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Escherichia coli F-18 Col- was previously shown to be a poor colonizer of the streptomycin-treated mouse large intestine, relative to its parent, E. coli F-18. Prior to attempting to clone genes responsible for the colonization phenotype of E. coli F-18 into E. coli F-18 Col-, a suitable cloning vector had to be found. In this investigation, we report that the commonly used cloning vectors pBR322, pHC79, and pBR329 all segregate from E. coli F-18 Col- both when grown in L broth under conditions of nonselection (i.e., in vitro) and when fed to streptomycin-treated mice (i.e., in vivo). Insertion of the cer region (which promotes resolution of replicating plasmids into monomeric forms) into pHC79 stabilized this plasmid in E. coli F-18 Col- in vitro and in vivo. In contrast, two independent cer insertions into pBR329 did not stabilize the plasmid completely in E. coli F-18 Col- in vitro, and feeding the strain to streptomycin-treated mice resulted in rapid segregation of the plasmids in vivo. Also, stability of all three plasmids in E. coli F-18 Col- in vitro was achieved by insertion of the parB region of plasmid R1, which encodes a cell-killing protein, Hok, that is active only postsegregationally. However, as with cer, complete in vitro and in vivo stabilization was achieved only in parB constructs of pBR322 and pHC79.

infection and immunity

Construction of stable cloning vectors that do not segregate from a human fecal Escherichia coli strain in the streptomycin-treated mouse large intestine