作用。引物延伸分析用于 DNA 聚合酶 (DNAP) 活性测试。噬菌体 lambda Redγβα 辅助同源重组用于构建噬菌体突变体。

结果

生物信息学分析表明，felixounoviruses 编码与 T7 DNAP 同源的 DNA 聚合酶。我们发现体外大肠杆菌硫氧还蛋白 A (TrxA)与预测的 Alf5 噬菌体 (gp096) 的 DNAP 相互作用并增强其活性。噬菌体 Alf5 和 VpaE1 不会在缺少trxA基因的大肠杆菌菌株上生长，除非它以反式形式提供。该特征用于构建felixounoviruses非必需基因的缺失/插入突变体。