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

Abstract

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.

Peer Review #1 of "A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein (v0.2)"

“…PCC 6803. Although copy numbers were determined by a relatively simple densitometric approach (Projan, Carleton & Novick, 1983), it is still regularly used (e.g., Valdelvira et al, 2021). Thus, our results provide compelling evidence that pMJc01 has an approximately 2-fold higher copy number than pPMQAK1 in E. coli.…”

pDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells

“…It may move from the original cancer in the mammary gland to numerous distant regions (Farhanji et al 2015 ). Moreover, there is a notable similarity between the progressive progression of MC4-L2 metastases in lymphatic arteries and tissues of mouse models of BALB/c and human breast tumors (Zhang et al 2021 ; Valdelvira et al 2021 ). Tumors were observed in untreated mice after a 14-day injection of MC4-L2 cells throughout the experiment.…”

The Facts and Family Secrets of Plasmids That Replicate via the Rolling-Circle Mechanism

“…In addition, one or more cofactors are needed to complete the reaction, namely, metal ions and even small auxiliary proteins (210)(211)(212). In the case of plasmid pMV158, a Tyr residue (Y99) is the active residue in RepB (213,214), whereas a His (H22) is the catalytic residue employed by MobM (215). Proteins RepC pT181 , RepD pC221 , and MobA pC221 require Mg 2+ (216,217).…”

“…(145,214).From the structural point of view, the landscape is discouraging because highresolution structures are available only for DNA-free representatives of the Rep_2 (RepB pMV158 ; (206)) and Rep_trans (Rep pSTK1 and plasmid pC221 chimeric RepDE and RepDN proteins; (207)) families, pointing to the lack of structural understanding on how…”