1. Regulation of the Pap Epigenetic Switch by CpxAR
Aaron D. Hernday, Bruce A. Braaten, Gina Broitman-Maduro, Patrick Engelberts, David A. Low 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
Activation of CpxAR Inhibits Pap Phase Variation and papBA Pilin Transcription
The effects of cpxA* alleles, PapI, and NlpE overexpression on Pap gene expression were analyzed as described in the Experimental Procedures. In (A), (B), and (C), the boxed areas below the x-axes indicate the pap genotype: wild-type, DL1504; + PapI, DL1504 (pDAL468); GCTCdist, DL1505; and site 3, DL2121. Writing below the boxed areas indicates the cpxA genotype: wt, wild-type cpxAR gene; wt + nlpE, wild-type cpxAR (pLD404). Error bars show the standard deviation of the mean.
(A) CpxAR activation inhibits Pap phase OFF to ON switching. The phase OFF to ON switch frequencies of the indicated E. coli isolates were measured as described in Experimental Procedures.
(B) CpxAR activation inhibits pap pilin transcription. Transcription from phase OFF [wt (OFF)]; phase ON [wt (ON)]; and NlpE overexpressing E. coli DL1504 (wt + nlpE) and DL1504 containing the cpxA* alleles shown were measured by β-galactosidase activity as described in the Experimental Procedures.
(C) The effects of CpxAR activation on pap phase-locked ON mutants. See (B).
(D) CpxAR activation inhibits Pap pili expression. Immunoblot analysis was carried out using anti-Pap17 pilin antiserum as described in the Experimental Procedures. E. coli strain MC4100 containing the pap17 operon on a single copy plasmid (denoted as “+ Pap17” at the top) and either wild-type cpxA (wt), cpxA9, or cpxA2.1 alleles were analyzed for Pap17 pilus expression. Corresponding control strains lacking the pap17 operon were also analyzed (denoted “− Pap17”).
(E) Schematic of the pap pilin promoter fusion used to obtain results shown in (A), (B), and (C).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Phosphorylation of CpxR Increases Its Affinity for pap Sites 1–3 and Sites 4–6
(A) The effect of nonspecific, herring sperm competitor DNA (HS-DNA) on CpxR (left) or CpxR-P (right) binding to pap DNA Lrp sites 4–6 was measured. CpxR or CpxR-P (440 nM) was added to 32P-labeled pap DNA and then immediately mixed with serial dilutions of herring sperm DNA to yield the final competitor DNA concentrations indicated above each lane.
(B) Same as in (A) but with 32P-labeled pap DNA Lrp sites 1–3.
(C) Analysis of CpxR (left) and CpxR-P (right) binding to 32P-labeled pap Lrp DNA sites 1–3 in the presence of 5 μg/ml herring sperm DNA.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 DNA Footprint Analyses of CpxR-P Binding to pap DNA Sites 1–6
(A) DNaseI protection footprinting of pap sites 1–6 (linear) regulatory region DNA. Analysis of the top (left) and bottom (right) pap DNA strands is shown (see [B] for orientation). Increasing CpxR-P levels were added as follows: lane A, 0 nM; lane B, 220 nM; lane C, 440 nM; lane D, 880 nM; lane E, 1760 nM. Lrp binding sites 1–6 are shown as vertical bars to the right of each footprint. Binding conditions are described in the Experimental Procedures.
