Journal: Molecular Microbiology
Article Title: Organization and architecture of AggR‐dependent promoters from enteroaggregative Escherichia coli
Figure Lengend Snippet: Analysis of the aafD 100 promoter fragment from EAEC strain 042. A. The panel shows the base sequence of the EAEC 042 aafD 100 regulatory region fragment, which includes the start of the aafD coding sequence. The sequence is flanked by upstream EcoRI and downstream HindIII sites and is numbered from the base immediately upstream of the HindIII site. The limits of the aafD 99, aafD 98, aafD 97, aafD 96, aafD 95 and aafD 94 nested deletions are indicated by flags. The proposed promoter −10 hexamer element is underlined, the experimentally determined transcript start sites are indicated by bent horizontal arrows and the initiating ATG codon is in bold. Potential AggR‐binding sites are indicated by horizontal arrows, with functional and non‐functional sites denoted by dark and light shading respectively. Each site is aligned with the AggR‐binding consensus (Morin et al. , 2010 ). The locations of the 65 C and 92 C /90 C substitutions, which disrupt the −10 element and the functional AggR‐binding site, respectively, are shown. B. The panel illustrates measured β‐galactosidase activities in E. coli K‐12 BW25113 ∆ lac cells, containing pRW50 carrying the aafD 100 fragment, shortened derivatives or no insert. Cells also carried either pBAD/ aggR (grey bars) or pBAD24 (black bars). C. The panel shows an autoradiogram of a denaturing polyacrylamide gel run to determine the primer extension products from RNA synthesis initiating at the aafD promoter in BW25113 cells carrying pRW50/ aafD 96. AggR (+) and AggR (‐) indicates cells carried pBAD/ aggR or pBAD24. Reactions are calibrated with the M13mp18 phage reference sequence (A, C, G and T), which serves as sequence ladder. Primer extension products, produced in the presence of AggR, are indicated by arrows. D. The panel shows the β‐galactosidase activities of BW25113 cells, containing pRW50 carrying either the aafD 96 fragment or mutant derivatives. Cells also carried either pBAD/ aggR (grey bars) or pBAD24 (black bars). In panels B. and D. cells were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min –1 mg –1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points.
Article Snippet: RNA isolation, rRNA depletion and cDNA synthesis for RNA‐seq Triplicate overnight cultures of EAEC 042 and EAEC 042 ΔaggR were used to inoculate 50 ml of Dulbecco’s modified Eagle’s medium with 0.45% glucose (DMEM high glucose) (Sigma) to an OD600 of 0.05.
Techniques: Sequencing, Binding Assay, Functional Assay, Produced, Mutagenesis, Activity Assay