FIG. 1.

Suicide mutagenesis using pRR10-250v vector. (A) Plasmid pCF305 (internal fragment of gadC inserted into pRR10-250v) was integrated into the chromosome of strain EF393 by homologous recombination at the gadC gene. The positions of the different primers used either to generate internal fragments or to verify the final insertion are shown. The sequences of these oligonucleotides are given in Table 2. The integration site was verified by PCR with primer pairs 43 and 101 (gadC), which produced a fragment of the predicted size, 1.4 kb. (B) The procedure was the same as in panel A, except that plasmid pCF312 containing an internal fragment of gadA was used. The integration site was verified by PCR with primer pairs 43 and 121 (gadA), which produced a fragment of the predicted size, 1.2 kb. Insertions that amplify with oligonucleotides 43 and 121 were distinguished as being within gadA or gadB by using oligonucleotides 121 and 106. If the insertion was in gadA, a PCR product was obtained which represented normal gadBC. If the insertion was in gadB, no PCR product was formed. Heavy arrows indicate the direction of transcription.