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 XbaI and SacI (pMHH6). A 390 bp sequence was deleted from the insert through restriction digest with enzymes DraIII and BsaAI leading to plasmid pMHH7. The mutated rpoS gene was then cloned into suicide vector pMHH1 after restriction digest with XbaI and SacI. The resulting plasmid was termed pMHH8. It was used in the construction of all rpoS deletion mutants described in this study.