Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors.
pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.
Takamatsu
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D
Laboratory of Molecular Bacteriology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan. p1013dt@niah.affrc.go.jp
Osaki
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M
Sekizaki
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T
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017011:Streptococcus suis / Q000235:genetics*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D011938:Rec A Recombinases