Thermosensitive suicide vectors for gene replacement in Streptococcus suis

Three thermosensitive (Ts) suicide vectors, pSET4s, pSET5s, and pSET6s, have been constructed for gene replacement in Streptococcus sids. Each vector contains an antibiotic-resistance gene (spc or cat), a Ts replication origin of pWV01 lineage, multiple cloning sites, lacZ', and the ColE1 replication origin of pUC19. These vectors could be propagated at 37 degreesC in Escherichia coli, but their replication was blocked above 37 degreesC in S. sids. 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 thiol-activated hemolysin of S. suis, pSLYK, in which the sly gene was interrupted by the cat gene, was constructed using pSET4s and introduced into S. sids DAT2. After growth at the nonpermissive temperature under the 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. Moreover, complementation of the sly gene by use of the previously reported S. suis-E. coli shuttle vector pSET2 was demonstrated. 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. (C) 2001 Academic Press.