Growth rate of Escherichia coli.

It should be possible to predict the rate of growth of Escherichia coli of a given genotype in a specified environment. The idea that the rate of synthesis of ATP determines the rate of growth and that the yield of ATP determines the yield of growth is entrenched in bacterial physiology, yet this idea is inconsistent with experimental results. In minimal media the growth rate and yield vary with the carbon source in a manner independent of the rate of formation and yield of ATP. With acetate as the carbon source, anapleurotic reactions, not ATP synthesis, limit the growth rate. For acetate and other gluconeogenic substrates the limiting step appears to be the formation of triose phosphate. I conclude that the rate of growth is controlled by the rate of formation of a precursor metabolite and, thus, of monomers such as amino acids derived from it. The protein-synthesizing system is regulated according to demand for protein synthesis. I examine the conjecture that the signal for this regulation is the ratio of uncharged tRNA to aminoacyl-tRNA, that this signal controls the concentration of guanosine tetraphosphate, and that the concentration of guanosine tetraphosphate controls transcription of rrn genes. Differential equations describing this system were solved numerically for steady states of growth; the computed values of ribosomes and guanosine tetraphosphate and the maximal growth rate agree with experimental values obtained from the literature of the past 35 years. These equations were also solved for dynamical states corresponding to nutritional shifts up and down.

Effect of temperature on the size of Escherichia coli cells.

The distributions of cell volumes of steady-state Escherichia coli ML30G cultures at various temperatures were measured. For cultures in a minimal medium, the distributions were indistinguishable at several temperatures between 15 and 30 C; at higher temperatures the cells were slightly smaller, and at lower temperatures they were slightly larger. For cultures in a complex medium, the cells were slightly larger at both high and low temperatures of growth. An abrupt change of temperature within the middle range led to a transient change in the distribution of cell volume, suggesting that the size of dividing cells is well regulated. No synchrony of division was induced by a change in temperature.

Estimation of growth rate from the mitotic index.

The growth rate of a eukaryotic population dividing at a constant rate can be estimated from the equation, t(m)/g ln 2 = ln (1 + R), in which t(m) is the time required for mitosis, g is the generation time, and R is the fraction of cells undergoing mitosis. Values for t(m) and R can be determined by direct microscope examination of the population. The validity of the derived equation has been checked with an exponentially growing culture of a prokaryote, Escherichia coli, in which chloramphenicol was administered to inhibit protein synthesis. Cells having enough protein completed the division process whereas the rest of the population was inhibited. From the plot of the growth curve before and after administration of chloramphenicol, t(m) and R were estimated. The calculated and actual growth rates were almost identical.

Effect of nutrient concentration on the growth of Escherichia coli.

The relationship between specific growth rate of Escherichia coli and the concentration of limiting nutrient (glucose or phosphate or tryptophan) has been determined for populations in a steady state. At high concentrations the specific growth rate is independent of the concentration of nutrient, but at low concentrations the specific growth rate is a strong function of the nutrient concentration. Such a relationship was predicted by Monod; however, Monod's equation does not predict the relationship over the entire range of nutrient concentration. If parameters of the equation are estimated from the results obtained at low concentrations, then at high concentrations of nutrient, the specific growth rate is significantly higher than that predicted by Monod's equation. These results were interpreted on the basis that the rate of growth is controlled by at least two parallel reactions and that the affinities of the enzymes catalyzing these reactions are different. The relationship between specific growth rate and mean cell volume was also measured, and the results indicate that mean cell volume depends not only on the specific growth rate but also on the nature of the limiting nutrient. There are different mean cell volumes at the same specific growth rate established by different limiting nutrients. Therefore, the mean cell volume is not uniquely determined by the specific growth rate.

Determination of the minimal temperature for growth of Escherichia coli.

The minimal temperature for growth of Escherichia coli ML30 in glucose minimal medium is between 7.5 and 7.8 C. After transfer to subminimal temperature, growth occurs but is not sustained.

Synchronous growth of enteric bacteria.

Helmstetter and Cummings devised a technique of synchronization in which cells are implanted on a membrane filter and the membrane is subjected to reverse flow of liquid medium. The cells in the effluent stream have predominantly the characteristics of newborn cells. The advantage of this technique is that the population experiences a minimum of physiological stress; hence, the behavior of the synchronous culture should reflect the normal divisional cycle. The disadvantage is that strains other than Escherichia coli B/r cannot be synchronized. We have found that a modification of the method makes it possible to synchronize several strains of E. coli, including both male and female strains, as well as Salmonella typhimurium LT2. The principal difference in technique is a prolonged period (>400 doublings) of cultivation in glucose minimal medium at 30 C and at low density (<5 x 10(6) cells/ml) prior to implantation. This precaution was taken to insure that the bacterial growth population is in a steady state of balanced growth. From the resulting synchronous growth, the distribution of interdivision times has been computed; these distributions have coefficients of variation in the range 0.18 to 0.22 and are not appreciably skewed.

Growth in volume of Euglena gracilis during the division cycle.

The distribution of volumes of Euglena gracilis cells was measured conductimetrically. The volume spectrum of cultures in balanced growth was analyzed by the method of Collins and Richmond. The kinetics of volume increase of Euglena is neither linear nor exponential; the growth rate of small and large cells is low, but intermediate size cells show the largest growth rate.

Kinetics of growth of individual cells of Escherichia coli and Azotobacter agilis.

Escherichia coli and Azotobacter agilis were grown in minimal media until a steady state was established. The distribution of cell size was determined electronically. From the equation of Collins and Richmond, the growth rate of individual cells was computed as a function of size. The main features of the growth of individual E. coli and A. agilis cells revealed by this work were: the specific growth rate decreased at the time of division, and both the absolute and specific growth rates increased between divisions. The frequency function of interdivision times was computed and was found to be positively skewed with a coefficient of variation of approximately 0.3. The results supported the hypothesis of Koch and Schaechter that the size of an individual cell at division is highly regulated.

Measurement of size distributions of bacterial cells.

Harvey, R. J. (University of California, Davis), and Allen G. Marr. Measurement of size distributions of bacterial cells. J. Bacteriol. 92:805-811. 1966.-Apparatus for the automatic determination of the volume distribution of particles by measurement of the amplitude of pulses generated in a Coulter transducer is described. Distributions of volume estimated by direct measurement of pulse amplitude are distorted by coincidence. Differentiation and integration of the pulses followed by automatic pulse-height analysis permit precise measurement of volume of latex spheres and of bacteria over a range of at least 0.25 to 20 mu(3). The apparatus is also capable of accurate determination of particle concentration over a wide range. Other advantages are the speed of both measurement and data processing.