Effects of temperature on the size and shape of Escherichia coli cells

Two substrains of Escherichia coli B/r were grown to steady-state in batch cultures at temperatures between 22 and 42° C in different growth media. The size and shape of the cells were measured from light and electron micrographs and with the Coulter channelizer. The results indicate that cells are shorter and somewhat thicker at the lower temperatures, especially in rich growth media; cell volume is then slightly smaller. A lower temperature was further found to increase the relative duration of the constriction period. The shapes of the cell size distributions are indistinguishable, indicating that the pattern of growth of the cells is the same at all temperatures. The adaptation of the cells to a temperature shift lasted several generations, indicating that the morphological effects of temperature are mediated by the cell's physiology.     

Isopycnography of intact cells. VI: Effect of temperature on steady state Escherichia coli

Steady state cultures of Escherichia coli W3110 possess constant cellular concentrations of ribosomes while above 22 C as measured isopycnographically. Below 22 C, ribosome levels are significantly diminished, equivalent to those found in dormant cells. Concommitantly, the energy of activation of steady state population growth in E. coli remains at a constant 18.6 kcal between 22 C and 37 C. This suggests that the thermal stability of the protein synthesis apparatus may play an important role in pathogenesis as well as in other areas of ecological niche acquisition and dominance.     

Effect of temperature on motility and chemotaxis of Escherichia coli.

The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature. The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C. The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature. When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed. A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling. These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition.     

Effect of temperature on the self-assembly of the Escherichia coli ClpA molecular chaperone

Protein quality control pathways rely upon ATP-dependent proteases, such as Escherichia coli ClpAP, to perform maintenance roles in the cytoplasm of the cell. ATP-dependent proteases remove misfolded and partially synthesized proteins. This action is particularly important in situations where an unregulated accumulation of such proteins will have a deleterious effect on the cell. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and the ATPase/protein unfoldase ClpA (84.2 kDa monomer). ClpA also uses its protein unfolding activity to remodel proteins and protein complexes; thus, in the absence of the proteolytic component, ClpA is considered a molecular chaperone. Previous reports, by others, suggested that ClpA exists in a monomer-dimer equilibrium at 4 °C. In contrast, using a combination of sedimentation velocity, sedimentation equilibrium, and dynamic light scattering, we recently reported that ClpA exists in a monomer-tetramer equilibrium at 25 °C. Here we report an investigation of the effect of temperature on the self-association of the E. coli ClpA protein unfoldase using analytical ultracentrifugation techniques. The results of sedimentation velocity and sedimentation equilibrium experiments performed at multiple loading concentrations of ClpA over a range of temperatures from 3.9 to 38.2 °C are discussed. Sedimentation velocity experiments show a decrease in weight average s(20,w) at the extremes of temperature. This result, along with extensive sedimentation equilibrium data and analysis, suggests the presence of a dimeric intermediate of ClpA that is differentially populated as a function of temperature. Further, analysis of sedimentation equilibrium data as a function of temperature led us to propose a monomer-dimer-tetramer equilibrium to describe the temperature dependence of ClpA self-assembly in the absence of nucleotide.     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.     

Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.     

Effect of temperature and htpR on the biosynthesis of superoxide dismutase in Escherichia coli

The synthesis of Mn- and FeSODs in response to temperature changes was examined in strains of Escherichia coli with different mutations in sod and htpR genes. Growth at or shift to elevated temperatures induced FeSOD but not MnSOD. The induction of FeSOD by heat was inhibited by chloramphenicol and was independent of the heat shock (htpR-controlled) regulon. FeSOD was more stable at 42 degrees C than was MnSOD.     

Effect of temperature on the ATP pool and adenylate energy charge in Escherichia coli

The effect of cultivation temperature on the ATP pool and adenylate energy charge (EC) in Escherichia coli has been studied in both batch and continuous cultures. In batch culture, μ_max and the ATP pool increased with increasing growth temperatures between 27–42°C (from 0.26 to 0.62 h⁻¹, and from 5.1 to 8.2 nmol/mg dry wt., respectively). In continuous culture at a constant dilution rate (D = 0.2 h⁻¹), with increasing growth temperatures between 28–43°C, the ATP pool increased about 2-fold (from 4.2 to 8.1 nmol/mg dry wt) and the EC from 0.80 to 0.99.     

Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli.

