Construction of stable cloning vectors that do not segregate from a human fecal Escherichia coli strain in the streptomycin-treated mouse large intestine.
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.
Burghoff
R L
RL
Department of Microbiology, University of Rhode Island, Kingston 02881.
Laux
D C
DC
Cohen
P S
PS
D000818:Animals; D003360:Cosmids; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D005243:Feces / Q000382:microbiology; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007420:Intestine, Large / Q000382:microbiology*; D051379:Mice; D010957:Plasmids; D013307:Streptomycin / Q000494:pharmacology
D013307:Streptomycin