Figure 3

Construction of suicide vector pMHH8. The rpoS gene from EHEC 86-24 was amplified by PCR with primers RpoS 3/RpoS 4 and cloned into pUC19 after restriction digest with enzymes Xba I and Sac I (pMHH6). A 390 bp sequence was deleted from the insert through restriction digest with enzymes Dra III and Bsa AI leading to plasmid pMHH7. The mutated rpoS gene was then cloned into suicide vector pMHH1 after restriction digest with Xba I and Sac I. The resulting plasmid was termed pMHH8. It was used in the construction of all rpoS deletion mutants described in this study.