Figure 4
Construction of complementation plasmids. Complementation plasmids pSC1, pSC2, pUD2, pUD4, pUD8, pUD6, pUD9, pUD10 and pDS4 were constructed using low copy vector pBR322 as backbone. Each of them harbors one complete rpoS gene and the rpoS p promoter [39] from EHEC/STEC strains 126814 (pSC1), EDL933a (pSC2), EDL933b (pUD2), 86-24 (pUD4), E-D53 (pUD8) and 288597 (pUD10), amplified by PCR with primers RpoS 8/RpoS 9 containing 5' Hind III restriction sites. RpoS 8 and RpoS 4c were taken to synthesize the respective DNA fragment from STEC E-D68 in order to generate the two independent plasmids pUD6 and pUD9. pDS4 contained the cloned rpoS gene from EcN, amplified with primers RpoS 8 and RpoS 22. pMH33 was made from pSC2 by PCR mediated site specific mutagenesis, employing the mutation rpoST721G to resolve the TAA stop codon in rpoS*. RpoS negative mutant strain E. coli MHH126-5 was transformed with each of the complementation vectors by electroporation.