Ohdate Kazuma, Sakata Minori, Maeda Kaisei, Sakamaki Yutaka, Nimura-Matsune Kaori, Ohbayashi Ryudo, Hess Wolfgang R, Watanabe Satoru
Department of Bioscience, Faculty of Life Science, Tokyo University of Agriculture, Tokyo, Japan.
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.
Front Microbiol. 2024 Feb 14;15:1311290. doi: 10.3389/fmicb.2024.1311290. eCollection 2024.
Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in 6803, and , both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The -containing plasmids exhibited lower copy numbers and instability in 7942 cells compared to the expression vector pYS. While recombination occurred in the case of , the engineered plasmid with coexisted with plasmids encoding CyRepA1 or Slr6090 in 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid." Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in 7942 and 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.
许多能够进行产氧光合作用的蓝细菌拥有多个大小超过100 kbp的大型质粒。这些质粒被认为具有独特的复制和分配机制，因为它们在细胞内共存而不会导致质粒之间的不相容性。然而，关于蓝细菌中质粒复制蛋白（Rep）的信息有限。集胞藻属PCC 6803宿主四个大型质粒，即pSYSM、pSYSX、pSYSA和pSYSG，但除了pSYSA上的CyRepA1外，这些质粒的Rep蛋白均未知。利用自主复制测序（AR-seq），我们在集胞藻6803中鉴定出两个潜在的Rep基因，即 和 ，它们都位于pSYSX上。相应的Rep候选蛋白Slr6031和Slr6090与其他细菌的Rep相关蛋白具有结构相似性，并且在各种蓝细菌中也鉴定出了同源物。我们通过将它们的基因与表达绿色荧光蛋白（GFP）的构建体融合，并通过转化引入集胞藻PCC 7942中，观察到了Slr6031和Slr6090的自主复制活性。与表达载体pYS相比，含有 的质粒在集胞藻7942细胞中表现出较低的拷贝数和不稳定性。虽然 在这种情况下发生了重组，但含有 的工程质粒在集胞藻7942细胞中与编码CyRepA1或Slr6090的质粒共存，这表明Slr6031和Slr6090与CyRepA1具有相容性。基于这些结果，我们分别将Slr6031和Slr6090命名为CyRepX1（编码在pSYSX上的蓝细菌Rep相关蛋白）和CyRepX2，这表明pSYSX是一个“一个质粒中有两个Rep”的质粒。此外，我们测定了集胞藻7942和集胞藻6803中含有蓝细菌Rep的质粒的拷贝数和稳定性，以阐明它们的潜在应用。本研究揭示的CyRepX1和2的新特性为开发蓝细菌中的创新基因工程工具带来了希望。
