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

R L Burghoff, D C Laux, P S Cohen
1990 Infection and Immunity  
Escherichia coli F-18 Colwas 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 Colboth when grown in L broth under conditions of
more » ... onselection (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 Colin vitro and in vivo. In contrast, two independent cer insertions into pBR329 did not stabilize the plasmid completely in E. coli F-18 Colin 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 Colin vitro was achieved by insertion of the parB region of plasmid Rl, 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.
doi:10.1128/iai.58.5.1141-1145.1990 fatcat:63mwlaadefc25d6ujtvoidxirq