A chaperone network controls the heat shock response in E. coli

Abstract

The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, sigma32. The DnaK chaperone machine regulates both the amount and activity of sigma32, thereby coupling sigma32 function to the cellular protein folding state. In this manuscript, we analyze the ability of other major chaperones in E. coli to regulate sigma32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of sigma32. We show that increasing the level of GroEL/S leads to a decrease in sigma32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of sigma32 by increasing the level of sigma32. In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to sigma32 and decreases sigma32-dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate sigma32 results in a more sensitive and accurate detection of the protein folding environment.

Figure 1.

Effects of GroEL/S and DnaK/J overexpression on σ³²-dependent transcription. An exponential phase culture of strain 594 with a σ³²-dependent lacZ reporter and carrying either a plasmid able to overexpress GroEL/S (pGro7) or a plasmid able to overexpress DnaK/J (pKJE7) was grown at 30°C with and without 0.2% arabinose to induce GroEL/S or DnaK/J overexpression. A standard differential rate of synthesis plot is shown. The uninduced (control) strains gave identical results; therefore, for simplicity, we have included the data for only one control strain. This experiment and every other differential rate of synthesis experiment was performed at least three times with similar results.

Figure 2.

GroEL/S overexpression results in inhibition of σ³² activity in vivo. An exponential phase culture of strain C600 carrying plasmid able to overexpress GroEL/S (pGro11) was grown in M9 minimal media containing all amino acids except for methionine and cysteine at 30°C. Anhydrotetracycline was added at time 0 to induce GroEL/S overexpression. The rate of synthesis of two σ³²-dependent proteins, HtpG and DnaK (A), and a σ^E-dependent protein, RseA, and a σ⁷⁰-dependent protein, RpoB (C), were measured. (B) The level of GroEL and σ³² were measured using Western analysis. All protein synthesis and Western data shown are the average from at least two independent experiments.

Figure 3.

The ratio of GroEL/S to substrates in vivo is important for determining the activity of σ³². An exponential phase culture of strain 594 carrying a plasmid able to overexpress GroEL/S (pGro7) and one able to overexpress HrcA (pJDW39) was grown in LB at 30°C in the absence of inducers (control) or in the presence of 0.2% arabinose to induce GroEL/S overexpression and/or 1 mM IPTG to induce HrcA expression. A standard differential rate of synthesis plot is shown.

Figure 4.

Depletion of GroEL/S in vivo increases σ³² activity. Strain CAG48176, whose chromosomal groELS gene is driven by the inducible Para promoter, was grown in exponential phase at 30°C in M9 minimal media containing all amino acids except methionine and cysteine with 0.2% fructose as the main carbon source and 0.1% arabinose to maintain near wild-type levels of GroEL/S. Depletion of GroEL/S was initiated at time 0 by removing arabinose from the media and adding 0.2% glucose. HtpG synthesis and GroEL levels were analyzed as in Figure 2.

Figure 5.

Changing the levels of GroEL/S increases the magnitude and duration of the heat shock response. An exponential phase culture of strain C600 or a derivative having the chromosomal groELS gene under control of the inducible Para promoter (CAG48176) was grown at 30°C and subjected to heat shock by increasing the temperature to 42°C. σ³² activity was measured by examining the rate of synthesis of HtpG.

Figure 6.

GroEL interacts directly with active σ³² in vitro. (A) Purified ³⁵S-labeled σ³² (500 nM) was incubated with GroEL (2 μM), core RNAP (2 μM), or a GroEL-binding mutant, GroELY203E (2 μM), for 30 min at 20°C in protein-binding buffer (PBB). The proteins were then fractionated on a Superose 12 gel filtration column with PBB at 4°C. Fractions were collected and counted on a scintillation counter to determine the level of σ³² in each fraction. (B) The free σ³² peaks from the GroEL and σ³²-binding reaction in A were pooled and additional unlabeled σ³² was added to bring the concentration to 500 nM as in A. This σ³² was then incubated with 2 μM GroEL or 2 μM core RNAP and analyzed as in A.

Figure 7.

GroEL inhibits σ³²-dependent in vitro transcription. Multiround in vitro transcription was performed with holoenzyme containing either σ³² (Eσ³²) or σ⁷⁰ (Eσ⁷⁰) (100 nM) incubated with GroEL (500 nM), GroES (1 μM), GroELY203E (500 nM), DnaK (2 μM), DnaJ (400 nM), or combinations thereof. An end-labeled oligo was added to each reaction as an internal control. Transcription reactions were phenol-chloroform extracted, ethanol precipitated, and analyzed on a 6% polyacrylamide gel. (A) Representative transcription gel showing duplicate reactions documenting GroEL inhibition of Eσ³². (B) Quantification and summary of transcription results from A as well as from additional transcription experiments.