Discovery of substance involved in plasmid replication in photosynthetic cyanobacteria — Expectations for a new genetic engineering tool

A collaborative research effort led by Graduate Student Kazuma Ohdate and Associate Professor Satoru Watanabe, both from the Faculty of Life Science, Tokyo University of Agriculture, along with the partners from the Laboratory of Chemistry and Life Science of the Institute of Innovative Research of the Tokyo Institute of Technology, the Faculty of Science of Shizuoka University, and the Faculty of Biology of the University of Freiburg in Germany, announced that they newly identified CyRepX, a protein involved in plasmid replication in photosynthetic cyanobacteria. They have also engineered vectors incorporating this protein, which are expected to be used as a new genetic engineering tool. The study findings were published in the February 14^th online issue of the international academic journal Frontiers in Microbiology.

Cyanobacteria, also known as blue-green algae and speculated to be the ancestors of plant chloroplasts, are prokaryotic microalgae that reproduce employing oxygen-evolving photosynthesis, similar to that of plants. These microalgae require no organic carbon source for reproduction, grow while absorbing CO₂ through photosynthesis, and reproduce rapidly. They are expected to be carbon-neutral hosts for the production of useful substances.

However, unlike general industrial microorganisms in which genetic modification is used to confer traits, the genetic engineering tools available for cyanobacteria are limited. In particular, plasmids (large DNA molecules that exist separately from chromosomal DNA and replicate independently) which have potential for use as vectors, are not well understood. Moreover, unlike model microorganisms such as Escherichia coli, many cyanobacterial species have been found to have multiple plasmids. Since plasmids usually cannot coexist in the same cell if they have the same replication mechanism (incompatibility), cyanobacteria with multiple plasmids are thought to have different replication mechanisms.

In this study, the research group examined Synechocystis sp. PCC 6803 (PCC6803), which is the first cyanobacterium to have its entire genome sequenced. This organism possesses four large plasmids (pSYSM, pSYSX, pSYSA, and pSYSG) in addition to its major chromosomes. Previously, the research group had identified CyRepA, a protein factor involved in pSYSA replication.

The research group identified two novel genes (slr6031 and slr6090) coding for proteins with replication activity through comprehensive screening of a genomic library. Located on pSYSX, these two genes were considered to be homologs with similar functions because of their high sequence similarity. Although the proteins encoded by these genes are widely conserved among cyanobacteria, their functions were unknown.

Analysis using a 3D structure prediction program revealed that the 3D structures were similar to those of DNA replication proteins from other microorganisms. Expression vectors p6031 and p6090 were constructed using these two genes, and the replication activity was assessed using a heterologous cyanobacterial strain (PCC7942). As a result, these proteins were retained in strain PCC7942. The researchers concluded that these proteins are novel replication factors, designating them as CyRepX.

The researchers further developed expression vectors carrying CyRepX. They can coexist with CyRepA and other plasmids in PCC7942 cells, mimicking the coexistence of multiple plasmids. It has been shown that foreign genes can be expressed in combination with existing vectors.

The mechanism of cyanobacterial reproduction remains to be clarified.

Watanabe said, "Cyanobacteria have been used for photosynthesis research for a long time, but there are still many things we do not know, such as their reproduction mechanism. We have discovered new DNA replication factors by combining classical genetic methods with advanced analytical techniques. Other DNA replication factors have been found, and we believe that future studies on the functions and coordination of these factors will help answer the fundamental question of how cyanobacteria reproduce."

Journal Information
Publication: Frontiers in Microbiology
Title: Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering
DOI: 10.3389/fmicb.2024.1311290