Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures

Abstract

As a typical mesophile, Escherichia coli can maintain balanced growth between approximately 10 and 49°C (19). In the range of approximately 21 to 37°C, the rate of E. coli growth varies as a simple function of temperature (19). Raising the temperature above 40°C leads to progressively slower growth rates and changes in the cellular content of many proteins (30, 31). The adaptation processes that occur on a shift to high temperature include an increased expression of a set of genes, called heat shock genes, many of which are highly conserved among procaryotic and eucaryotic organisms. These genes are dispersed throughout the chromosome, and their products perform various functions in the cell, most of which are not clearly understood thus far (12,16,30,31,34). The transcription of the majority of these genes is positively regulated by the product of the rpoH gene (previously known also as htpR or hin; for a review, see references 30 and 31), the ar32 subunit of the RNA polymerase holoenzyme (6,17). Some of these genes are also essential for bacterial growth under normal temperature conditions, for example, the rpoD gene, which codes for the cr70 subunit of RNA polymerase (30, 31), or grpE, which is essential for the replication of bacteriophage X but which plays an otherwise unknown role in E. coli physiology (D. Ang and C. Georgopoulos, submitted for publication). Several heat shock genes, like lysU, which codes for an alternate form of lysyl-tRNA synthetase (30,31,38), or lon, which codes for an ATP-dependent protease (26), are not absolutely essential for bacterial growth. There is also a class of heat shock genes that is conditionally dispensable at low temperatures; i.e., deletion mutants can be constructed at low temperatures but they grow poorly and rapidly accumulate extragenic suppressors (e.g., dnaK [7a] and dnaJ [S. Sell and C. Georgopoulos, unpublished data]).Apart from the canonical heat shock genes, the rpoH regulatory gene itself is indispensable for cell adaptation to high temperatures (9). It is also known from two-dimensional electrophoresis of total E. coli proteins that there are other * Corresponding author.

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

“…6A. MCKH21 cells began to lyse at 60 min after the shift to 43ЊC, as observed with the htrA mutant cells (9). The colony-forming ability of the rpoE mutant cells began to decline as the optical density of the cells decreased (data not shown).…”

“…The htrA mutants were originally isolated as mini-Tn10 transposon insertion mutants which could grow at 30ЊC but could not grow at temperatures above 42ЊC (9,10). Since expression of the htrA gene was very strictly regulated by E in vitro and in vivo, we thought that the rpoE-deficient strain could not grow at high temperature.…”

The HtrA family of serine proteases

“…HtrA, also known as DegP and probably identical to the Do protease (Seol et al, 1991), is a heat shock-induced envelope-associated serine protease that was first described in Escherichia coli (Lipinska et al, 1989;Strauch et al, 1989) but is now known to have homologues in bacteria as diverse as cyanobacteria and mycobacteria, as well as in some eukarya (humans, yeast) (Zumbrunn and Trueb, 1996;Ponting, 1997). HtrA is one of several dozen proteases in E. coli (for a review, see Gottesman, 1996).…”

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype

“…The htrA gene was discovered independently by two different approaches. One approach involved a search for genes whose products are required at high temperature (above 42°C) but are not required at low temperature (30°C) (19). The other strategy involved the characterization of a mutant which was defective in proteolysis of certain periplasmic fusion proteins (33).…”