Table 1.
Primers and plasmids used in generating the E. coli strain BW25113ΔptsGΔmalE::km double mutant.
Figure 1.
Fluorescence spectra of purified Peak 1 CFP-IIGlc plus Peak 1 YFP-IIGlc mixture, purified Peak 1 CFP-IIGlc and purified Peak 1 YFP-IIGlc.
(A) The two fluorescent proteins were not detergent-treated, but were mixed before purification at a 1:6 ratio (Both; dark line). Alternatively, the spectrum of pure Peak 1 CFP-IIGlc (CFP, light line) or pure Peak 1 YFP-IIGlc (YFP, dotted line) alone was measured. (B) The spectrum of the YFP-IIGlc protein was subtracted from the spectrum of the Peak 1 CFP-IIGlc - Peak 1 YFP-IIGlc mixture (Both corrected, dark line). The purified CFP-IIGlc spectrum is also shown (CFP, light line). (No FRET was observed).
Table 2.
Summary of FRET analyses demonstrating interactions between MBP-IIGlc-CFP and MBP-IIGlc-YFP fused proteins of peaks 1 and 2.
Figure 2.
Fluorescence spectra of purified Peak 1 CFP-IIGlc plus Peak 1 YFP-IIGlc mixture, purified Peak 1 CFP-IIGlc and purified Peak 1 YFP-IIGlc.
(A) The two fluorescent proteins were mixed before purification at a 1:6 ratio, detergent treated and then purified (Both, dark line). Alternatively, the spectrum of pure Peak 1 CFP-IIGlc (CFP, light line) or Peak 1 YFP-IIGlc (YFP, dotted line) alone was treated similarly before the spectrum was measured. (B) The spectrum of the YFP-IIGlc protein was subtracted from the spectrum of the Peak 1 CFP-IIGlc - Peak 1 YFP-IIGlc mixture (Both (corrected), dark line). The purified CFP-IIGlc spectrum is also shown (light line). FRET was observed due to detergent treatment preceding purification.
Figure 3.
Fluorescence spectra of Peak 2 CFP-IIGlc (hereafter designated “CFP”) plus Peak 2 YFP-IIGlc (hereafter designated “YFP”).
(A) The two fluorescent proteins were mixed at a ratio of 1∶10 before purification (dark line). The light and dotted lines show the spectra for purified Peak 2 CFP and Peak 2 YFP, respectively as indicated. (B) Corrected fluorescence spectra of Peak 2 CFP plus Peak 2 YFP (mixed at a ratio of 1:10 before purification) (both, dark line) and purified Peak 2 CFP alone (light line). FRET was observed in the absence of detergent treatment.
Figure 4.
Fluorescence spectra of Peak 2 CFP plus Peak 1 YFP.
(A) The two fluorescent proteins were mixed at a ratio of 1∶6 before purification (dark line), and purified Peak 2 CFP and Peak 1 YFP were examined alone as indicated (light and dotted lines, respectively). (B) Fluorescence spectra of corrected Peak 2 CFP plus Peak 1 YFP (Both corrected, dark line) and Peak 2 CFP alone (CFP, light line).
Table 3.
Summary of FRET analyses of interactions between MBP-IIGlc-CFP and MBP-IIGlc-YFP fused proteins of peak 1 in the presence of different concentrations of un-labeled IIGlc-MBP.
Table 4.
FRET analyses of positive FRET samples of MBP-IIGlc-CFP/YFP of peak 1 after digestion with factor Xa.
Figure 5.
SDS PAGE of factor Xa digested samples of MBP-IIGlc-CFP/MBP-IIGlc-YFP.
M. W., molecular weight markers; C, factor Xa undigested sample; 12 h a & b, samples were digested for 12 h with 0.1 and 0.2 µg enzyme/µg sample protein, respectively; 34 h a & b, samples were digested for 34 h with 0.1 or 0.2 µg enzyme/µg of sample protein, respectively.
Figure 6.
SDS PAGE of FRET positive samples digested with factor Xa of MBP-IIGlc-CFP/MBP-IIGlc-YFP.
Samples were applied at 1 µg protein per lane. Digestion was carried out in two stages, the first stage at 0.3 µg of enzyme protein per µg of sample protein for 3 days, and the second stage at 0.2 µg of enzyme protein per µg of sample protein for a further 3 days.
Figure 7.
Effect of in vivo cross linking with different concentrations of paraformaldehyde (PFA) on the protein concentration of different preparations obtained from the cell lysate of E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG).
Low speed pellets, white bars; high speed pellets, black bars; high speed supernatants, grey bars. Thin lines above the histograms represent the standard deviations (S.D).
Figure 8.
Effect of in vivo cross linking with different concentrations of paraformaldehyde (PFA) on transphosphorylation (TP) activity of different preparations obtained from the cell lysate of E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG).
