Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering
Open accessAbstract
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. 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 (Cy anobacterial Rep-related protein encoded on pSYS X) 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.
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Replicative plasmids generally contain origins of replication that facilitate autonomous maintenance within the cyanobacterial host.
“Replicative plasmids generally contain origins of replication that facilitate autonomous maintenance within the cyanobacterial host [89].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [89]
The Introduction (page not available) describes an autonomous replication region in a cyanobacterial plasmid, and the Results describe engineered plasmids that replicate autonomously in a cyanobacterial host. The paper supports the link between replication machinery and autonomous replication, but it does not establish the broader claim that replicative plasmids generally contain identified origins of replication that ensure their maintenance. (LLM, conf=0.77)
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Cites
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Saitou, N., & Nei, M. (1987) · Molecular Biology and Evolution · doi:10.1093/oxfordjournals.molbev.a040454 · Cited by 59,962 · see citing works
UCSF Chimera—A visualization system for exploratory research and analysis
Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004) · Journal of Computational Chemistry · doi:10.1002/jcc.20084 · Cited by 45,260 · see citing works
MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013) · Molecular biology and evolution · doi:10.1093/molbev/mst197 · Cited by 41,174 · see citing works
MEGA11: Molecular Evolutionary Genetics Analysis Version 11
Tamura, K., Stecher, G., & Kumar, S. (2021) · Molecular Biology and Evolution · doi:10.1093/molbev/msab120 · Cited by 23,008 · see citing works
Varadi, M., Anyango, S., Deshpande, M., Nair, S., Natassia, C., Yordanova, G., Yuan, D., Stroe, O., Wood, G., Laydon, A., et al. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkab1061 · Cited by 7,850 · see citing works
Fast and accurate protein structure search with Foldseek
van Kempen, M., Kim, S. S., Tumescheit, C., Mirdita, M., Lee, J., Gilchrist, C. L. M., Söding, J., & Steinegger, M. (2022) · doi:10.1101/2022.02.07.479398 · Cited by 2,598 · see citing works
Fast and accurate protein structure search with Foldseek
van Kempen, M., Kim, S. S., Tumescheit, C., Mirdita, M., Lee, J., Gilchrist, C. L. M., Söding, J., & Steinegger, M. (2024) · Nature Biotechnology · doi:10.1038/s41587-023-01773-0 · Cited by 2,596 · see citing works
SMART: recent updates, new developments and status in 2020
Letunic, I., Khedkar, S., & Bork, P. (2020) · Nucleic Acids Res. · doi:10.1093/nar/gkaa937 · Cited by 2,121 · see citing works
VARNA: Interactive drawing and editing of the RNA secondary structure
Darty, K., Denise, A., & Ponty, Y. (2009) · Bioinformatics · doi:10.1093/bioinformatics/btp250 · Cited by 1,375 · see citing works
Improving the coverage of the cyanobacterial phylum using diversity driven genome sequencing
Shih, P. M., Wu, D., Latifi, A., Axen, S. D., Fewer, D. P., Talla, E., Calteau, A., Cai, F., Tandeau de Marsac, N., Rippka, R., et al. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1217107110 · Cited by 800 · see citing works
Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants
Villa, L., García-Fernández, A., Fortini, D., & Carattoli, A. (2010) · Journal of Antimicrobial Chemotherapy · doi:10.1093/jac/dkq347 · Cited by 652 · see citing works
Shintani, M., Sanchez, Z., & Kimbara, K. (2015) · Frontiers in Microbiology · doi:10.3389/fmicb.2015.00242 · Cited by 368 · see citing works
AlphaFold2 and the future of structural biology
Cramer, P. (2021) · Nature Structural & Molecular Biology · doi:10.1038/s41594-021-00650-1 · Cited by 325 · see citing works
AlQuraishi, M. (2019) · Bioinformatics · doi:10.1093/bioinformatics/btz422 · Cited by 312 · see citing works
Gruber, A. R., Bernhart, S. H., & Lorenz, R. (2014) · Methods in molecular biology · doi:10.1007/978-1-4939-2291-8_19 · Cited by 245 · see citing works
Comparative Analysis of the Primary Transcriptome of Synechocystis sp. PCC 6803
Kopf, M., Klähn, S., Scholz, I., Matthiessen, J. K. F., Hess, W. R., & Voß, B. (2014) · DNA Research · doi:10.1093/dnares/dsu018 · Cited by 242 · see citing works
