Regulon and promoter analysis of theE. coliheat-shock factor, σ³², reveals a multifaceted cellular response to heat stress

Abstract

The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative factor, 32 . To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by 32 and screened ORF overexpression libraries to identify 32 inducers. We triple the number of genes validated to be transcribed by 32 and provide new insights into the cellular role of this response. Our work indicates that the response is propagated as the regulon encodes numerous global transcriptional regulators, reveals that 70 holoenzyme initiates from 12% of 32 promoters, which has important implications for global transcriptional wiring, and identifies a new role for the response in protein homeostasis, that of protecting complex proteins. Finally, this study suggests that the response protects the cell membrane and responds to its status: Fully 25% of 32 regulon members reside in the membrane and alter its functionality; moreover, a disproportionate fraction of overexpressed proteins that induce the response are membrane localized. The intimate connection of the response to the membrane rationalizes why a major regulator of the response resides in that cellular compartment.[Keywords: Heat-shock response; 32; transcription; microarray] Supplemental material is available at http://www.genesdev.org. Received March 13, 2006; revised version accepted April 25, 2006. When cells are shifted from low to high temperature, synthesis of the heat-shock proteins (hsps) is rapidly and selectively induced. The heat-shock response (HSR), was first identified by Ritossa (1963), who showed that exposure to heat lead to transient changes in the puffing pattern of salivary chromosomes in Drosophila; Tissieres et al. (1974) demonstrated that these changes reflected the transient induction of several proteins. Initially, hsp function was unclear; however, experiments in several organisms revealed that many hsps were chaperones that promote protein folding (Pelham 1986;Beckmann et al. 1990;Gaitanaris et al. 1990;Skowyra et al. 1990). These studies not only suggested that a major function of the HSR is to maintain the protein folding state of the cell, but also indicated that some of these chaperones, such as Hsp70 and Hsp90, are present in all organisms Craig 1984, 1987). Thus, both the HSR and some hsps are universally conserved among organisms.In Escherichia coli, 32 , an alternative factor, controls the HSR by directing RNA polymerase to transcribe hsps (Yamamori and Yura 1980;Grossman et al. 1984;Taylor et al. 1984;Cowing et al. 1985). Synthesis of hsps is induced upon temperature upshift and repressed upon temperature downshift (Lemaux et al. 1978;Yamamori et al. 1978;Straus et al. 1987 Straus et al. , 1989Taura et al. 1989), thereby allowing a rapid cellular response to changes in temperature.32 is controlled by negative feedback loops controlling its activity (Straus et al. 1989;Blaszczak et al. 1999) and stabili...

YrhB is a highly stable small protein with unique chaperone‐like activity in Escherichia coli BL21(DE3)

“…The measurement of time-course b-galactosidase activity at 37, 42, and 48°C (Fig. S4) indicates that transcription of yrhB seems to be regulated by r 70 -dependent promoter, which also corresponds to the following previous results [18][19][20][21]: the transcription level of yrhB was not increased by the overexpression of rpoH [18,19] and was not upregulated by heat shock [20,21]. Consequently, it seems that YrhB cannot be defined as a traditional heat shock protein.…”

“…In this study, we also found that YrhB is indispensable for sustaining growth of E. coli BL21(DE3) at high culture temperature. A result of this study and other previous reports [18][19][20][21] show that the transcription of yrhB is not regulated by RpoH, indicating that YrhB cannot be defined as a traditional heat shock protein. This study reports that YrhB is extremely thermostable and keeps monomer conformation under heat shock condition unlike other small heat shock proteins that form multimeric or oligomeric structures.…”

Translation quality control is critical for bacterial responses to amino acid stress

“…To assess further how increased cellular levels of m-TyrtRNA Phe affect the global integrity of the proteome, transcript levels of several components of the sigma 32-(rpoH)-and sigma E-(rpoE)-dependent protein stress regulons were measured (Table 1). Consistent with increased levels of misfolded proteins, genes encoding components of the general heat shock response (rpoH, dnaK, and mopA) were up-regulated in the presence of m-Tyr in the PheRS editing-deficient strain (31). In contrast, transcript levels of two components of the sigma E envelope stress response (rpoE and ptr) were unchanged under the conditions studied, but a third Sigma E-dependent transcript, htrA, was 32-fold higher in the editing-deficient strain in the presence of m-Tyr (32).…”

Analysis of σ 32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

“…Multiround in vitro transcription was carried out as reported previously (16), and single-round transcription was done as described (44).…”