Isolation and characterization of the Escherichia coli htrD gene, whose product is required for growth at high temperatures

Abstract

Those genes in Escherichia coli defined by mutations which result in an inability to grow at high temperatures are designated htr, indicating a high temperature requirement. A new htr mutant of E. coli was isolated and characterized and is designated htrD. The htrD gene has been mapped to 19.3 min on the E. coli chromosome. Insertional inactivation of htrD with a mini-TnlO element resulted in a pleiotropic phenotype characterized by a severe inhibition of growth at 42°C and decreased survival at 50°C in rich media. Furthermore, htrD cells were sensitive to H202. Growth rate analysis revealed that htrD cells grow very slowly in minimal media supplemented with amino acids. This inhibitory effect has been traced to the presence of cysteine in the growth medium. Further studies indicated that the rate of cysteine transport is higher in htrD cells relative to the wild type. All of these results, taken together, indicate that the htrD gene product may be required for proper regulation of intracellular cysteine levels and that an increased rate of cysteine transport greatly affects the growth characteristics of E. coli. The heat shock response is a highly conserved cellular response to environmental stress, especially rapid changes in temperature (for a review, see reference 30). In E. coli, about 20 genes are transiently expressed at increased levels following a shift up in growth temperature. These genes form a regulon under the coordinate control of the positive transcriptional regulator C32. At least some of the heat shock genes are essential for E. coli growth at all temperatures, indicating that these genes, and indeed the heat shock response itself, play a fundamental role in bacterial physiology.In order to better understand how E. coli survives and adapts to exposure to high temperatures, a study has been initiated to define new genes whose products are required for growth at high temperatures. Libraries of E. coli cells harboring transposon insertions were created at 30°C. These libraries were subsequently screened at 42°C for the inability to grow well at this higher temperature. The genes defined by these insertions are designated htr (for high temperature requirement). Some of the genes isolated by this method have been described and fall into two broad classes based upon regulation of expression. The first class of htr genes is composed of previously unidentifi ( heat shock genes and * Corresponding author. includes the htrA and htrC genes (22,32 MATERIALS AND METHODSBacteria, bacteriophages, and plasmids. The bacteria, bacteriophages, and plasmids used in this work are described in Table 1.Media and bacterial growth. Bacteria were routinely cultured in either L broth or M9 minimal medium (27). When required, antibiotics were added to the following final concentrations: kanamycin, 100 ,uglml; ampicillin, 50 ,ugIml; tetracycline, 15 ,ug/ml. Glucose and glycerol were added to a final concentration of 0.4% each. Sodium acetate was added to a concentration of 0.2%. Sucrose, when used for suppression of the H...

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

“…Cyd 2 mutants have a complex phenotype, including sensitivity to: (i) zinc and azide, inhibitors of the remaining cytochrome o-mediated respiratory chain (Poole et al, 1989); (ii) iron chelators (Cook et al, 1998) and (iii) H 2 O 2 (Delaney et al, 1992;Lindqvist et al, 2000). We exploited these sensitivities to test strain AN2343 (cydD1) that had been transformed with pRKP1602 (bearing the wild-type cydDC genes) or site-directed cydD mutant derivatives, by plating on ZABC medium (containing Zn 2+ and azide ions) or plates supplemented with EDDHA or H 2 O 2 .…”

“…Loss of cytochrome bd causes a complex phenotype that extends beyond the lack of a functional oxidase. The Cyd 2 phenotype in E. coli includes (i) an inability to exit aerobically from stationary phase at 37 uC (Siegele et al, 1996); (ii) sensitivity to high temperature (Delaney et al, 1992), (iii) increased sensitivity to H 2 O 2 (Delaney et al, 1992); (iv) sensitivity to iron chelators (Cook et al, 1998); and loss of motility and increased benzylpenicillin sensitivity (Pittman et al, 2002). The need to understand cytochrome bd assembly is underlined by the importance of this oxidase in many bacteria.…”

A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered

“…For example, cytochrome 6d is induced when E. coli is grown under unfavourable conditions (Cotter et al, 1990;Avetisyan et al, 1991;Bogachev et al, 1995). Mutants that cannot synthesize cytochrome bd have a pleiotropic phenotype: they are sensitive to H,O,, temperature sensitive (Delaney et al, 1992(Delaney et al, , 1993Wall et al, 1992), and are unable to exit from stationary phase and resume aerobic growth at 37 "C (Siegele & Kolter, 1993 ;Siegele et al, 1996). Macinga & Rather (1996) have shown that the growth of Cyd-mutants (cydAB or cydD) is inhibited by an extracellular factor excreted by wild-type cells or Cydmutants.…”

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon

“…No significant differences were observed in the growth kinetics of DW114 compared with those of its B178 parental strain (data not shown). Sensitivity to hydrogen peroxide treatment was also monitored as described by Delaney et al (6). Figure 3 shows that DW114 was much more sensitive to the presence of 10 mM hydrogen peroxide in L broth supplemented with 100 ,uM CuS04 at 30°C for 30 min than the B178 parental strain.…”