Specific Growth Rate Determines the Sensitivity of Escherichia coli to Thermal, UVA, and Solar Disinfection

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h⁻¹) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T₉₀ (time until 90% of the population is inactivated) values (T_{90, chemostat} = 2.66 h; T_{90, batch} = 2.62 h), whereas T₉₀ for cells growing at a μ of 0.9 h⁻¹ was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (μ = 1.0 h⁻¹) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (μ = 0.3 h⁻¹), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.     

Codon usage determines translation rate in Escherichia coli

We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure. We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged. The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other. The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures. The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second. We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons. In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol. In this way we could investigate the influence of mRNA structure on translation rate. This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments. We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase. Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate. In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.     

Interrelation of DNA replication, specific growth rate and growth temperature in the sensitivity of Escherichia coli to cold shock.

Escherichia coli W3110 was grown in a chemostat under conditions of carbon limitation at various temperatures and specific growth rates (mu). Exponential survivor-time curves following cold osmotic shock were biphasic. These could be described by the sum of two exponential functions representing the survival of sensitive and resistant fractions of the population where the size of the sensitive fraction was directly proportional to mu. Decimal reduction times for the more resistant fraction were unaffected by mu yet decreased with increasing growth temperature. Sensitivity to cold shock was evaluated for an E. coli CR34 mutant, temperature-sensitive in initiation of DNA replication. When grown in the chemostat at the non-restrictive temperature (30 degrees C) sensitivity was directly proportional to mu. Following a rise in the incubation temperature to 42 degrees C, sensitivity decreased markedly and reached a minimum 45 to 60 min after the temperature increase. Sensitivity of the E. coli mutant grown at 30 degrees C and raised to 42 degrees C for 1 h was low and relatively unaffected by growth rate.     

Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli

Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon- and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinone-sufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.     

Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae

AIMS: To determine the efficacy of solar disinfection (SODIS) for enteric pathogens and to test applicability of the reciprocity law. METHODS AND RESULTS: Resistance to sunlight at 37 degrees C based on F99 values was in the following order: Salmonella Typhimurium>Escherichia coli>Shigella flexneri>Vibrio cholerae. While F90 values of Salm. Typhimurium and E. coli were similar, F99 values differed by 60% due to different inactivation curve shapes. Efficacy seemed not to be dependent on fluence rate for E. coli stationary cells. Sensitivity to mild heat was observed above a temperature of 45 degrees C for E. coli, Salm. Typhimurium and Sh. flexneri, while V. cholerae was already susceptible above 40 degrees C. CONCLUSIONS: Salmonella Typhimurium was the most resistant and V. cholerae the least resistant enteric strain. The reciprocity law is applicable for stationary E. coli cells irradiated with sunlight or artificial sunlight. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli might not be the appropriate indicator bacterium to test the efficacy of SODIS on enteric bacteria and the physiological response to SODIS might be different among enteric bacteria. The applicability of the reciprocity law indicates that fluence rate plays a secondary role in SODIS efficacy. Stating inactivation efficacy with T90 or F90 values without showing original data is inadequate for SODIS studies.     

Evaluation of the influence of deep geomagnetic impacting on growth rate and sensitivity to antibiotics in Escherichia coli

The short-term (1-7 days) and prolonged (30-90 days) adaptation of bacteria to hypomagnetic conditions was studied by observing changes in growth rate and resistance to antibiotics in E. coli used as an experimental model. The decrease of the growth rate of bacterial cells in the exponential phase was found to occur. The effect disappeared when the screened culture was placed under normal geomagnetic conditions for 1 hour daily. After prolonged screening an increase in the resistance of these bacteria to kanamycin and carbenicillin was registered. These changes were not hereditary: after the cessation of screening the sensitivity of the bacteria to antibiotics was restored.     

