Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli

Abstract

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i). the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii). heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii). basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

FIG. 1.

Induction of GFP expression by treatment with 75 MPa (♦), 150 MPa (▴), or 50°C (—) for 15 min. Noninduced control (▪) cells were kept at 20°C during pretreatment. Transcription was assayed from rpoH, lon, clpPX, and dnaK promoter fusions with gfp. Rfu, relative fluorescence units.

FIG. 2.

HHP reduction factors (250 MPa for 15 min) of heat-shocked and non-heat-shocked cells at different time intervals after pretreatment (bars). dnaK expression was assayed at the same time (line with black squares, control cells; line with gray triangles, heat-shocked cells) by pAA212. Rfu, relative fluorescence units.

FIG. 3.

Basal expression levels of rpoH, lon, clpPX, and dnaK promoter fusions with gfp in wild-type MG1655 (▪) and its pressure-resistant mutants LMM1010 (♦), LMM1020 (▴), and LMM1030 (—). Rfu, relative fluorescence units.

FIG. 4.

Two-dimensional autoradiograms of protein expression profiles in MG1655 (A), LMM1010 (B), LMM1020 (C), and LMM1030 (D) in late exponential phase at 37°C in supplemented M9 medium. Numbers mark heat shock proteins: 1, DnaK; 2, GroEL; 3, GrpE; 4, GroES; 5, ClpB; 6, HtpG. The letters a and b mark unidentified proteins with altered expression in the mutants. The presence of two ClpB spots is due to the presence of two translational starts on the clpB mRNA (50).