Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo

Abstract

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46 degrees C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50 degrees C. When cultures were allowed to recover from transient exposure to 50 degrees C, all mutations except Deltaibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42 degrees C when combined with the dnaK756 or groES30 alleles, while the Deltaibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46 degreesC compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.

FIG. 1

Structure of the ibp operon. An ibp null mutant was constructed by insertion of a kanamycin resistance cartridge (Kan) between the indicated BstXI sites. This operation removes about 60% of the ibpA open reading frame and the entire ibpB gene. The presence of the Δibp1::kan mutation in E. coli JGT1 and JGT17 was confirmed by PCR analysis of chromosomal DNA with the primer set shown. The same primers were used for PCR amplification of the ibp operon. The figure is not drawn to scale.

FIG. 2

Growth and viability of chaperone mutants at high temperatures. (A) Stationary-phase cells grown at 30°C were diluted directly into fresh medium held at 46°C. Average variation (coefficient of variation [CV]) between duplicate cultures was 6.2%. (B) Cells containing the indicated mutations were grown to mid-exponential phase at 30°C and shifted to 50°C. Data shown represent the ratio of viable cells after high-temperature incubation to that of viable cells before the shift to 50°C. The average CV for duplicate cultures was 23%.

FIG. 3

Recovery of chaperone mutants following transient incubation at 50°C. Cells grown to mid-exponential phase at 30°C were shifted to 50°C for 1 h and diluted into fresh medium held at 42°C (A) or 45°C (B). The average coefficient of variation for duplicate cultures was 7.2%. Note the difference in scales for culture absorbance and incubation time.

FIG. 4

Effect of double chaperone mutations on growth at 42°C. (A) dnaK756 single or double mutants grown overnight at 30°C were diluted into fresh medium held at 42°C. The average coefficient of variation (CV) for duplicate cultures was 5.9%. (B) groES30 single or double mutants were grown overnight at 30°C and diluted into fresh medium held at 42°C. The average CV for duplicate cultures was 6.8%. Note the difference in scale for culture absorbances.

FIG. 5

Effect of chaperone mutations on host protein aggregation at 46°C. Cells containing the indicated mutations were grown to mid-exponential phase at 30°C and shifted to 46°C for 1 h. Soluble (upper gel) and insoluble (lower gel) cellular fractions from identical absorbance units (A₆₀₀) of culture were separated by reducing SDS-PAGE (12.5% polyacrylamide). Insoluble fractions were loaded at three times the absorbance units of soluble fractions. Markers (M [Bio-Rad]) correspond to the molecular masses 104, 81, 47.7, 34.6, 28.3, and 19.2 kDa. The positions of the following proteins are indicated by arrows: upper gel from top to bottom, ClpA and ClpB, DnaK and HtpG, and GroEL; lower gel, GroES, IbpA, and IbpB. Gels were digitized with a Sharp JX-325 high-resolution scanner and the NIH Image 1.60 software for PowerPC.