Self-citationCyanobacteria: Promising biocatalysts for sustainable chemical production
Knoot, C. J., Ungerer, J., Wangikar, P. P., & Pakrasi, H. B. (2018) · Journal of Biological Chemistry · doi:10.1074/jbc.r117.815886 · Cited by 225 · see citing works
Klotz, A., Georg, J., Bučinská, L., Watanabe, S., Reimann, V., Januszewski, W., Sobotka, R., Jendrossek, D., Hess, W. R., & Forchhammer, K. (2016) · Current Biology · doi:10.1016/j.cub.2016.08.054 · Cited by 169 · see citing works
Self-citationMicroevolution in Cyanobacteria: Re-sequencing a Motile Substrain of Synechocystis sp. PCC 6803
Trautmann, D., Voß, B., Wilde, A., Al‐Babili, S., & Hess, W. R. (2012) · DNA Research · doi:10.1093/dnares/dss024 · Cited by 144 · see citing works
Self-citationKaneko, T. (2003) · DNA Research · doi:10.1093/dnares/10.5.221 · Cited by 133 · see citing works
Kanesaki, Y., Shiwa, Y., Tajima, N., Suzuki, M., Watanabe, S., Sato, N., Ikeuchi, M., & Yoshikawa, H. (2011) · DNA Research · doi:10.1093/dnares/dsr042 · Cited by 125 · see citing works
Self-citationOsborn, A. M., Tatley, F. D. S., Steyn, L. M., Pickup, R. W., & Saunders, J. R. (2000) · Microbiology · doi:10.1099/00221287-146-9-2267 · Cited by 125 · see citing works
Light‐dependent and asynchronous replication of cyanobacterial multi‐copy chromosomes
Watanabe, S., Ohbayashi, R., Shiwa, Y., Noda, A., Kanesaki, Y., Chibazakura, T., & Yoshikawa, H. (2012) · Molecular Microbiology · doi:10.1111/j.1365-2958.2012.07971.x · Cited by 73 · see citing works
Self-citationWhat's in a name? The case of cyanobacteria
García‐Pichel, F., Zehr, J. P., Bhattacharya, D., & Pakrasi, H. B. (2019) · Journal of Phycology · doi:10.1111/jpy.12934 · Cited by 66 · see citing works
Biocatalysis in Green and Blue: Cyanobacteria
Jodlbauer, J., Rohr, T., Oliver Spadiut, A., Mihovilovič, M. D., & Rudroff, F. (2021) · Trends in biotechnology · doi:10.1016/j.tibtech.2020.12.009 · Cited by 63 · see citing works
Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria
Watanabe, S., Ohbayashi, R., Kanesaki, Y., Saito, N., Chibazakura, T., Soga, T., & Yoshikawa, H. (2015) · PLoS ONE · doi:10.1371/journal.pone.0136800 · Cited by 55 · see citing works
Self-citationYang, X., & McFadden, B. A. (1993) · Journal of Bacteriology · doi:10.1128/jb.175.13.3981-3991.1993 · Cited by 47 · see citing works
Jin, H., Wang, Y., Idoine, A., & Bhaya, D. (2018) · Frontiers in Microbiology · doi:10.3389/fmicb.2018.01662 · Cited by 46 · see citing works
Functional analysis of pSM19035-derived replicons in Bacillus subtilis
Cegłowski, P., Lurz, R., & Alonso, J. C. (1993) · FEMS Microbiology Letters · doi:10.1111/j.1574-6968.1993.tb06159.x · Cited by 43 · see citing works
Plasmid pSM19035, a model to study stable maintenance in Firmicutes
Lioy, V. S., Pratto, F., de la Hoz, A. B., Ayora, S., & Alonso, J. C. (2010) · Plasmid · doi:10.1016/j.plasmid.2010.04.002 · Cited by 40 · see citing works
Xu, W., & McFadden, B. A. (1997) · Plasmid · doi:10.1006/plas.1997.1281 · Cited by 33 · see citing works
Yang, X., & McFadden, B. A. (1994) · Plasmid · doi:10.1006/plas.1994.1014 · Cited by 33 · see citing works
Nagy, C., Thiel, K., Mulaku, E., Mustila, H., Tamagnini, P., Aro, E.-M., Pacheco, C. C., & Kallio, P. (2021) · Microbial Cell Factories · doi:10.1186/s12934-021-01622-2 · Cited by 33 · see citing works
Berla, B. M., & Pakrasi, H. B. (2012) · Applied and Environmental Microbiology · doi:10.1128/aem.01174-12 · Cited by 32 · see citing works
Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector
Itaya, M. (2003) · The Journal of Biochemistry · doi:10.1093/jb/mvg168 · Cited by 30 · see citing works
Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria
Opel, F., Siebert, N. A., Klatt, S., Tüllinghoff, A., Hantke, J. G., Toepel, J., Bühler, B., Nürnberg, D. J., & Klähn, S. (2022) · ACS Synthetic Biology · doi:10.1021/acssynbio.1c00605 · Cited by 30 · see citing works
Gärtner, K., Klähn, S., Watanabe, S., Mikkat, S., Scholz, I., Hess, W. R., & Hagemann, M. (2019) · Frontiers in Microbiology · doi:10.3389/fmicb.2019.01233 · Cited by 28 · see citing works
Self-citationFinished Genome Sequence of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6714
Kopf, M., Klähn, S., Voß, B., Stüber, K., Hüettel, B., Reinhardt, R., & Hess, W. R. (2014) · Genome Announcements · doi:10.1128/genomea.00757-14 · Cited by 14 · see citing works