Low speed pellets, white bars; high speed pellets, black bars; high speed supernatants, grey bars. Thin lines above the histograms show the error bar (S.D). Values are expressed in percentage of original samples.
Figure 9.
Transphosphorylation (TP) activities of gel filtration fractions of 2 h high speed supernatants prepared from cultures of E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG) that were subjected to in vivo cross linking with paraformaldehyde (PFA) at 0.3, 0.6 and 1.2%.
Control, x's; 0.3% PFA, squares; 0.6% PFA, triangles; 1.2% PFA, diamonds.
Figure 10.
Protein profile of gel filtration fractions of a 2 h high speed supernatant prepared from a culture of E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG) that were subjected to in vivo cross linking with paraformaldehyde (PFA) at 0.3, 0.6 and 1.2%.
Control, x's; 0.3% PFA, squares; 0.6% PFA, triangles; 1.2% PFA, diamonds.
Figure 11.
Effect of in vitro cross linking with different concentrations of paraformaldehyde on transphosphorylation activity of IIGlc peaks 1 (white bars) and 2 (black bars) from E. coli BW25113ΔptsGΔmalE::km(pMALE-ptsG).
The 2 h HSS of a crude cell lysate was gel filtered to get the two activity peaks 1 and 2. Both peaks were subjected to in vitro cross linking as described in the Methods section. The thin vertical lines above the histograms represent error bars (S.D).
Figure 12.
Cryo-electron micrographs of purified IIGlc-MBP preparations.
E. coli strain BL21DE3(pMALE-ptsG) was used (A) pellet, (B) peak 1, (C) peak 2, (D) buffer control.
Figure 13.
Percentage distribution of IIGlc-MBP molecular sizes of pellet (top), peak 1 (middle) and peak 2 (bottom) preparations of E. coli strain BL21DE3(pMALE-ptsG) as determined by DLS.
Values are expressed in volume (diamonds), intensity (squares) and number (triangles).
Table 5.
Summary of DLS analyses.
Figure 14.
[31P]NMR spectra of purified IIGlc preparations of pellet (Pt), initial, middle and tail parts of peak 1 (P1-A, P1-B and P1-C, respectively) and peak 2 (P2) of E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG).
The spectra were recorded at 37°C, 46,000 scans and at an acquisition time of 1.6 seconds. Peak 2 was prepared from cells subjected to in vivo cross linking. For details see Materials and Methods. The spectra of these tested preparations when examined at 25, 37 and 45°C and at different scans were shown in Figs. S1 to S5 (Saier's web site).
Figure 15.
Transphosphorylation (TP) activities of pellet (Top; A; squares) and Peak 2 (Top; A; triangles), as well as Peak 1 (A (leading edge; triangles), B (central part; x's) and C (trailing edge; diamonds)) fractions (Bottom; B).
Purified IIGlc-MBP was prepared from E. coli strain BW25113ΔptsGΔmalE::km(pMALE-ptsG). The radioactive substrate was 100 µM [14C]methyl α-glucodside, used with different concentrations of glucose-6-phosphate as indicated.
Figure 16.
LC-ESI-MS negative ion mode spectra of phosphatidylethanolamine (PE) molecular species of lipid extract of peak 1 fraction of E. coli strain BL21DE3(pMALE-ptsG).
Arrows indicate different PE molecular species detected in peak 1 and standard preparations (for standard preparation see Figure S6 at Saier web site). I.S., internal standard; right handed numbers at the top of the Y axis indicate full-scale intensity in arbitrary units. The lipid contents of E. coli strain BL21DE3(pMALE-ptsG) were extracted and analyzed as described in Materials and Methods.
Figure 17.
LC-ESI-MS negative ion mode spectra of phosphatidylglycerol (PG) molecular species of lipid extract of peak 1 fraction of E. coli strain BL21DE3(pMALE-ptsG).
Arrows indicate different PG molecular species detected in peak 1 and standard preparations (for standard preparation see Figure S9 at Saier web site). Right handed numbers at the top of the Y axis indicate full-scale intensity in arbitrary units. The lipid contents of E. coli strain BL21DE3(pMALE-ptsG) were extracted and analyzed as described in Materials and Methods.
Figure 18.
LC-ESI-MS negative ion mode spectra of cardiolipin (CA) molecular species of lipid extract of peak 1 fraction of E. coli strain BL21DE3(pMALE-ptsG).
Arrows indicate different CA molecular species detected in peak 1 and standard preparations (for standard preparation see Figure S12 at Saier web site). I.S., internal standard; right handed numbers at the top of the Y axis indicate full-scale intensity in arbitrary units. The lipid contents of E. coli strain BL21DE3(pMALE-ptsG) were extracted and analyzed as described in Materials and Methods.
Table 6.
Relative ratios of PE, PG and CA and lipid/protein ratios of different EIIGlc-MBP preparations.
Table 7.
Approaches used in this study and results obtained.