Antagonistic Roles for GcvA and GcvB in hdeAB Expression in Escherichia coli
Figure 1
(a) The hdeAB control region. The hdeAB promoter −35, −10 and transcription start site and the SD sequence and translation start site are indicated above the sequence [31]. The transcription start site for the divergently transcribed hdeD gene is also shown [32]. Binding sites for H-NS [16] and MarR/SoxS [15] are indicated above the sequence with arrows. The binding site for GadX/W [33] is below the sequence in blue and for GadE above the sequence in green [18, 34]. In addition, there are putative binding sites for the transcriptional regulators Lrp and TorR (not shown) [34]. The consensus GcvA binding site is T-N11-A containing a 5′-CTAAT-3′ sequence [35]. Two putative GcvA binding sites are indicated in red. The fusion sites for the λhdeA::lacZ translational fusion, the λ hdeA−36::lacZ transcriptional fusion, and the λPBAD::hdeA::lacZ fusion (see below) are indicated with black, red, and green arrows, respectively. (b) Construction of a λPBAD::hdeA::lacZ promoter fusion. The WT PBAD and PhdeA promoters are shown in the top line. The transcription start sites are in red [31, 36]. Small case letters show bases added during PCR amplification of the PBAD and PhdeA promoters. The PBAD promoter was amplified with an upstream primer containing an EcoRI site at bp −272 relative to the transcription start site (not shown) and a downstream primer with a BsaI site (blue). The PhdeA promoter was amplified with an upstream primer containing a BsaI site (blue) and a downstream primer containing a SmaI site at codon 10 in the hdeA gene (not shown). The arrows indicate cut sites for BsaI. The amplified products were cut with BsaI, mixed, and ligated, generating a fusion of the PBAD promoter with the +1G residue of the PhdeA promoter. The fragment was then digested with EcoRI + SmaI and ligated into the EcoRI-SmaI sites of plasmid pMC1403, and subsequently subcloned into λgt2 as described [19]. (c) The gcvA gcvB region of the E. coli chromosome. The regions amplified by PCR and cloned into pACYC184 to generate pGS611 (p) and pGS624 (p) are indicated with bars. See Section 2.2 for details.