Self-citationKaltenbrunner, A., Reimann, V., Hoffmann, U. A., Aoyagi, T., Sakata, M., Nimura-Matsune, K., Watanabe, S., Steglich, C., Wilde, A., & Hess, W. R. (2023) · Frontiers in Microbiology · doi:10.3389/fmicb.2023.1112307 · Cited by 12 · see citing works
Self-citationPhotosynthetic 1,8-cineole production using cyanobacteria
Sakamaki, Y., Ono, M., Shigenari, N., Chibazakura, T., Shimomura, K., & Watanabe, S. (2023) · Bioscience Biotechnology and Biochemistry · doi:10.1093/bbb/zbad012 · Cited by 11 · see citing works
Self-citationSakamaki, Y., Maeda, K., Nimura-Matsune, K., Chibazakura, T., & Watanabe, S. (2023) · Frontiers in Microbiology · doi:10.3389/fmicb.2023.1111979 · Cited by 11 · see citing works
Self-citationFunctional analysis of pSM19035-derived replicons in Bacillus subtilis
Cegłowski, P. (1993) · FEMS Microbiology Letters · doi:10.1016/0378-1097(93)90011-p · Cited by 10 · see citing works
Itaya, M., Kusakabe, H., Sato, M., Tomita, M., & Sato, R. (2018) · The Journal of Biochemistry · doi:10.1093/jb/mvy026 · Cited by 9 · see citing works
Estimation of the Chromosomal Copy Number in Synechococcus elongatus PCC 7942
Watanabe, S., & Yoshikawa, H. (2016) · BIO-PROTOCOL · doi:10.21769/bioprotoc.1855 · Cited by 3 · see citing works
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Klähn, S., Opel, F., & Hess, W. R. (2024) · Green Carbon · doi:10.1016/j.greenca.2024.05.002 · Cited by 27 · see citing works
Self-citationChen, M., Dhakal, D., Eckhardt, C. W., Luesch, H., & Ding, Y. (2025) · Natural Product Reports · doi:10.1039/d5np00009b · Cited by 21 · see citing works
Improving viral annotation with artificial intelligence
Flamholz, Z., Li, C., & Kelly, L. (2024) · mBio · doi:10.1128/mbio.03206-23 · Cited by 12 · see citing works
Prathima, R., Basalingappa, K. M., Sai Kavya, D., Arjun, K. R., Girish Kanavi, K., Suresh, J., Murugesan, K., Radhakrishnan, A., Kandaswamy, D., Roy, B., et al. (2024) · Luminescence · doi:10.1002/bio.4898 · Cited by 4 · see citing works
Walshe, J., & Saha, S. K. (2026) · Current Issues in Molecular Biology · doi:10.3390/cimb48060638 · Cited by 1 · see citing works
“Replicative plasmids generally contain origins of replication that facilitate autonomous maintenance within the cyanobacterial host [89].”
Cyanobacterial Biofertilizer Production by Guanidine-Producing Enzymes
Lee, H., Sebesta, J., Schaedig, E., Wu, C., Pakrasi, H. B., & Yu, J. (2026) · ACS Synthetic Biology · doi:10.1021/acssynbio.5c00801 · Cited by 1 · see citing works
Genetic Modifications in Cyanobacteria: Current Status and Future Prospects
Tripathi, K., & Mishra, Y. (2026) · IntechOpen eBooks · doi:10.5772/intechopen.1014041 · Cited by 1 · see citing works
Antibiotic Resistance Genes Detection in Several Local Cyanobacteria Isolates
Buniya, H. K., Mohammed, N. A. R. K., & AlHayani, D. A. (2024) · Limnological Review · doi:10.3390/limnolrev24040033 · Cited by 1 · see citing works
Milman, P. Y., Зайцев, Г. А., & Ясаков, Т. Р. (2026) · Current Issues in Molecular Biology · doi:10.3390/cimb48111128
- Authors
- Ohdate, K., Sakata, M., Maeda, K., Sakamaki, Y., Nimura-Matsune, K., Ohbayashi, R., Hess, W. R., & Watanabe, S.
- Affiliations
- Tokyo University of Agriculture (JP)
- Tokyo Institute of Technology (JP)
- Shizuoka University (JP)
- University of Freiburg (DE)
- Corresponding author
- Watanabe, Satoru
- Venue
- Frontiers in Microbiology
- Publisher
- Frontiers Media SA
- Published
- 2024-02-14
- Volume
- 15
- Pages
- 1311290-1311290
- ISSN
- 1664-302X (ISSN-L)
- PMC
- PMC10899382
- OpenAlex
- W4391820144
- Cited by
- 9
- References
- 42
- Citations by year
- 2026: 4 · 2025: 1 · 2024: 4
- FWCI
- 2.39
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Microbiology (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- Deutsche Forschungsgemeinschaft (322977937, GRK 2344)
- Ministry of Education, Culture, Sports, Science and Technology (20K05793, JP22J13447)
- Tokyo University of Agriculture
- Japan Society for the Promotion of Science (21K15142)
- Japan Science and Technology Agency
- NODAI Genome Research Center, Tokyo University of Agriculture
- Advanced Low Carbon Technology Research and Development Program
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- plasmid replication proteins, large plasmids, cyanobacteria, Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, plasmid copy number, plasmid stability, plasmid incompatibility, plasmid distribution, plasmid recombination
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