naica® digital PCR system facilitates quantitative detection of key genes for Vibrio cholerae replication and verifies conjectures
2024-05-06
2024-05-06
Introduction
Vibrio cholerae is the pathogen of human cholera, which is one of the most ancient and widespread infectious diseases. It has caused many pandemics in the world, mainly manifested by severe vomiting, diarrhea, dehydration, and a high mortality rate. It is an international quarantine infectious disease. Understanding the replication principle of Vibrio cholerae can help people explore its infection mechanism more systematically.
Scientists from the Pasteur Institute in France and the Department of Bacterial Genetics at the University of Warsaw published an academic paper in the internationally renowned journal Nucleic Acids Research, revealing the replication mechanism of Vibrio cholerae. (IF=16.971)

Application highlights:
▶ Revealed the replication coordination mechanism of the two chromosomes of Vibrio cholerae.
▶ The key regulatory genes of Vibrio cholerae replication were quantitatively detected and the hypothesis was verified by the naica® droplet chip digital PCR system.
▶ The replication mechanism of Vibrio cholerae discovered in this study may exist in all Vibrio species, which has reference significance for the replication research of other Vibrio.
Research background:
Vibrio cholerae is the pathogen that causes cholera. It replicates in a carefully arranged order with two chromosomes (Chr1 and Chr2). The study found that Chr2 initiation is triggered only after the crtS site on Chr1 is replicated. This study provides new ideas on how crtS triggers the initiation of Chr2 replication and conducts an in-depth discussion on the replication coordination mechanism of Chr1-Chr2.
Research results:
❶, crtS (located on Chr1, promoter binding site) and 39m site (located on Chr2, promoter binding site) affect the replication of Chr2 by competing with the promoter RctB for binding. The presence of crtS reduces the binding of RctB to 39m.

▲crtS can counteract the inhibitory effect of the 39m site. Sequence alignment of the Chr2 replication origin (ori2) and the 39m site. RctB binding sites are indicated in green (iterons, promoter binding sites) and red (39m).
❷, RctB is divided into 4 structural thresholds, and studies have found that it interacts with crtS and 39m through the same DNA binding domain. Further studies have shown that RctB domain IV is indispensable for the regulation of 39m and crtS at the ori2 (Chr2) start site, and the C-terminus of RctB domain IV is essential for crtS to coordinate the replication of the two chromosomes.

Based on this regulatory model, the article used the naica® droplet chip digital PCR system (Stilla Technologies) to quantify (ori1/ori2) in Vibrio cholerae and (oriC/pORI2) in control Escherichia coli. At the same time, the naica® multiplex digital RT-dPCR (Stilla Technologies) was used to quantify RctB mRNA in Vibrio cholerae from exponentially growing cultures (OD600 0.4) and confirmed the correctness of the above hypothesis.

▲B. Copy number (CN) of pORI2 relative to the chromosome of E. coli strains, and various pORI2 deletion expression vectors were constructed by inserting a stop codon in rctB. C. Ratio of pORI2 copy number in E. coli with and without a chromosomal crtS site (+/- crtS). D. Copy number of Chr2 relative to Chr1 (ori2/ori1) in non-replicating V. cholerae. In all mutants, the crtS site was knocked out and the RctB binding site was inserted into the attTn7 insertion site of Chr1 (mean ± standard deviation).
Finally, the article explains the replication mechanism model of V. cholerae:

▲Model of crtS coordinating replication between Chr1 and Chr2. RctB binding sites are shown in green (iterons), red (39m), and blue (crtS).
OFF = Chr1: crtS is not replicated. Chr2: RctB bound to the 39m site inhibits replication initiation of ori2 primarily as indicated by the red arrow.
ON = Chr1: crtS is replicated. Transient binding of RctB to the replicated crtS site results in reduced affinity for the 39m site (blue arrow), thereby releasing ori2. Binding of RctB to methylated iterons results in opening of the DNA unwinding element (DUE) and oligomerization of RctB onto single-stranded DNA (green arrow).
Journal Introduction:
Nucleic Acids Research (NAR): Founded in 1974, it is a peer-reviewed, openly published scientific journal by Oxford University Press. The journal mainly publishes cutting-edge research results on the physics, chemistry, biochemistry and biology of nucleic acids and proteins involving nucleic acid metabolism and/or interactions. The latest impact factor is 16.971.