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Thermosensitive suicide vectors for gene replacement in Streptococcus suis
D Takamatsu1, M Osaki, T Sekizaki
1Molecular Bacteriology Section, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan. p1013dt@niah.affrc.go.jp
Plasmid
|October 10, 2001
Summary
New thermosensitive suicide vectors enable precise gene replacement in Streptococcus suis. These tools facilitate genetic analysis and manipulation in important veterinary pathogens through homologous recombination.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus suis is a significant veterinary pathogen.
- Efficient genetic manipulation tools are crucial for understanding S. suis virulence factors, such as the sly gene encoding a thiol-activated hemolysin.
- Existing genetic tools may have limitations for allelic exchange in S. suis.
Purpose of the Study:
- To construct and characterize novel thermosensitive (Ts) suicide vectors for gene replacement in Streptococcus suis.
- To demonstrate the efficacy of these vectors for targeted gene inactivation via homologous recombination.
- To facilitate genetic analysis of S. suis and related streptococci.
Main Methods:
- Construction of three Ts suicide vectors (pSET4s, pSET5s, pSET6s) containing antibiotic-resistance genes, Ts replication origins, and multiple cloning sites.
- Propagation in Escherichia coli at 37°C and replication blocking above 37°C in S. suis.
- Construction of a specific vector (pSLYK) to inactivate the sly gene in S. suis DAT2 using a double-crossover event.
- Confirmation of thermosensitivity in other streptococcal species (S. equi, S. dysgalactiae).
Main Results:
- The Ts suicide vectors were successfully constructed and functional in S. suis, with replication blocked at non-permissive temperatures.
- The sly gene in S. suis DAT2 was replaced with a sly::cat gene construct at a rate of 2.6% among resistant cells.
- The thermosensitivity of the replication origin was confirmed across multiple streptococcal species.
- Complementation of the sly gene using a previously established shuttle vector was also demonstrated.
Conclusions:
- The developed thermosensitive suicide vectors are effective tools for gene replacement in Streptococcus suis.
- These vectors enable allelic exchange through homologous recombination, significantly advancing the genetic analysis of S. suis and other veterinary streptococci.
- The findings provide a valuable resource for researchers studying streptococcal pathogens.