The expression of kanamycin nucleotidyltransferase (KNTase) in Escherichia coli results in different forms of the protein, depending on the temperature; soluble active enzyme is synthesized at 23 degrees C but the protein is mostly aggregated and inactive in inclusion bodies when made at 37 degrees C. However, active enzyme can be recovered by solubilization of the inclusion bodies with 8 M urea followed by dilution of the denaturant, indicating that the polypeptide is not damaged covalently but is present in a misfolded state. The availability of thermostable mutants of KNTase allows a test of the hypothesis that formation of inclusion bodies when proteins are highly expressed in E. coli is due to the accumulation of a folding intermediate that is prone to temperature-dependent aggregation. Because these mutants were isolated by cloning the KNTase gene into the thermophile Bacillus stearothermophilus and selecting for kanamycin resistance at high growth temperatures, they must be thermostable for both synthesis and activity and must have folding intermediates that are less susceptible to the formation of aggregates. Indeed, whereas decreasing the temperature from 37 to 23 degrees C increased the KNTase specific activity 10-fold in cells expressing the wild-type enzyme, this change resulted in only a 2.1-fold increase for the TK1 (Asp80----Tyr) mutant and a 1.7-fold increase for the TK101 (Asp80----Tyr and Thr130----Lys) double mutant. The strategy of cloning in thermophiles and selecting or screening for mutants that fold correctly to yield biological activity at high growth temperatures may be useful in overcoming the problem of the insolubility of some proteins when expressed in heterologous hosts.     

Mechanism of thermoresistance in Escherichia coli cells exposed to temperature elevation

The influence of media with different osmotic pressure (NaCl water solution) and chloramphenicol (10 micrograms/ml) on the survival, permeability, and survival curve shape of Escherichia coli B/r and E. coli Bs-1 cells, heated up to 50, 52, and 60 degrees C was investigated. As shown, the survival curve of cells heated up to 60 degrees C in isotonic conditions was characterized by exponential shape, while the survival curves of cells heated up to 50 and 52 degrees C consisted of two components characterizing thermosensitive and thermoresistant parts of cell population. Hypertonic conditions of heat at 52 degrees C decreased cell lethality and permeability. In this case, survival curves were characterized by exponential shape. Chloramphenicol was shown to protect against damaging action of heat at 50 degrees C and not to affect the viability of cells heated at 52 and 60 degrees C. It is proposed that the increase of cell thermoresistance with heat dose elevation at 50 and 52 degrees C in isotonic conditions, which is accompanied by the appearance of thermotolerant components on survival curves, may be associated with accommodational cell reactions. The essence of these reactions consists in stabilization of the osmotic cell homeostasis.     

Light-scattering study of the temperature dependence of Escherichia coli motility.

Two light-scattering techniques are used to study the temperature dependence of translational and rotational motility in Escherichia coli. The method of number fluctuation spectroscopy is developed theoretically and experimentally to measure the translational swimming speed of a smooth swimming strain of E. coli. Interference fluctuation techniques are used to study the rotational component of the motion. The results demonstrate that the thrust remains proportional the the torque generated by the flagella throughout the range studied and also show that relative changes in translational swimming speed may be inferred from the dynamics of rotational motion.     

Effect of destruction of Escherichia coli cells on the catalytic ability of catalase

The possibility for investigation of catalase (CAT) activity under the conditions of intact E. coli cells was estimated. This approach is based on the possibility of hydrogen peroxide freely cross biological membranes. CAT activity of native cells had a broad maximum between pH values 4.5 and 7.5. Desintegration of cells by freezing--thawing and ultrasonication indicated that there were two CAT activity peaks at pH values about 3.5 and 7.0. Activity of CAT with acid pH-optimum decreased at cell desintegration, but one with neutral pH-optimum was rather stable under this procedure. The enzyme in native conditions was less sensitive to the inhibition by high concentrations of hydrogen peroxide than its counterpart from destroyed cells. Activity of CAT in native and desintegrated cell preparations had different sensitivity to heating and inhibition by reduced glutathione, but it was inhibited by azide similarly. Difference in the CAT properties of native and desintegrated bacteria preparations may be explained by different possibility to penetrate cell membrane by reagents and/or by possible modification of the enzyme properties at destruction of native microenvironment.     

Number and size distribution of the DNA chains in Escherichia coli.

The essential replication protein encoded by gene O of bacteriophage λ (O-λ) is one of the major polypeptides produced in vitro in a DNA-dependent protein synthesizing system with λ DNA as template (Yates et al., 1977). We have used this system to identify the proteins encoded by lambdoid phages φ80 and 82 and equivalent in function to O-λ. The O protein of each phage type differs slightly in polypeptide molecular weight. Hybrid λ-φ80 and λ-82 phages derived by recombination within gene O direct synthesis of hybrid O proteins with the aminoterminal segment characteristic of one parent, and the carboxyl-terminal segment characteristic of the other. Differences in structure among O-λ, O-80 and O-λ82 segregate together with specificity determinants for interactions between the O protein and the control site ori, and between the O protein and the product of replication gene P. The coding region for the O protein includes ori.