We describe here the development and use of two new allelic exchange v
ectors, pKAS32 and pKAS46. These vectors can be used for allelic excha
nge in a wide variety of bacterial species because their R6K origin of
replication functions only in bacteria engineered to produce the repl
ication protein pi. In addition, these vectors express the Escherichia
coli rpsL gene, encoding ribosomal protein S12, which provides a posi
tive selection for bacteria that have exchanged cloned plasmid sequenc
es with the corresponding chromosomal sequences. In this report, we sh
ow that these vectors can be used to efficiently introduce point mutat
ions and deletions into the chromosome of Vibrio cholerae.