FIG. 6.
Loss of mapped AlgZ binding sites at algZ results in loss of AlgZ binding to the algZ promoter. EMSAs were used to test binding interactions between His-AlgZ and end-labeled fragments (−258 to +79 bp of algZ) containing the wild-type algZ promoter or allelic variants. (A) Illustrations of the deletions and substitution of the 5′ regulatory region of the algZ promoter are shown. The wild-type algZ promoter contains two mapped AlgZ binding sites centered at −93 and −161 bp upstream of the transcriptional start site, designated algZ1 and algZ2, respectively. Deletion of the algZ1 binding site resulted in a loss of 36 bp (−111 to −76 bp) and was designated the algZ47 allele. Deletion of the algZ2 binding site resulted in a loss of 18 bp (−153 to −170 bp) of algZ and was designated the algZ67 allele. Removal of a 110-bp fragment (−180 to −71 bp) resulted in a deletion of both algZ1 and algZ2 binding sites and was designated the algZ57 allele. A fragment containing 110 bp of algB DNA was used to replace the 110-bp fragment (−180 to −71 bp) containing both mapped AlgZ binding sites, generating the algZ58 allele. (B to F) EMSAs containing labeled DNAs from the wild-type algZ promoter or allelic variants (algZ47, algZ67, algZ57, or algZ58) were incubated with His-AlgZ in the following amounts: lane 1, 0 pmol; lane 2, 0.61 pmol; lane 3, 1.22 pmol; lane 4, 1.83 pmol; lane 5, 2.44 pmol; lane 6, 3.66 pmol; lane 7, 4.88 pmol; lane 8, 6.10 pmol; lane 9, 7.32 pmol; lane 10, 8.54 pmol.