697308.fig.001a
(a)
697308.fig.001b
(b)
697308.fig.001c
(c)
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 𝑔 𝑐 𝑣 𝐴 3 + ) and pGS624 (p 𝑔 𝑐 𝑣 𝐴 3 + 𝑔 𝑐 𝑣 𝐵 3 + ) are indicated with bars. See Section 2.2 for details.