Figure 1.
Overview of the hipBA locus of E. coli based on Schumacher et al.
(A) Model of the hipBA operon. One of four operator sites is shown. (B) View of the crystal structure of the HipB dimer bound to a 21 base pair hipBA operator site (from reference [15]. One HipB subunit is colored green and the other red. The α helices are shown as coils and the α strands as arrows. The amino termini of each subunit are labelled N and the carboxy termini, C. The 16 C-terminal residues (73–88) are unstructured and residues 75–88, which are disordered in the structure of the HipA-HipB-DNA complex, are depicted as dashes and could easily extend from the body of HipB by more than 50 Å.
Figure 2.
HipB proteolysis in E. coli wild type and protease deficient strains.
HipB was expressed from pBRhipB in BW25113 (KLE901) and its lon::kan (KLE902), clpP::kan (KLE903) or hslVU::FRT (KLE904) derivate. The strains were grown in LB medium, and at an OD600 of 0.3 1 mM IPTG was added. After 1 h induction, protein synthesis was inhibited by the addition of 100 µg/ml Cam, and samples for Western blots were removed over the course of 30 min. (A) The presence of HipB in whole cell lysates was detected with an anti-his antibody. (B) The rate of degradation was calculated from at least 3 independent experiments. Closed squares, KLE901 (wild type); open squares, KLE902 (Δlon); closed triangles KLE904 (ΔhslVU); open triangles, KLE903 (ΔclpP).
Figure 3.
Lon degradation of HipB in vitro.
0.6 µM His6-Lon and 0.48 µM His6-HipB were incubated in reaction buffer at 37°C (50 mM Tris-HCl (pH 8.0), 4 mM ATP, 7.5 mM MgCl2) for indicated times with or without the component specified and subjected to SDS-PAGE and silver staining followed by analysis (at least 3 independent experiments were used to calculate HipB turn over). (A) In vitro degradation of His6-HipB by His6-Lon. (B) ATP or MgCl2 were omitted in the assay. Closed squares, no ATP; open squares no MgCl2. (C) Addition of an oligodeoxynucleotide encompassing the 21 bp hip operator (closed squares) or control oligo (open squares) and (D) addition of His6-HipA (closed squares) or control protein (lysoszyme) (open squares) to the degradation assay.
Figure 4.
The 16 C-terminal amino acid residues of HipB are required for degradation.
(A) Degradation of HipB72 in vivo. HipB72 was expressed from a pBRlacitac promoter in BW25113 (KLE905) and its lon::kan derivate (KLE906). Both strains were grown in LB medium, and at an OD600 of 0.3 1 mM IPTG was added. After 1 h of induction, protein synthesis was inhibited by the addition of 100 µg/ml Cam, and samples for Western blots were removed over the course of 30 min. (B) Degradation of HipB 72 in vitro. His6-HipB72 was purified and added to the Lon degradation assay. At least 3 independent experiments were performed to calculate HipB72 turnover.
Figure 5.
In vivo degradation of GFP and a GFP-HipB hybrid.
GFP and GFP with C-terminal fusion to the C terminus of HipB were expressed from a pBRlacitac promoter in BW25113 (KLE907 and KLE908, respectively). The strains were grown in LB medium, and at an OD600 of 0.3 1 mM IPTG was added. After 1 h of induction, protein synthesis was inhibited by the addition of 100 µg/ml Cam, and samples for Western blots were removed over the course of 60 min. Closed squares, GFP; open squares GFP-H (GFP-HipB(73–88)). The graph represents the average of five independent experiments.
Figure 6.
Multiple sequence alignment of selected HipB proteins from a variety of Gram-negative bacteria.
CLUSTALW and CLC Main Workbench were used for the alignment and graphic representation, respectively. HipB sequences were downloaded from NCBI database.
Figure 7.
HipB, HipB(W88A) and HipB72 bind hipBA operator DNA or HipA identically.
(A) Wild type HipB protein (red closed circle), HipB(W88A) protein (blue closed square) or the HipB72 C-terminal truncation protein (green closed triangle) was titrated into fluoresceinated hipBA O1O2 operator sequence and the change in fluorescence polarization (normalized millipolarization, mP) plotted as a function of the concentration of the titrant. The typical change in mP of each titration was between 60 and 80 units. The correlation coefficients for each curve fitting were 0.98, 0.99 and 0.99, respectively. (B) Wild type HipA protein was titrated into hipBA O1O2 DNA after the DNA was prebound by 20 nM wild type HipB monomer. Note that the concentration range is different in the left half and right half of the binding isotherms, with HipB titrations in the nanomolar range and HipA titrations in the micromolar range. (C) Wild type HipA protein was titrated into solutions containing 1 nM fluorescently labelled hipBA O1O2 DNA and titrated up to 50 nM wild type HipB monomer, 50 nM HipB(W88A) protein or HipB72 protein. This ensures stoichiometric binding of these HipB proteins to the DNA. Thus, the resulting binding affinity is formally between HipA and HipB that is bound specifically to hipBA DNA. The change in mP of each titration was between 88 and 150 units. The correlation coefficients for each curve fitting were 0.95, 0.99 and 0.99, respectively. A representative binding isotherm is shown for each protein binding to DNA or to the HipB-hipBA O1O2 complex.
Table 1.
Dissociation constants of HipB-DNA and HipA-(HipB-DNA)*.
Table 2.
Strains and plasmids used in this study.