Valdelvira Rafael, Bordanaba-Ruiseco Lorena, Martín-Huestamendía Cristina, Ruiz-Masó José Angel, Del Solar Gloria
Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Front Mol Biosci. 2021 Mar 5;8:634461. doi: 10.3389/fmolb.2021.634461. eCollection 2021.
Plasmid vectors constitute a valuable tool for homologous and heterologous gene expression, for characterization of promoter and regulatory regions, and for genetic manipulation and labeling of bacteria. During the last years, a series of vectors based on promiscuous replicons of the pMV158 family have been developed for their employment in a variety of Gram-positive bacteria and proved to be useful for all above applications in lactic acid bacteria. A proper use of the plasmid vectors requires detailed knowledge of their main replicative features under the changing growth conditions of the studied bacteria, such as the acidification of the culture medium by lactic acid production. Initiation of pMV158 rolling-circle replication is catalyzed by the plasmid-encoded RepB protein, which performs a sequence-specific cleavage on one of the parental DNA strands and, as demonstrated in this work, establishes a covalent bond with the 5'-P end generated in the DNA. This covalent adduct must last until the leading-strand termination stage, where a new cleavage on the regenerated nick site and a subsequent strand-transfer reaction result in rejoining of the ends of the cleaved parental strand, whereas hydrolysis of the newly-generated adduct would release the protein from a nicked double-stranded DNA plasmid form. We have analyzed here the effect of pH on the different reactions catalyzed by RepB and on the replication ability of plasmid pMV158. We show that acidic pH greatly impairs the catalytic activity of the protein and reduces hydrolysis of the covalent RepB-DNA adduct, as expected for the nucleophilic nature of these reactions. Conversely, the ability of pMV158 to replicate , as monitored by the copy number and segregational stability of the plasmid in , remains almost intact at extracellular pHs ranging from 7.0 to 5.0, and a significant reduction (by ∼50%) in the plasmid copy number per chromosome equivalent is only observed at pH 4.5. Moreover, the RepB to pMV158 molar ratio is increased at pH 4.5, suggesting the existence of compensatory mechanisms that operate to allow pMV158 replication at pH values that severely disturb the catalytic activity of the initiator protein.
质粒载体是用于同源和异源基因表达、启动子和调控区域表征以及细菌基因操作和标记的重要工具。在过去几年中，一系列基于pMV158家族混杂复制子的载体已被开发出来，用于多种革兰氏阳性细菌，并被证明对乳酸菌的上述所有应用都很有用。正确使用质粒载体需要详细了解它们在被研究细菌不断变化的生长条件下的主要复制特征，例如乳酸产生导致培养基酸化的情况。pMV158滚环复制的起始由质粒编码的RepB蛋白催化，该蛋白对一条亲代DNA链进行序列特异性切割，并且如本研究所示，与DNA中产生的5'-P末端建立共价键。这种共价加合物必须持续到前导链终止阶段，此时在再生的切口位点进行新的切割以及随后的链转移反应导致切割的亲代链末端重新连接，而新产生的加合物的水解会使蛋白质从带切口的双链DNA质粒形式中释放出来。我们在此分析了pH对RepB催化的不同反应以及质粒pMV158复制能力的影响。我们表明，酸性pH极大地损害了蛋白质的催化活性，并减少了共价RepB-DNA加合物的水解，正如这些反应的亲核性质所预期的那样。相反，通过质粒在细胞中的拷贝数和分离稳定性监测，pMV158的复制能力在细胞外pH值从7.0到5.0范围内几乎保持完整，仅在pH 4.5时观察到每个染色体当量的质粒拷贝数显著减少（约50%）。此外，在pH 4.5时RepB与pMV158的摩尔比增加，这表明存在补偿机制，其作用是允许pMV158在严重干扰起始蛋白催化活性的pH值下进行复制。
