The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature

Abstract

In vitro transcription analysis has shown that only RNA polymerase containing an alternative sigma subunit, E , activates transcription from one of the rpoH promoters and the htrA promoter. The location of the rpoE gene encoding E on the Escherichia coli chromosome has recently been established, but no rpoE mutant has yet become available for phenotypic testing. We cloned the rpoE gene from the -ordered clones of the E. coli genome and confirmed that the reconstituted RNA polymerase containing the gene product (E E ) can transcribe htrA in vitro. We constructed an rpoE-defective strain by gene disruption using the cloned rpoE gene. We demonstrate that expression of htrA is completely dependent on the rpoE gene in vivo and that the rpoE gene is essential for bacterial growth at high temperature.In Escherichia coli, RNA polymerase consists of the subunits of 2␣, ␤, ␤Ј, and a specific . The subunit directs RNA polymerase to initiate transcription at promoter sites on the DNA (6). The primary factor, 70 encoded by rpoD, is responsible for transcription of the majority of the genes expressed during exponential growth. In addition, alternative factors direct transcription of sets of genes whose products are needed for specific functions, such as nitrogen fixation, flagellum synthesis, heat shock response, and stationary-phase growth (4).The activities of two alternative factors, 32 and E , increase after the cell is exposed to a high temperature or ethanol (2,3,5,21,25,28). RNA polymerase containing 32 (E 32 ) transcribes the heat shock genes whose products are related to chaperones and proteases (29). The functions of many of these heat shock proteins are involved in binding to cytoplasmic proteins and assist the cytoplasmic proteins in folding or unfolding (7,15,22,26) and in protecting the cell from severe stresses such as exposure to 10% ethanol or a 50ЊC temperature (18). Using an in vitro transcription assay, Erickson and Gross (2) and Wang and Kaguni (25) have independently identified the E subunit that is essential for transcription from one (rpoHp 3 ) of the promoters of rpoH encoding 32 and the promoter of htrA encoding a periplasmic endopeptidase essential for growth at high temperatures. Mecsas et al. (16) have proposed that the E regulon is involved in the processes that function in the extracytoplasmic compartments.

Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

“…In a subsequent study, Martin et al (1994) showed that the ¹35 and ¹10 regions of promoters transcribed by AlgU shared considerable similarity with the known promoter consensus sequence for E , the second heat-shock sigma factor identified at the biochemical level in Escherichia coli (Erickson and Gross, 1989). These and subsequent sequence analyses permitted the identification of E. coli and Salmonella typhimurium rpoE genes based on striking homologies of their gene products with AlgU (Hiratsu et al, 1995;Martin et al, 1994;Raina et al, 1995;Rouviè re et al, 1995). These observations and additional analyses have uncovered the existence of a broader family of novel alternative sigma factors termed ECF (Lonetto et al, 1994) or E -like factors .…”

Efficient export of alkaline phosphatase overexpressed from a multicopy plasmid requires degP, a gene encoding a periplasmic protease of Escherichia coli.

“…E. coli MC4100 lF [htrA-lacZ] (Hiratsu et al, 1995) (Kadokura et al, 1994) is a pBR322-derived plasmid that expresses phoA under its own promoter. Plasmid pRS414 and pRS415 (Simons et al, 1987) are generous gifts of R. W. Simons, Department of Microbiology, University of California, Los Angeles, USA.…”

“…The level of PhoA synthesis increased in keeping with the IPTG concentration, confirming the regulated expression of phoA from the trc promoter. To examine the level of degP transcription in these cells, we used a htrA-lacZ fusion gene (Hiratsu et al, 1995), in which degP promoter is immediately followed by a lacZ gene lacking its own promoter. As Fig.…”

Cell lysis directed by σ E in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli

“…s E , first identified as a transcription factor for the rpoH gene, which encodes a main heat-shock s factor (Erickson & Gross, 1989;Wang & Kaguni, 1989), is involved in the expression of several E. coli genes (De Las Peñas et al, 1997a;Rouviëre et al, 1995) whose products deal with unfolded periplasmic or membrane proteins caused by heat shock or environmental stresses Raina et al, 1995;Rouviëre et al, 1995). s E has been demonstrated to be an essential sigma factor in the organism at both high and low temperatures (De Las Peñas et al, 1997b;Hiratsu et al, 1995;Lipinska et al, 1989). The accumulation of unfolded extracytoplasmic proteins triggers intracellular active s E molecules (Mecsas et al, 1993) via a unique mechanism of s E modulation, in which RseA, RseB and RseC, encoded by the rpoE-rseABC operon, may be involved (De Las Peñas et al, 1997a;.…”