LexA Cleavage Is Required for CTX Prophage Induction Mariam Quinones, Harvey H. Kimsey, Matthew K. Waldor  Molecular Cell  Volume 17, Issue 2, Pages 291-300 (January 2005) DOI: 10.1016/j.molcel.2004.11.046

Figure 1 Influence of UV Treatment on CTXφ and lexA Transcript Levels Induction of CTX-Knφ and lexA mRNAs in cells treated with 25 J/m2 UV. Transcript levels were measured by quantitative RT-PCR in BD81 (black bars), BD206 (dark gray bars), and MQ163 (light gray bars), respectively. The data is the average value from two experiments. Fold increase was calculated by dividing the amount of the indicated mRNA measured in UV-treated cells by the amount of mRNA measured in untreated cells. Molecular Cell 2005 17, 291-300DOI: (10.1016/j.molcel.2004.11.046)

Figure 2 Regulation of PrstA Activity (A) LexA cleavage is required for UV-induced activation of rstA::lacZ and lexA::lacZ fusions. β-galactosidase activity of the rstA::lacZ fusion in pHK102 (top) and the lexA::lacZ fusion in pMQLZ (bottom) was measured at the indicated times after UV treatment (25 J/m2) of BD81 (squares), or MQ163 (triangles). (B) LexA and RstR regulate the activity of an rstA::lacZ fusion. The β-galactosidase activity from the rstA::lacZ reporter plasmid pHK102 (Kimsey and Waldor, 1998) was measured in E. coli strains RW118 (pHK1) and in RW542 (pHK1). Cultures were grown overnight in the presence or absence of 0.02% arabinose to induce the expression of rstR from pHK1. The mean and standard deviation are shown. (C) RpoA augments transcription of an rstA reporter containing sequences upstream of the −35 of PrstA. The β-galactosidase activity of the F2 and F4 rstA::lacZ fusions was measured in E. coli strain RW542 containing either pHTF1αwt or pHTF1αR265A (Tang et al., 1994). Cultures were grown overnight, then diluted 1:100 and grown for 5 hr in the presence of 1 mM IPTG to induce expression of rpoA or rpoAA265A. The mean and standard deviation are shown. The boundaries of the inserts used to construct the different rstA::lacZ fusions are shown in (D). Molecular Cell 2005 17, 291-300DOI: (10.1016/j.molcel.2004.11.046)

Figure 3 Immunoblot Analyses of RstR and LexA Levels after UV Treatment RstR (top) and LexA (bottom) abundance were assessed in the absence (−) or presence (+) of UV treatment. Immunoblots of whole-cell extracts from BD81, BD206, and MQ163 with anti-RstR (top) or anti-LexA (bottom) antisera. An extract from 2740-80 that does not harbor the CTX prophage (CTXφ−) was used as a negative control for the RstR immunoblot. Molecular Cell 2005 17, 291-300DOI: (10.1016/j.molcel.2004.11.046)

Figure 4 Comparison of the Sequence of the Putative LexA Binding Site in the CTXφ ig-2 Region with the Sequences of Other Likely V. cholerae LexA Binding Sites (A) Bold letters are used when a base is found in either all of the sequences or in six out of seven sequences. lexA1 and lexA2 are the two putative SOS boxes found upstream of lexA in V. cholerae. The consensus E. coli SOS box from (Walker, 1984) is shown below the V. cholerae sequences. (B) DNA sequence of CTXφ intergenic sequence 2 (ig-2) and part of the rstA and rstR coding sequences. The RstR operators sequences O1, O2, and O3 are boxed. The rstA promoter and coding sequence is shown in dark gray shading. The rstR promoter and coding sequence is shown in light gray shading. Bases inside the black box indicate the putative LexA binding sequence in ig-2. The DNA region protected from DNaseI cleavage by LexA (see Figures 5C and 5D) is indicated by brackets over the LexA binding site. Molecular Cell 2005 17, 291-300DOI: (10.1016/j.molcel.2004.11.046)

Figure 5 LexA-His6 Binds to the CTXφ ig-2 Region (A) Schematic of the intergenic region (ig-2) between rstA and rstR and flanking sequence. O1, O2, and O3 (in rstR) are RstR binding sites shown as black boxes (Kimsey and Waldor, 2004). The transcription start sites of rstA (PrstA) and rstR (PrstR) are shown. The boundaries of the 873 and 835 probes are shown below the map. (B) Autoradiograms of band shift experiments testing LexA-His6 binding to the lexA SOS box (region upstream of V. cholerae lexA) as well as to two probes derived from the CTXφ ig-2 region. Lanes 1, probe alone; lane 2, 30 μM LexA; lane 3, 15 μM LexA; lane 4, 7.5 μM LexA. (C) DNase I footprint of RstR and/or LexA bound to O1 and O2 DNA (bottom strand, complement of sequence depicted in Figure 4B). G+A refers to the sequencing ladder. Probe refers to DNase I digestion of probe DNA with no protein added. RstR was used at a concentration of 31.5 μM, and LexA was used at a concentration of 60.0 μM. The numbers showing the boundaries of the LexA binding site are relative to the start site of rstA transcription. (D) DNase I footprint of RstR and LexA bound to O2 and O3 DNA (top strand). Reactions in lanes 1–4 contained RstR in the following increasing concentrations: 16, 31.5, 63, and 126 μM. Reactions in lanes 5–7 contained 30 μM LexA + RstR in the following concentrations: 16, 63, and 126 μM, respectively. Molecular Cell 2005 17, 291-300DOI: (10.1016/j.molcel.2004.11.046)