table1_Acidic pH Decreases the Endonuclease Activity of Initiator RepB and Increases the Stability of the Covalent RepB-DNA Intermediate while Has Only a Limited Effect on the Replication of Plasmid pMV158 in Lactococcus lactis.docx

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 in vitro reactions catalyzed by RepB and on the in vivo 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 in vivo, as monitored by the copy number and segregational stability of the plasmid in Lactococcus lactis, 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 in vivo to allow pMV158 replication at pH values that severely disturb the catalytic activity of the initiator protein.

质粒载体（plasmid vectors）是一类极具应用价值的研究工具，可用于同源与异源基因表达、启动子及调控区域的功能表征，以及细菌的遗传操作与标记。近年来，一系列基于pMV158家族宽宿主域复制子（promiscuous replicons）的载体被开发出来，可应用于多种革兰氏阳性菌（Gram-positive bacteria），并已被证实在乳酸菌（lactic acid bacteria）中可完美适配上述全部应用场景。要合理使用这类质粒载体，需要深入了解其在目标细菌生长条件变化下的核心复制特性，例如因乳酸产生导致的培养基酸化过程。

pMV158的滚环复制（rolling-circle replication）起始过程由质粒编码的RepB蛋白（RepB protein）催化完成：该蛋白可对亲本DNA链中的一条进行序列特异性切割，且如本研究所示，其会与DNA产生的5′-磷酸末端形成共价结合。该共价加合物需持续存在直至前导链终止阶段：此时在重新生成的切口位点进行新一轮切割，并通过后续的链转移反应使被切割的亲本DNA链末端重新连接；而新生成加合物的水解则可将蛋白从带切口的双链DNA质粒形式中释放出来。

本研究分析了pH值对RepB蛋白催化的各类体外（in vitro）反应，以及质粒pMV158体内（in vivo）复制能力的影响。研究结果表明，酸性pH会显著抑制该蛋白的催化活性，并降低RepB-DNA共价加合物的水解效率——这与两类反应的亲核反应本质相符。

相反，通过质粒拷贝数（copy number）与乳酸乳球菌（Lactococcus lactis）中质粒分离稳定性（segregational stability）监测得到的pMV158体内复制能力，在细胞外pH 7.0至5.0的范围内几乎不受影响；仅当pH降至4.5时，每染色体当量（chromosome equivalent）对应的质粒拷贝数才会出现约50%的显著下降。此外，在pH 4.5条件下，RepB与pMV158的摩尔比值有所升高，这提示体内存在补偿机制，可在严重干扰起始蛋白催化活性的pH条件下，保障pMV158的正常复制。