Article Figures & Data
Figures
- Fig. 1.
Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase (A600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
- Fig. 2.
Comparison of anti-Hsp90 and anti-HtpG reactivities withP. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase (A600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti-A. ambisexualis Hsp90 (A. a.), rabbit anti-E. coli HtpG (E. c.), and rabbit anti-human Hsp90 (H. s.). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
- Fig. 3.
Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log (A600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
- Fig. 4.
Kinetics of expression of HtpG following heat shock ofP. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log (A600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
- Fig. 5.
Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log (A600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
- Fig. 6.
Comparison of HtpG expression by five strains ofP. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis. Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpGmRNA transcript following heat shock of P. gingivalis. Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the32P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
- Fig. 7.
Alignments of deduced amino acid sequences of P. gingivalis, E. coli, and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli (1), A. actinomycetemcomitans (43), and P. gingivalis ATCC 33277. Shaded residues are identical between at least two of the sequences. Boxed residues are conserved between at least two of the sequences (34).
