Three thermosensitive (Ts) suicide vectors, pSET4s, pSET5s, and pSET6s have been constructed for gene replacement in Streptococcus suis. Each vector contains an antibiotic-resistance gene (spc or cat), a Ts replication origin of pG+host3, as well as multiple cloning sites, lacZ' and the ColE1 replication origin of pUC19. These vectors could be propagated at 37ºC in Escherichia coli, but their replication was blocked above 37ºC in S. suis. Moreover the thermosensitivity of the replication origin was confirmed in S. equi ssp. equi, S. equi ssp. zooepidemicus, and S. dysgalactiae by using pSET4s. For inactivation of the sly gene, which encodes a cholesterol-binding cytolysin of S. suis, pSLYK, in which the sly gene was interrupted by the cat gene, was constructed using pSET4s and introduced into S. suis DAT2. After growth at the nonpermissive temperature under antibiotic pressure, the chromosomal sly gene was replaced with the sly ::cat gene of pSLYK by a double-crossover event at a rate of 2.6% among chloramphenicol-resistant cells. These results indicate that the Ts suicide vectors described here will facilitate the genetic analysis of S. suis and other streptococci of veterinary importance by means of allelic exchange of the genes of interest via homologous recombination. (Molecular Bacteriology Section, Department of Infectious Diseases, TEL +81-298-38-7743)
Reference:
Takamatsu et al. (2001) Plasmid 46:140-148.
