POSITIVE SELECTION VECTORS FOR ALLELIC EXCHANGE

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.