Hyperthermic sensitivity and growth stage in Escherichia coli

Hyperthermic sensitivities of Escherichia coli B/r and Bs-1 were determined for lag-, midlog-, and stationary-phase cells at 47, 48, and 49 degrees C. In both strains midlog-phase cells were strikingly more heat sensitive (100-fold greater killing after 4 h at 48 degrees C) than stationary-phase cells, with intermediate sensitivity for lag-phase cells. In contrast to the reported difference in the radiation sensitivity between these two strains, very little difference in heat sensitivity was seen. Patterns of fatty acid composition of both strains were very similar at each phase of growth. From midlog to stationary phase, 16:1 and 18:1 unsaturated fatty acids decrease from 16 and 30% to 0.5 and 3%, respectively, while the C17 and C19 cyclopropane fatty acids increase from 7 and 3% to 22 and 25%, respectively. Concomitant with these changes in fatty acid composition, substantially higher membrane microviscosity values were recorded for stationary-phase cells. Total membrane microviscosity was positively associated with the C17 and C19 cyclopropane fatty acid composition and with cell survival following hyperthermia. In contrast to hyperthermic sensitivity, radiation survival differences between B/r and Bs-1 are little affected by growth stage. We propose that these results are consistent with a critical influence of membrane lipids on cellular hyperthermic sensitivity and further that the target sites for radiation and hyperthermia are different in these cells.     

Influence of specific growth rate and nutrient limitation upon the sensitivity of Escherichia coli towards chlorhexidine diacetate

The sensitivity of Escherichia coli to chlorhexidine has been assessed for cells grown in a chemostat at a variety of specific growth rates, under conditions of carbon, nitrogen, phosphorus and magnesium limitation. At slow rates of growth ( ca 0.08/h) little difference in sensitivity was observed. As growth rate was increased, however, the sensitivity of nitrogen- and carbon-limited cells increased whilst that of magnesium- and phosphate-limited cells decreased. It was not possible to correlate the observed patterns of chlorhexidine sensitivity with any single measure of cell envelope composition (phospholipid content, lipopolysaccharide, envelope proteins, etc.). The results presented are not consistent, therefore, with any simple model for chlorhexidine binding or action and more probably reflect subtle interaction between chlorhexidine, phospholipid-lipopolysaccharide complexes and cations within the envelope.     

Influence of specific growth rate and nutrient limitation upon the sensitivity of Escherichia coli towards chlorhexidine diacetate

The sensitivity of Escherichia coli to chlorhexidine has been assessed for cells grown in a chemostat at a variety of specific growth rates, under conditions of carbon, nitrogen, phosphorus and magnesium limitation. At slow rates of growth (ca 0.08/h) little difference in sensitivity was observed. As growth rate was increased, however, the sensitivity of nitrogen- and carbon-limited cells increased whilst that of magnesium- and phosphate-limited cells decreased. It was not possible to correlate the observed patterns of chlorhexidine sensitivity with any single measure of cell envelope composition (phospholipid content, lipopolysaccharide, envelope proteins, etc.). The results presented are not consistent, therefore, with any simple model for chlorhexidine binding or action and more probably reflect subtle interaction between chlorhexidine, phospholipid-lipopolysaccharide complexes and cations within the envelope.     

Relationship of Escherichia coli density to growth rate and cell age.

The cell densities of Escherichia coli strains B/rA, BrF, and K-12 (OV-2) were measured at several growth rates and found to be very near 1.105 g/ml in all cases. Ninety percent of the cells of any exponentially growing population banded at densities differing less than 0.75% from the mean. Synchronized populations of B/rA selected as newborn cells were found to keep their density constant for longer than one generation time. However, if selection was based on cell size, by sedimentation through a sucrose gradient, cell density was found to be almost 2% lower than that of newborn cells, but it reached normal values before the first division had taken place. These results meant that mass and volume during the lifetime of the bacterial cell followed parallel kinetics. It was unlikely that density could regulate any event of the lifetime of a cell; on the contrary, density seemed to be a physical parameter that was well controlled during the bacterial growth.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.