[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-401] Construction of Versatile Shuttle Vectors for Pseudomonas via Replication Initiation Proteins Identification

○Hui Xie¹, Maho Tokuda², Nanako Isogai², Chiho Suzuki-Minakuchi^1,3, Yosuke Nishimura⁴, Haruo Suzuki^5,6, Yoshitaka Moriwaki^1,3,7, Yasuhiro Tanizawa⁸, Masato Suzuki⁹, Masaki Shintani^2,10,11, Hideaki Nojiri^1,3 (1. Grad. Sch. Agric. Life Sci., UTokyo (Japan), 2. Dept. Eng., Shizuoka Univ. (Japan), 3. CRIIM, UTokyo (Japan), 4. CeBN, JAMSTEC (Japan), 5. Fac. Environ. Info. Stud., Keio Univ. (Japan), 6. Inst. Adv. Biosci., Keio Univ. (Japan), 7. Lab. Med. Res., Science Tokyo (Japan), 8. Dept. Informatics, NII (Japan), 9. AMRRC, NIID (Japan), 10. RIGST, Shizuoka Univ. (Japan), 11. JCM, RIKEN BRC (Japan))

Keywords:

Pseudomonas,plasmid,replication initiation proteins

[Purpose] Plasmid vectors are essential tools in bacterial genetic engineering. However, most currently available vectors are designed for model bacteria such as Escherichia coli, limiting their applicability to non-model bacteria. The genus Pseudomonas comprises a wide range of species found in soil and aquatic environments, including plant-associated strains and opportunistic human pathogens. Due to their environmental and clinical importance, expanding genetic tools for Pseudomonas is highly desirable. This study aimed to systematically identify functional replicons from Pseudomonas-derived plasmids and to construct a collection of shuttle vectors that can function in both Pseudomonas species and E. coli. [Method] Complete nucleotide sequences of 846 plasmids derived from Pseudomonas species were obtained from the public database PLSDB. Among these, 537 plasmids containing identifiable replication initiation protein (RIP) were subjected to further analysis. Then GC/AT content was visualized to identify AT-rich regions and repeat sequences around the rep gene, which were defined as candidate origins of replication (oriV). Based on this data, mini plasmids containing both the RIP and its associated oriV were designed and then chemically synthesized into pUC vectors commonly used in E. coli, carrying either kanamycin or ampicillin resistance markers. In total, 152 mini plasmids were constructed, comprising 76 plasmid groups. The constructed mini plasmids were then introduced into two representative Pseudomonas strains—P. aeruginosa PAO1 and P. putida KT2440—via electroporation to assess their replication capability. [Results] Among the 152 constructed mini plasmids, 55 were capable of replication in P. putida KT2440, while 53 replicated in P. aeruginosa PAO1. Overall, approximately 72% of the mini plasmids were able to replicate in at least one of the tested hosts, indicating a high success rate in identifying functional replicons. Some plasmids exhibited host-specific replication, whereas others were functional in both strains, suggesting variability in host compatibility. [Consideration] The results demonstrate that combining RIP genes with their associated oriV regions enables the successful construction of plasmid vectors that function in both E. coli and Pseudomonas. Experimental validation further confirmed that these replicons are capable of stable replication in Pseudomonas hosts. In addition, mini plasmids derived from different groups might be able to coexist within the same cell, which could be important for multi-plasmid vector systems. [Conclusion] This study successfully establishes a diverse and functional shuttle vector collection for Pseudomonas species, significantly expanding the available molecular tools beyond traditional model bacteria. These vectors can be used flexibly, including introducing multiple plasmids into a single host for independent gene expression. These features make the system highly valuable for applications in environmental microbiology and biotechnology involving Pseudomonas.