(B) Summary of DNaseI and UV footprinting results. CpxR-P binding sites identified by both DNaseI (see [A]) and UV footprinting (data not shown) at 880 nM CpxR-P are shown. UV footprinting was performed on a covalently closed circular (negatively supercoiled) pap DNA template as described in the Experimental Procedures. Lrp binding sites 1–6 are outlined by gray boxes, and “GATC” sites are in bold. DNaseI protection sites are underlined, UV protection sites are overscored, and DNaseI hypersensitive sites are indicated by vertical arrows. The 5′-GTAAA-3′ consensus CpxR-P binding sites are shown by horizontal arrows.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Effect of CpxAR Activation on pap DNA Methylation Patterns
Methylation protection analysis of pap GATCprox and GATCdist sites was carried out by digestion of pap DNA samples with MboI and EcoRI and Southern blot analysis (Experimental Procedures). Lanes 1 and 2, phase OFF E. coli DL1504; lanes 3 and 4, lrp mutant DL1609; lanes 5 and 6, hns mutant DL1947; lanes 7 and 8, lrp− hns− mutant E. coli E. coli analyzed in lanes 2, 4, 6, and 8 contained plasmid pLD404 (NlpE) to activate CpxAR. Arrows at the left of the blot indicate pap DNA fragments shown in the schematic in the bottom panel. DNA fragments “b” and “e” result from digestion at GATCprox, characteristic of phase OFF cells, whereas DNA fragments “c” and “d” result from digestion at GATCdist, characteristic of phase ON cells.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Pap Pilin Transcription Is Inhibited by CpxR-P In Vitro and by Alkaline Conditions In Vivo in a CpxAR-Dependent Manner
(A) Competitive binding analysis. The effect of CpxR-P on Lrp binding to pap sites 1–3 and sites 4–6 was analyzed by EMSA. CpxR-P was incubated with pap DNA at the concentrations shown at the bottom and mixed with serial dilutions of Lrp. Samples were analyzed by EMSA to separate CpxR-P bound, Lrp bound, and unbound pap DNA's. The fraction of total DNA bound to Lrp was quantitated to calculate a relative Kd (compared to Lrp in the absence of CpxR-P) for Lrp-DNA binding at each concentration of CpxR-P. Relative affinities of Lrp for sites 1–3 (open bars) and sites 4–6 (filled bars) are shown at 0 nM, 110 nM, and 220 nM CpxR-P.
(B) In vitro transcription analysis. CpxR-P was incubated with covalently closed circular pap DNA (plasmid pDAL337 containing the papI-B regulatory region). Binding mixtures were incubated at 37°C for 30 min and then analyzed by UV footprinting to measure specific binding of CpxR-P (gray squares) and in vitro transcription reactions to measure papBA transcription (black circles) as described previously (Weyand et al., 2001).
(C) CpxAR-dependent repression of pap pilin transcription in response to alkaline conditions. Phase-locked ON E. coli (papBA-lacZYA, GCTCdist) containing either wild-type cpxAR (cpxARwt, left panel) or a cpxAR deletion (cpxARΔ, right panel) was grown in minimal medium at the pH indicated on the x-axis. PapBA transcription was quantitated by a β-galactosidase assay as described in the Experimental Procedures and is shown on the y-axis.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Model for Repression of the pap Phase OFF to ON Switch by CpxR-P
The model for Pap phase variation shown in steps A, B, and C is based on our recent work (Hernday et al., 2002, 2003). Steps D and E show the postulated mechanism (see Discussion) by which CpxR-P inhibits transition of cells to the phase ON state. (Step A) The Pap phase OFF transcription state (top) is stabilized by methylation at GATCdist, which inhibits binding of PapI/Lrp and Lrp to sites 4–6. DNA replication generates two hemimethylated intermediate DNAs designated as Hemi-T (methylated on the top strand relative to orientation of pap shown) and Hemi-B (methylated on the bottom DNA strand). Only Hemi-T is shown. (Step B) Under conditions in which PapI expression is induced, PapI forms a ternary complex with Lrp and pap sites 4–6 DNA. Methylation at GATCprox (step C) is required for the phase ON transition by inhibiting PapI-dependent binding of Lrp to sites 1–3. Binding of PapI/Lrp at sites 4–6, together with cAMP-CAP binding at −215.5 relative to the papBA transcription start (Weyand et al., 2001), activates papBA transcription. PapI transcription is maintained at a high level by the PapB positive feedback loop shown. Under conditions of CpxAR activation, CpxR-P binds at sites 1–6 (step D), competing with PapI/Lrp for binding at sites 4–6 and inhibiting transcription of papBA by binding at sites 1–3. In step E, the pap GATCdist site has been fully methylated by Dam, stabilizing the phase OFF state.
Molecular Cell  , DOI: ( /j.molcel )