Biology subjects

Nimura-Matsune, K.

Publications and source records attributed to Nimura-Matsune, K..

3 recordsLinked to original sources

bioRxiv2023

Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering

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 Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited. Synechocystis 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 Synechocystis 6803, slr6031 and slr6090, 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 Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 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 Synechococcus 7942 and Synechocystis 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.

microbiology↗

bioRxiv2022

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The [~]100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

microbiology↗

bioRxiv2022

Exploration of the autonomous replication region and its utilization for expression vectors in cyanobacteria.

Due to their photosynthetic capabilities, cyanobacteria is expected to be an ecologically friendly host for the production of biomaterials. However, compared to other bacteria, there is little information of autonomous replication sequences, and tools for genetic engineering, especially expression vector systems, are limited. In this study, we established an effective screening method, namely AR-seq (Autonomous Replication sequencing), for finding autonomous replication regions in cyanobacteria and utilized the region for constructing expression vector. AR-seq using the genomic library of Synechocystis sp. PCC 6803 revealed that a certain region containing Rep-related protein (here named as Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. The reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in a wide range of multiple cyanobacterial species, not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, the GFP expression in pYS-based system can be tightly regulated by IPTG, achieving 10-fold higher levels than that of chromosome-based system. Furthermore, pYS can be used together with conventional vector pEX, which was constructed from an endogenous plasmid in 5. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

synthetic biology↗